Niveles de antitoxina tetánica y diftérica relacionadas con el alelo HLA-B27 en pacientes cubanos con uveítis anterior no infecciosa / Tetanus and diphtheria antitoxin levels related to the HLA-B27 allele in Cuban patients with non-infectious anterior uveitis

La uveítis anterior no infecciosa es una enfermedad inflamatoria del ojo que afecta al tracto uveal y que puede causar ceguera total y otras discapacidades visuales. Esta enfermedad se ubica en el espectro de enfermedades autoinmunes y autoinflamatorias. Se han descrito respuestas no adecuadas a la vacunación en enfermedades mediadas por el sistema inmune, por lo que se evaluaron los niveles de antitoxina tetánica y diftérica en pacientes cubanos con uveítis anterior no infecciosa, relacionada con el alelo HLA-B27. Se determinaron los niveles de antitoxina tetánica y diftérica mediante ELISA en 190 pacientes con uveítis anterior no infecciosa y controles supuestamente sanos. El 97,37 por ciento de los pacientes con uveítis mostraron niveles de protección de antitoxina tetánica mayor o igual a 0,1 UI/mL, similar a lo observado en los controles sanos (98,95 por ciento) (p=0,4385). Las proporciones de pacientes con uveítis anterior no infecciosa y sus controles en los diferentes niveles de protección de antitoxina tetánica fueron similares (p>0,05), al igual que los títulos medios geométricos (p=0,2907). En los pacientes con uveítis, de 65 años o más, se detectó una mayor proporción de individuos con títulos protectores de larga duración (>1,0 UI/mL) de antitoxina diftérica (p=0,0065). En los pacientes con uveítis no se observó asociación entre la presencia del alelo HLA-B27 y la respuesta de anticuerpos frente al toxoide tetánico (p=0,6196) y diftérico (p=0,1917). El 37,9 por ciento de los pacientes con uveítis y el 42 por ciento de los controles, presentaron títulos no protectores (<0,1 UI/mL) de antitoxina diftérica (0,1148). La mayoría de los pacientes con uveítis anterior no infecciosa y los controles supuestamente sanos presentaron protección frente al toxoide tetánico; mientras que, en los pacientes con uveítis, así como en los controles supuestamente sanos, con edad igual o más de 18 años, se debe reevaluar incluir refuerzos con toxoide diftérico para alcanzar mayores niveles de protección frente a la difteria(AU)

Non-infectious anterior uveitis is an inflammatory disease of the eye that affects the uveal tract and can cause total blindness and other visual disabilities. Autoimmune and inflammatory diseases are associated with qualitative and quantitative alterations in the immune response; therefore, the levels of tetanus and diphtheria antitoxin related to the HLA-B27 allele were evaluated in Cuban patients with non-infectious anterior uveitis. Tetanus and diphtheria antitoxin levels were determined by ELISA in 190 patients with non-infectious anterior uveitis and healthy control individuals. 97.37 percent of patients with uveitis showed protective tetanus antitoxin levels greater than and equal to 0.1 IU/mL as well as healthy controls (98.95 percent) (p=0.4385). The proportions of patients with non-infectious anterior uveitis and presumably healthy controls in the different levels of protective tetanus antitoxin were similar (p>0.05) at all levels of protection, as were the geometric mean titers for this antitoxin (p=0.2907). Patients with uveitis aged 65 years or older had a higher proportion of individuals with long-term reliable protective titers (>1.0 IU/mL) of diphtheria antitoxin (p=0.0065). In uveitis patients, no association was observed between the presence of the HLA-B27 allele and the antibody response against tetanus toxoid (p=0.6196) and diphtheria (p=0.1917). Similarly, 37.9 percent of patients with uveitis and 42 percent of their controls had non-protective titers (<0.1 IU/mL) of diphtheria antitoxin (0.1148). Most patients with anterior uveitis and control subjects were protected against tetanus (p>0.05), while in patients with uveitis and supposedly healthy controls, aged 18 years or older, the administration of booster doses with diphtheria toxoid should be reevaluated to achieve higher levels of protection against diphtheria(AU)

Validación de un ensayo inmunoenzimático tipo ELISA para cuantificar niveles de antitoxina diftérica en suero humano / Validation of an ELISA-type immune-enzymatic test to quantify levels of diphtheria antitoxin in human serum

Introducción: La difteria aún persiste en numerosos países. En Cuba, estudios realizados en diferentes grupos etarios han demostrado que existen niveles no protectores de antitoxina diftérica en la población, por lo que es necesario contar con métodos que permitan la estimación serológica de la inmunidad poblacional. La cuantificación de anticuerpos contra antígenos vacunales como la toxina diftérica es además un método útil, rápido y económico para evaluar la respuesta inmune. Objetivo: Validar un ensayo inmunoenzimático tipo ELISA para cuantificar los niveles de antitoxina diftérica en suero humano. Material y Método: Se realizó un estudio experimental de desarrollo tecnológico, en el cual se determinaron los valores óptimos de las variables que influyen en el resultado de un ensayo inmunoenzimático heterogéneo indirecto para la cuantificación de antitoxina diftérica, desarrollado en el laboratorio de Inmunología del Centro Nacional de Genética Médica de Cuba. La curva de calibración se evaluó contra el estándar de la OMS (Diphtheria Antitoxin Human Serum 00/496). Se realizó la validación analítica del método estandarizado. Resultados: Los coeficientes de variación intraensayo e interensayo fueron inferiores a 10 por ciento y 20 por ciento, respectivamente. En la exactitud y selectividad se encontraron valores de recobrado entre 90 y 110 por ciento. El paralelismo entre la curva estándar y las muestras estudiadas presentó un coeficiente de variación menor o igual a 10 por ciento. El límite de cuantificación fue 0,015 UI/mL y el de detección 0,0039 UI/mL. Conclusiones: El resultado obtenido en la precisión, exactitud y selectividad del ensayo inmunoenzimático tipo ELISA desarrollado demostró que puede ser utilizado en la práctica clínica para cuantificar los valores de antitoxina diftérica en suero humano(AU)

Introduction: Diphtheria still persists in many countries. In Cuba, studies conducted in different age groups have demonstrated that there are non-protective levels of diphtheria antitoxin in the population, so it is necessary to have methods that allow the serologic survey of population immunity. The quantification of antibodies against vaccine antigens such as diphtheria toxin is also a useful, rapid and economic method to evaluate the immune response. Objective: To validate an ELISA-type immune-enzymatic test to quantify the levels of diphtheria antitoxin in human serum. Material and Method: An experimental study of technological development was carried out in the Immunology Laboratory of the National Medical Genetics Center, Havana, Cuba. The optimal values ​​of the variables that influence on the result of the indirect heterogeneous immune-enzymatic test for the quantification of diphtheria antitoxin were determined. The calibration curve obtained was evaluated against the WHO standard (Diphtheria Antitoxin Human Serum 00/496). The analytical validation of the standardized method was performed. Results: The intra-assay and inter-assay coefficients of variation were less than 10 percent and 20 percent, respectively. Recovery values ​​between 90 and 110% were found in accuracy and selectivity. The parallelism between the standard curve and the samples studied showed a coefficient of variation lower or equal to 10 percent. The limit of quantification was 0,015 IU/mL and the one of detection was 0,0039 IU/mL. Conclusions: The result obtained in the precision, accuracy and selectivity of the ELISA-type immune-enzymatic test developed and validated in the National Medical Genetics Center demonstrated that it can be used in the clinical practice to quantify the values ​​of diphtheria antitoxin in human serum(AU)

Seroprevalence of Corynebacterium diphtheriae among vaccinated population of Rawalpindi/Islamabad, Pakistan

Diphtheria is a communicable disease of global significance, and its outbreaks have to be reported to the world community under the International Health Regulations [IHR]. A pilot seroepidemiological survey was conducted to assess immunity status of diphtheria among healthy individuals of Rawalpindi/Islamabad [Pakistan], who had been administered at least one dose of the vaccine against the disease, as part of childhood vaccination. The study group comprised of 128 healthy subjects, grouped according to the decade representing their age. Antidiphtheria IgG levels were measured by Enzyme Linked Immunosorbent Assay [ELISA] method. The studied sample showed 100% prevalence of diphtheria antitoxin, confirming prior vaccination; however 49.2% exhibited only minimal protection against diphtheria. Full protection was observed in a significantly higher [p=0.013] percentage of males [54.45%] as compared to female subjects [33.33%]. Maximum level of serum antibodies were seen in 1-10 year age group [0.195+0.031 IU/mL], which was significantly higher than that recorded in the age group of 11-20 [p=0.024] and above 30 years [p=0.0064]. The present results emphasize the need for periodical booster immunization in adolescents and adults, after primary childhood immunization

A diphtheria outbreak in Buri Ram, Thailand.

In May 1996 there was an outbreak of diphtheria in Buri Ram, Thailand which infected 31 patients, 8 males and 23 females. The mean age of the patients was 8 +/- 5 years. Seventy-four percent had a history of childhood vaccinations. Common signs and symptoms included fever (100%) which was low grade in 61%, sore throat (90%), upper airway obstruction (3%), and hoarseness (10%). Pseudomembranes (seen in 100%) were located on the tonsils (71%), pharynx (22%), larynx (9.6%), and uvula (6%). The mean duration of symptoms prior to admission was 2 days with a range of 1 to 5 days. Complications included upper airway obstruction (10%) and cardiac complications (10%). There were no neurological complication or deaths. There were negative associations between cardiac complications, severity of disease and previous diphtheria vaccination. The ages varied from children to adults. Early recognition and prompt treatment decreased complications and mortality in this group of patients when compared with Chiang Mai and Queen Sirikit National Institute of Child Health (QSNICH) studies.

Determination of low tetanus or diphtheria antitoxin titers in sera by a toxin neutralization assay and a modified toxin-binding inhibition test

A method for the screening of tetanus and diphtheria antibodies in serum using anatoxin (inactivated toxin) instead of toxin was developed as an alternative to the in vivo toxin neutralization assay based on the toxin-binding inhibition test (TOBI test). In this study, the serum titers (values between 1.0 and 19.5 IU) measured by a modified TOBI test (Modi-TOBI test) and toxin neutralization assays were correlated (P < 0.0001). Titers of tetanus or diphtheria antibodies were evaluated in serum samples from guinea pigs immunized with tetanus toxoid, diphtheria-tetanus or triple vaccine. For the Modi-TOBI test, after blocking the microtiter plates, standard tetanus or diphtheria antitoxin and different concentrations of guinea pig sera were incubated with the respective anatoxin. Twelve hours later, these samples were transferred to a plate previously coated with tetanus or diphtheria antitoxin to bind the remaining anatoxin. The anatoxin was then detected using a peroxidase-labeled tetanus or diphtheria antitoxin. Serum titers were calculated using a linear regression plot of the results for the corresponding standard antitoxin. For the toxin neutralization assay, L+/10/50 doses of either toxin combined with different concentrations of serum samples were inoculated into mice for anti-tetanus detection, or in guinea pigs for anti-diphtheria detection. Both assays were suitable for determining wide ranges of antitoxin levels. The linear regression plots showed high correlation coefficients for tetanus (r² = 0.95, P < 0.0001) and for diphtheria (r² = 0.93, P < 0.0001) between the in vitro and the in vivo assays. The standardized method is appropriate for evaluating titers of neutralizing antibodies, thus permitting the in vitro control of serum antitoxin levels.

Study on the immunity of diphtheria in children at county level with different 4-vaccine coverage rates based on the results from "National Immunization Program Review" in 2004 / 中华流行病学杂志

<p><b>OBJECTIVE</b>To investigate the immunity level of diphtheria antibody among children living in the areas where different coverage rates of 4-vaccines stratified by results of national immunization program (NIP) reviewed in 2004.</p><p><b>METHODS</b>According to data from 4-vaccine coverage rates of NIP reviewed in 2004, 3 levels could be set. We randomly selected 2 counties at each level and then 10 villages from each county with 42 children involved who were born between 1992 and 2003. ELISA quantitative method was used to test IgG of diphtheria antitoxin.</p><p><b>RESULTS</b>(1) The positive rate of diphtheria antitoxin was only 49.6% with the highest as 78.1% and lowest as 33.0%. There was a significant decreasing trend of this positive rate with the increase of age. The highest (61.2%) fell in the group that were born in 2003 and the lowest (37.6%) was seen among children born in 1992 to 1995. (2) Geometric mean concentrations (GMCs) was only 0.48 IU/ml with a trend of decrease when age was increasing. There was no GMCs peak seen in children who were at the age of boosting, as expected. (3) Positive rates of children born between 2001 and 2003 were lower than 62% while the diphtheria-pertussis-tetanus (DPT) vaccine coverage rates were all higher than 90%. (4) There was no significant difference of diphtheria antitoxin positive rates between children with eligible routine immunization (58.1%) and those were ineligible (59.6%).</p><p><b>CONCLUSION</b>Other than some specific ones, children from most of the investigated counties had a low level of antibody against diphtheria. The coverage rate of DPT vaccine did not necessarily reflect the immunity against diphtheria, suggesting the increase of immunity against diphtheria an urgent task to be taken care of.</p>

Age Related Seroepidemiological Study of Diphtheria among Koreans / 감염

BACKGROUND: The incidence of diphtheria has been markedly reduced and almostly eradicated by widespread use of DTP vaccines in developed countries. However, outbreaks of this disease may be occurred under some circumstances of ineffective immunization. In recent time, some studies reported persistent outbreaks of diphtheria in developed countries and indicated the existence of a large pool of susceptible individuals with potential for epidemic infection. In Korea, diphtheria vaccination has been well maintained since 1956 with high acceptant vaccination rates. So, there has been no reported diphtheria patient since 1987. But, there has been few study to diphtheria serosuvey, and no assessment of diphtheria immunization. Also, we do not use Td vaccine in adults for maintenance of diphtheria immunity. In this aspect, we conducted age related seroepidemiology of diphtheria and indirectly assessed the immunity of diphtheria vaccines, used in Korea. METHODS: For the evaluation of age related serosurvey of diphtheria immunity in Korean populations, study subjects below 10 years old aged children were classified into 10 groups (A~J) with one year interval, and beyond this age to 60 years old aged adults were classified into 5 group (K~O) with 10 years interval. And the adults over 60 years old age was classified into the last group (P). The numbers of each group were 100, and sex distributions of each group were almostly equal. And for the indirect assessment of diphtheria immunization in Korean children, children under 15 years old were classified into 6 groups (I~ VI) according to the status of DTaP vaccination. The numbers of this each group were 30, and sex ratio was almostly equal. Detection of specific IgG antibody to diphtheria toxin were determined by ELISA (contained fragment A & B toxin; IBL, Germany). RESULTS: In age related groups, the antibody titers to diphtheria toxin were well maintained until 10 years old age group, thereafter the titers abruptly decreased below 0.1 IU/mL and then slightly elevated after 30 years old age group and then maintained with low levels. In the groups related DTaP vaccine status, the antibody titers were very low (below 0.07 IU/mL) in prevaccination status, but the titers after primary vaccinations were markedly increased and maintained (above 0.6 IU/mL) until 15 years. And diphtheria antitoxin levels in the groups (L, M, N) showed no significant differences between males and females. CONCLUSION: The results of our study showed that the antibody titers to diphtheria toxin in the 20~50 years old aged groups dramatically decreased. This result indicated that vaccine induced diphtheria immunity did not last throughout life, and Td vaccination program in adults should be considered for maintenance of diphtheria immunity. And the immunity to diphtheria in Korean children indicated that 3 timesprimary and 2 times booster diphtheria immunizations were optimal.

Quantitation of tetanus and diphtheria antitoxins in mouse sera by indirect haemagglutination.

Serum samples obtained from 75 groups of mice immunized with various doses of adsorbed tetanus vaccine, adsorbed diphtheria-tetanus vaccine and adsorbed diphtheria-tetanus-pertussis vaccine were titrated for tetanus antitoxin content by an in-vitro indirect haemagglutination (IHA) and by toxin neutralization test (TN) in mice. From these serum samples of 49 groups of mice which were immunized with combined vaccine containing diphtheria toxoid were titrated for their diphtheria antitoxin content by IHA and by i.d. toxin neutralization test (TN) in guinea pigs. Good correlations were found between the estimates obtained by in-vitro IHA and in vivo TN tests in both tetanus and diphtheria antitoxin titrations. The minimum level of tetanus or diphtheria antitoxin detectable by IHA was 0.00039 IU/ml. It is concluded that IHA is a simple, sensitive and reproducible alternative test which can replace the animal TN tests for the estimation of tetanus and diphtheria antitoxins and could reliably be used in the potency assay of tetanus and diphtheria toxoids of combined vaccines based on antibody induction in mice.

Serological Immunity to Diphtheria among Korean Population / 감염

BACKGROUND: Diphtheria epidemics in Russia have spread to all the other independent states of the former Soviet Union and East European countries around 1990s. One of the most important measures in preventing diphtheria is to maintain high levels of immunity in the population. We studied the diphtheria antibody levels of 1,086 participants to investigate herd immunity in Korea. METHODS: The tested 1,086 serum specimens were collected from healthy individuals from September 1995 to March 1996. Diphtheria antitoxin titers were measured by a micro cell culture method using Vero cells. Antibody titer of 0.01 IU/ml to neutralize diphtheria toxin is an internationally accepted protective level. RESULTS: We studied the diphtheria antitoxin titer levels of 1,086 cases consisting of 579 males and 507 females. The proportion of protective antitoxin level to diphtheria is 69.2%. Diphtheria antitoxin levels showed no significant difference between males and females. The highest seropositive rate was observed in the 5 to 9-year old age group(95.8%). The seropositivity rate declined with age. The lowest seropositive rate was observed in the 20~39 years of age, maximally 43.4 %. Over 40 years of age, the seropositive rates increased again. CONCLUSION: The antibody titers in the Korean population declined from 95.8% to below 50% with age in the 1~39 year-old age group. To maintain the rate of population with protective antibodies to diphtheria, we recommend Td booster immunization to adults with low antitoxin titers and continuous survey for antitoxin titers.

Immunity to diphtheria in women of childbearing age in Delhi in 1994: evidence of continued Corynebacterium diphtheriae circulation.

Blood samples from 171 full-term pregnant women (aged 18-38 years) of middle socioeconomic status from Delhi were tested for diphtheria antitoxins by indirect hemagglutination (IHA) test. History of primary immunization/clinical diphtheria during childhood was not ascertainable, but none had been revaccinated against diphtheria at any time. About 94% women had very high antitoxin titers (> or = 0.125 IU/ ml); none had antitoxin titer less than 0.015 IU/ml, the minimum protective level. The titers were uniformly high in all age groups. However, women having 2 or more children had significantly higher antitoxin titers than those having no or one child (p < 0.01). The results from this study and historical data on diphtheria in Delhi are compatible with continued transmission of C. diphtheriae in recent times in Delhi which is of sufficient magnitude to boost the antitoxin levels in adults, especially mothers having two or more children. The study highlights the need of increasing the immunization coverage with DPT among children to reduce the transmission of Corynebacterium diphtheriae.

Potency assay of diphtheria antitoxin in vero cell micro-cultures

Se establecieron condiciones para llevar a cabo la titulación de antitoxina diftérica en células Vero (TCC), y comparar los resultados con la prueba intradérmica (TID) tradicionalmente empleada en todo el mundo para determinación de potencia de toxoide diftérico y de antitoxina de uso terapéutico. Los resultados de titulaciones en pruebas intradérmicas y en cultivos celulares mostraron gran repetibilidad y detectabilidad a concentraciones muy bajas de toxina en la prueba de cultivos celulares, así como una correlación satisfactoria en la titulación de antitoxinas preparadas por inmunización con Patrón Internacional de OMS y con antitoxina de uso terapéutico, pero en los sueros de cobayo se observó un título aproximadamente 10 veces menor al de la prueba intradérmica. Se discuten las posibles causas de estas diferencias de títulos. Esta prueba se podrá adoptar en la forma propuesta para determinar potencia de antitoxina diftérica. En la prueba de potencia del toxoide diftérico, se podría usar si el límite de título en los cobayos se ajusta a 0.2 UIAD en lugar de 2.0 UIAD/ml que se emplea actualmente, considerando la equivalencia obtenida entre las dos pruebas y tomando en cuenta una relación 1:10 entre las pruebas TCC y TID

Diphtheria in the eighties: experience in a south Indian district hospital.

Epidemiological and clinical analysis of 228 cases of diphtheria treated during a 10-year period from 1980-1989 is presented. No statistically significant change in the incidence over a decade was noticed. A high percentage of cases (44.74%) were noted in the age group of 5 years and above. Bilateral tonsillar and pharyngeal membranes together were seen in 60% cases. All patients were treated with diphtheria antitoxin and benzyl penicillin. Severe myocarditis (9 cases), fulminant haemorrhagic complications (2 cases), palatal palsies (11 cases) and polyneuritis and cranial nerve paralysis (2 cases) were the significant complications. Case fatality rate was 13.21%. Statistically significant increase in the mortality (p less than 0.05) was noticed in the year 1987 and onwards. In this period, myocarditis (8 out of 16 deaths) is gradually replacing laryngeal obstruction, the major cause of death in early eighties or post-tracheostomy complications (6 out of 16 deaths). Only 2 patients died of haemorrhagic diphtheria in this period.

Immunity against diphtheria in the age group 15 to 25 years.

Levels of diphtheria antitoxin in 254 serum samples were estimated in subjects in the age group 15 to 25 years with no previous history of immunisation against diphtheria using indirect haemagglutination test. All the individuals showed more than 0.03 IU/ml of diphtheria antitoxin level. This indicates that the age group studied was not susceptible to diphtheria. No significant relationship could be, however, ascertained between diphtheria antitoxin level and epidemiological factors like age, area (rural/urban), socioeconomic status, family size, education status and religion.

Esterilizaçäo de soros e vacinas por radiaçäo gama de cobalto / Sterilization of sera and vaccines by cobalt gamma radiation

Os soros antitetânico, antidiftérico e antibotrópico-crotálico e, ainda, a vacina diftérica-pertussis-tetânica foram submetidos a diferentes intensidades de radiaçäo gama, com os seguintes intuitos: verificaçäo da resistência das atividades específicas destes produtos à açäo dos raios gama; avaliaçäo das possibilidades de utilizaçäo deste tipo de energia radiante para a esterilizaçäo de alguns soros hiperimunes heterólogos e vacinas, mais comumente utilizados em saúde pública. Os resultados obtidos, segundo os parâmetros empregados, mostraram a possiblidade de esterilizaçäo sem alteraçöes das atividades específicas, de ordem biológica ou química, dos produtos experimentados.

Difteria faríngea: revisión de 307 enfermos / Pharyngeal diphteria: review of 307 patients

Si bien en la última década, la morbimortalidad de la difteria en Chile ha disminuído considerablemente, desde 1984 se ha observado un aumento, como consecuencia de brotes geográficamente limitados. Ha parecido interesante comunicar el análisis de 307 enfermos, bacteriológicamente confirmados, en el Hospital de Enfermedades Infecciosas de Santiago de Chile, entre enero 1972 y diciembre 1984; 174 mujeres (56,6 por ciento) y 133 hombres (43,4 por ciento), con edades entre 5 y 14 años, en el 54,5 por ciento. Un 26 por ciento tuvo mas de 25 años, lo que estaría de acuerdo al desplazamiento paulatino de la difteria a edades mayores. La mitad presentó formas graves. El 84,7 por ciento ingresó dentro de los primeros 4 días. Recibieron antitoxina y la mayoría penicilina sódica, junto con corticoides en los cuadros severos. se comprobó complicaciones únicas o asociadas: miocarditis en 78 (29 por ciento) de 269 enfermos con electrocardiograma; parálisis, mas frecuente velopalatina (22,5 por ciento); nefritis, en 23 (14,5 por ciento) de 159 con uremia y/o creatinemia mas examen de orina. Las complicaciones fueron casi excepcionales en la forma común. La letalidad global fue de 5,5 por ciento ya que no hubo fallecidos con forma leve. En cambio, aquella alcanzó a 36,5 por ciento en la maligna, estando determinada por el daño miocárdico y renal

Hemorrhagic diphtheria.

Two cases of hemorrhagic diphtheria, a rare entity and severest form are reported. Both of them have been mildly ill for more than 3 days and did not seek medical attention, suddenly developed bleeding episodes with clinical evidence of diphtheria in association with severe thrombocytopenia. Inspite of antitoxin, antibiotics therapy and supportive care one died on the third day. The second case was given additional steroid therapy to the regimen and survived with diphtheria myocarditis.