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PubMed2002

Receptor-based antidote for diphtheria.

Although equine diphtheria antitoxin may be an effective therapy for human diphtheria, its use often induces serum sickness. We describe here a strategy for developing an alternative treatment based on the human diphtheria toxin (DT) receptor/heparin-binding epidermal growth factor-like growth factor (HB-EGF) precursor. Recombinant mature human HB-EGF acts as a soluble receptor analog, binding radioiodinated DT and preventing its binding to the cellular DT receptor/HB-EGF precursor. However, the possibility existed that radioiodinated DT-HB-EGF complexes associate with cells due to the binding of the heparin-binding domain of recombinant HB-EGF to cell surface heparan sulfate proteoglycans. This possibility was confirmed by performing DT binding studies in the presence of heparin. A recombinant truncated HB-EGF (residues 106 to 149), which lacks most of the heparin-binding domain, showed an essentially heparin-independent binding of radioiodinated DT to cells. Furthermore, it was a more effective inhibitor of DT binding than was recombinant mature HB-EGF. Since mature HB-EGF is a known ligand for the EGF receptor and is thus highly mitogenic (tumorigenic), we then changed amino acid residues in the EGF-like domain of the recombinant truncated HB-EGF and demonstrated that this DT receptor analog (I117A/L148A) displayed a low mitogenic effect. The truncated (I117A/L148A) HB-EGF protein retained high DT binding affinity, as confirmed by using surface plasmon resonance. Our results suggest that the truncated (I117A/L148A) HB-EGF protein could be an effective, safe antidote for human diphtheria.

3T3 Cells↗
PubMed1963

IDENTIFICATION OF CORYNEBACTERIUM DIPHTHERIAE WITH FLUORESCENT ANTIBACTERIAL REAGENTS.

Moody, Max D. (Communicable Disease Center, Atlanta, Ga.) and Wallis L. Jones. Identification of Corynebacterium diphtheriae with fluorescent antibacterial reagents. J. Bacteriol. 86:285-293. 1963.-Conditions are described whereby fluorescent-antibody reagents could be prepared and used to identify Corynebacterium diphtheriae in pure and mixed cultures and in clinical materials. The use of O and OK antigens for immunization of rabbits to prepare the antibody was compared. The most satisfactory reagents were those made from serum of rabbits injected with live (OK) suspensions of C. diphtheriae. Such fluorescent reagents were used successfully in direct and indirect fluorescent-antibody tests to identify both toxinogenic and atoxinogenic C. diphtheriae but not to differentiate the two kinds of organisms.

Animals↗
PubMed1998

Antitoxin-in-membrane and antitoxin-in-well assays for detection of toxigenic Corynebacterium diphtheriae.

The Elek culture plate precipitin test is routinely used for the detection of exotoxin from toxigenic strains of Corynebacterium diphtheriae. Recently, the World Health Organization standardized this test to ensure accuracy, reliability, and reproducibility. In this study, we further modified the standard Elek test by using the antitoxin-in-membrane (AIM) and antitoxin-in-well (AIW) approaches. In the AIM tests, each strain was stabbed and streaked backwards and away from a point approximately 7 mm from the edge of a sterile cellulose acetate-cellulose nitrate filter membrane disk (pore size, 0.45 microm; diameter, 25 mm) containing 25 IU of diphtheria antitoxin. For AIW tests, a central well (diameter, 5 mm) containing 9 microl of antitoxin (4.5 IU) was surrounded by eight equidistant stab-streaks of each strain placed 10 mm from the well. In both methods, precipitin bands of identity typically were noted after 24- and 48-h incubations at 37 degrees C. Both toxigenic and weak toxigenic strains gave clear and reproducible results. Compared with the standard Elek test, the AIM and AIW tests each use 50% less medium and 75 and 87% less antitoxin, respectively. AIM has the potential to test up to 14 isolates and AIW has the potential to test up to 24 isolates on the same plate. Furthermore, clearer positives were noted with weak toxigenic strains. In a blinded test of 209 verified C. diphtheriae isolates, a 99.5% agreement with the standard Elek test was obtained overall. Both modifications conserve reagents and medium, permit the simultaneous testing of a larger number of strains, and may be particularly suitable for reference laboratories or hospitals involved in diphtheria epidemic settings.

Antitoxins↗
PubMed1999

Use of molecular subtyping to document long-term persistence of Corynebacterium diphtheriae in South Dakota.

Enhanced surveillance of patients with upper respiratory symptoms in a Northern Plains community revealed that approximately 4% of them were infected by toxigenic Corynebacterium diphtheriae of both mitis and gravis biotypes, showing that the organism is still circulating in the United States. Toxigenic C. diphtheriae was isolated from five members of four households. Four molecular subtyping methods-ribotyping, multilocus enzyme electrophoresis (MEE), random amplified polymorphic DNA (RAPD), and single-strand conformation polymorphism-were used to molecularly characterize these strains and compare them to 17 archival South Dakota strains dating back to 1973 through 1983 and to 5 isolates collected from residents of diverse regions of the United States. Ribotyping and RAPD clearly demonstrated the household transmission of isolates and provided precise information on the circulation of several distinct strains within three households. By MEE, most recent and archival South Dakota strains were identified as closely related and clustered within the newly identified ET (electrophoretic type) 215 complex. Furthermore, three recent South Dakota isolates and eight archival South Dakota isolates were indistinguishable by both ribotyping and RAPD. All of these molecular methods showed that recent South Dakota isolates and archival South Dakota isolates were more closely related to each other than to the C. diphtheriae strains isolated in other parts of the United States or worldwide. The data also supported the improbability of importation of C. diphtheriae into this area and rather strongly suggest the long-term persistence of the organism in this region.

Bacterial Typing Techniques↗
PubMed1978

Determination of Corynebacterium diphtheriae toxigenicity by a colorimetric tissue culture assay.

Chinese hamster ovary (CHO) cell cultures in microtiter wells are sensitive to growth inhibition and killing by picogram quantities of diphtheria toxin. In the absence of biologically active toxin, the CHO cell culture produces sufficient acidic metabolites to change the phenol red pH indicator from pink to yellow within 56 h. In the presence of 10 pg of toxin per well, growth inhibition can be observed microscopically within 24 h. Diphtheria toxin can be qualitatively assayed from culture supernatants of Corynebacterium diphtheriae or from beta-phage agar plaque plugs. The colorimetric CHO cell assay method for determining toxigenicity allows for the large-scale screening of either diphtheria toxigenicity or antitoxin titration of sera.

Animals↗
PubMed2005

Diphtheria with polyneuropathy in a closed community despite receiving recent booster vaccination.

INTRODUCTION AND METHODS: We report 20 patients aged 18-24 years from Latvia with diphtheritic polyneuropathy. All lived in a closed community and 80% were known to have been fully vaccinated against diphtheria until at least 14 years old. Diphtheria antitoxin had been administered within 3 days of the onset of upper respiratory tract infection in 16 patients and 15 received antibiotics. RESULTS: Neurological symptoms developed after a median of 43 days (range 35-58) compared to only 10 days in previous studies of unvaccinated patients. All showed evidence of mild limb polyneuropathy with electrophysiological evidence of polyneuropathy. Only 30% showed early bulbar abnormalities compared to the usual rate of over 95% in diphtheritic polyneuropathy. However, 45% had later bulbar deterioration coinciding with the limb polyneuropathy. CONCLUSIONS: These patients show that an attenuated form of polyneuropathy of later onset, with less prominent early bulbar features, can occur in patients vaccinated against diphtheria according to schedule but living in a closed community in a country where diphtheria remains endemic.

Adolescent↗
PubMed2004

Optimization of casein-based semisynthetic medium for growing of toxigenic Corinebacterium diphtheriae in a fermenter.

Diphtheria toxin is produced by growing Corinebacterium diphtheriae either in a semisynthetic casein-based medium or in the Pope-Lingood meat extract based medium. The World Health Organization advises the use of the semisynthetic one, as it has important advantages. Data on the composition of casein-based media and their ability to support high toxin production are not freely available. Important factors affecting toxin production during C. diphtheriae cultivation are the pH of the culture medium and the concentration of casein hydrolysate and of Fe2+. We established that the optimal pH for toxin production is 7.2. The highest yield of toxin was obtained using a casein hydrolysate concentration of 35.0 g/L and a Fe2+ concentration of 0.05-0.41 microg/mL. Under these conditions, diphtheria toxin with higher purity and yield compared with the batches obtained using the meat-based medium of Pope-Lingood was produced.

Caseins↗
PubMed2006

Reactogenicity of meningococcal C conjugate vaccines when administered at the same time as, or a month prior to or after, tetanus and diphtheria booster vaccinations.

INTRODUCTION: This single blind study was conducted to address safety concerns regarding coadministration of diphtheria/ tetanus (DT,Td) boosters and meningococcal C conjugate (MCC) vaccines containing diphtheria or tetanus conjugate proteins. METHODS: 1754 subjects (835 preschoolers and 919 school leavers) were randomised to receive one of three MCC products a month before, after or at the same as routine DT or Td boosters. They (or their parents) completed a health diary for 10 days to record local reactions and systemic symptoms after each vaccination. The effect of antibody levels pre and post-vaccination, preexisting allergies and medication taken before and for up to ten days after vaccination on reactogenicity was assessed. RESULTS: No relationship between prevaccination antibody levels and local reactions or systemic symptoms was found. Local reactions were more common after DT/Td than MCC vaccination and were related to post-vaccination diphtheria and tetanus antibody levels in younger children and to the post-vaccination tetanus only antibody levels in older children. Preexisting allergies were not related to reactogenicity. Use of analgesics/antipyretic medication significantly reduced the incidence of local reactions and of some systemic symptoms. In the first three days after vaccination there was an excess of some systemic symptoms including fatigue/malaise and headache for all ages, and crying/ irritability for younger children and nausea/vomiting and dizziness/faintness for older subjects. Eleven serious adverse events were reported, none of which was deemed related to vaccination. CONCLUSION: MCC vaccines can be given under the above schedules and safely in the presence of raised diphtheria and tetanus antibody levels without materially altering reactogenicity profiles of either vaccine.

Adolescent↗
PubMed2000

[Diphtheria in a highly immunized population].

Although diphtheria vaccination is routine world-wide, outbreaks of the disease continue to occur in supposedly vaccinated populations. The incidence of diphtheria in Israel is very low, with only 3 cases reported in the past 24 years (all in unvaccinated children). In 2 of the 3 an asymptomatic carrier was identified among the patients' close contacts, presumable the source of the infection. We describe a recent case of diphtheria. It is important for physicians to be aware of the possibility of diphtheria occurring despite the high rate of vaccination in our population.

Child↗
PubMedDate not supplied

[Antibacterial immunity in diphtheria. Experimental research].

A number of 700 guinea pigs were immunized by parenteral route with non-toxigenic Corynebacterium diphtheriae strains, then inoculated with various doses of homologous and heterologous toxigenic C. diphitheriae strains. The results showed that non-toxigenic C. diphtheriae strains confer a certain degree of protection when the challenge is done with homologous toxigenous strains, especially when not more than 500 million germs/ml are used for the challenge dose. This suggests that the carriage of non-toxigenic diphtheriae bacilli is a useful phenomenon for maintaining an immune balance in diphtheria in the present epidemiological context, and that their sterilization is not opportune.

Animals↗
PubMed2000

[Immunity against diphtheria and tetanus in various age groups after 40 years since implementation of the mass vaccination program].

The aim of this study was to determine immunity against diphtheria and tetanus in various age groups and to compare it with the results of seroepidemiological studies conducted during previous 40 years. The level of diphtheria and tetanus antibody was determined by means of ELISA tests in 1534 sera obtained from persons aged 1-81 years. As elsewhere in Europe, the present study confirms the insufficiency of humoral immunity to diphtheria among Polish adults. Groups with the lowest levels of diphtheria antibody include persons aged 30-60 years (62% protected). Tetanus immunity was highest during the first three decades of life (above 90% protected), and then declined with increasing age. The comparison with results of several serologic surveys performed earlier showed gradually increasing immunity level against tetanus in all age groups.

Adolescent↗
PubMed1992

[The characteristics of population immunity to measles and diphtheria in adolescent collectives].

Population immunity to measles and diphtheria was evaluated among adolescents of a professional-technical school. Low indices of immunity were found. There was a clear difference in the number of girls and boys with antibody titers to measles virus and diphtheria anatoxin (dilution 1:40) as well as in the number of non-immune to diphtheria. A clear increase of the number of students with protective titers by the end of the school year evidences presence of latent epidemic process of measles and diphtheria.

Adolescent↗
PubMed2004

Collaborative study for the validation of serological methods for potency testing of diphtheria toxoid vaccines - extended study: correlation of serology with in vivo toxin neutralisation.

Phase I of BSP034 collaborative study was extended in two laboratories to include correlation of serology with in vivo toxin neutralisation test (TNT) using 2 separate sets of 20 serum pools, produced in-house. The study investigated the extent to which the in vitro methods for diphtheria antibodies, Vero cell assay and diphtheria enzyme-linked immunosorbent assay for diphtheria antitoxin (D-ELISA), can detect neutralising antibodies by comparison with TNT in guinea pigs. The study was also performed to compare the antibody neutralising potency obtained in relation to guinea pig (GP) or equine (DI) antitoxin standard. In addition, the study provided an opportunity to compare ELISA for tetanus antitoxin (T-ELISA) and TNT assay for detection of anti-tetanus antibodies, from the same set of serum pools. The data obtained show that antitoxin potency obtained by Vero cell assay, D-ELISA and T-ELISA using the same GP standard, highly correlated with neutralising potency as determined in respective TNT assays. Vero cell assay with DI provided estimates that also correlated with neutralising potency, but were of significantly lower titre. Since reference to DI standard is widely used in serodiagnosis, as well as in clinical studies where diphtheria antitoxin titres obtained in the Vero cell method are taken as surrogate markers for vaccine efficacy, it should be investigated if a similar difference is also observed for human serology.

Animals↗
PubMed2004

Seroepidemiologic study on pertussis, diphtheria, and tetanus in the Fukuoka area of southern Japan: seroprevalence among persons 0-80 years old and vaccination program.

In Japan, mass vaccination for diphtheria, pertussis, and/or tetanus has been mandated by the Vaccination Law since 1948. In order to evaluate the efficacy of this vaccination policy, we conducted seroepidemiological studies on pertussis, diphtheria, and tetanus among individuals aged 0 - 80 years. The pertussis toxin seropositive rates of the vaccine-eligible groups and vaccine-ineligible groups were 55.0 and 57.9%, respectively. The seropositive rate of each group for diphtheria antitoxin was 76.3 and 75.7%, respectively. The tetanus antitoxin seropositive rates were 91.7 and 10.5%, respectively, showing a significant difference between the two groups (P < 0.001). For the three diseases, variations were seen between age groups in the geometric mean antibody titers due to changes of the vaccination program. The results of this study show that natural Bordetella pertussis infection has occurred more frequently than expected. In order to establish the most appropriate vaccination program for the control of pertussis, diphtheria, and tetanus in Japan, further evaluation is necessary.

Adolescent↗
PubMed2006

Immunogenicity and reactogenicity of a reduced-antigen-content diphtheria-tetanus-acellular pertussis vaccine as a single-dose booster in Singaporean adults.

INTRODUCTION: Older children and adults, susceptible to pertussis because of waning immunity, may serve as a reservoir of infection, leading to severe disease among young unvaccinated infants. Booster diphtheria-tetanus-acellular pertussis (dTpa) vaccination in older age groups is rare in Singapore, one reason being the increase in reactogenicity with each successive dose. The aim of this study was to assess the immunogenicity, safety and reactogenicity of a reduced antigen, combined dTpa vaccine as a single booster dose in healthy adults aged 18 years or older. METHODS: A total of 150 healthy adults, 18 to 60 years of age, received a single dose of GlaxoSmithKline Biologicals' dTpa vaccine with reduced content for diphtheria and pertussis, with measurement of pre- and post-vaccination antibody titres. RESULTS: Prior to vaccination, 71.6 percent and 92.6 percent of the subjects had anti-diphtheria and anti-tetanus antibody levels greater than or equal to 0.1 IU/mL, respectively. 46.7 percent, 98.5 percent and 44.4 percent of subjects were seropositive for pertussis toxin (PT), filamentous haemagglutinin (FHA) and pertactin (PRN) antibodies, respectively. One month after vaccination, there was an increase in geometric mean titres from pre-vaccination to post-vaccination blood samples for anti-diphtheria (greater than seven-fold), anti-tetanus (greater than five-fold), anti-PT (greater than 11-fold), anti- FHA (greater than 25-fold) and anti-PRN (greater than 31-fold) antibodies. Solicited grade three local symptoms (pain, redness and swelling) were reported in 14.1 percent, 8.1 percent and 10.4 percent of subjects, respectively. No serious adverse events were reported. CONCLUSION: In summary, the dTpa vaccine is immunogenic, safe and well-tolerated in Singaporean adults.

Adolescent↗
PubMed1990

[The cellular immune response in different forms of diphtheria in adults].

In 109 adult diphtheria patients with different forms of the disease (toxic, subtoxic, localized) and carriers, the latter including 6 persons having had the localized form of diphtheria, 12 different subpopulations of T and B lymphocytes and neutrophils were studied by the methods of rosette formation with sheep, mouse and bovine red blood cells. The study showed that, starting from week 2 of the disease, an increase in the content of the subpopulations under study was registered in all forms of diphtheria; the disease, as well as in patients with the localized form of the disease accompanied by complications and in long-term carriers. In all forms of diphtheria a decrease in the content of neutrophil rosette-forming cells was shown. The marker tests for prognostication of the complicated course of the disease, viz. the elevated content of M-rosette-forming cell and T gamma-rosette-forming cells during the first week of then disease, were established.

Adolescent↗
PubMed1990

The changing age structure of diphtheria patients: evidence for the effectiveness of EPI in the Sudan.

During an outbreak of diphtheria in Khartoum, Sudan, in 1988, only 19.1% of patients admitted to hospital were under 5 years of age. This is considerably less than the proportion of such patients seen during a similar outbreak in Khartoum in 1978 (49.5%) and also less than the proportion (55.2%) of under-5-year-olds reported for all inpatients with diphtheria in the Sudan during 1979-86. Cluster surveys carried out between 1981 and 1989 demonstrate that vaccination coverage was much higher for under-5-year-olds (about 65% for the third dose of diphtheria-pertussis-tetanus vaccine (DPT3] than for children of school age (less than 20% for DPT3) at the time of the 1988 outbreak. These results indicate that improved vaccination coverage led to the shift in the age distribution of diphtheria patients seen during the 1988 outbreak. It is unlikely that these data are affected by the type of biases that usually plague disease surveillance systems and can therefore be used as a simple way of assessing the effectiveness of the Expanded Programme on Immunization (EPI).

Adolescent↗
PubMed1986

[The main trends in a diphtheria epidemic process and the potentials for its elimination in a large city].

In recent years the tendency towards the activation of the epidemic process of diphtheria, i.e. the appearance of cases of diphtheria simultaneously with the increased levels of circulation of toxigenic and nontoxigenic Corynebacterium diphtheriae and with antitoxic immunity characteristics in children and adults constantly remaining low, has been registered. For the effective control of diphtheria, the profound study of such problems as bacterial carriership, antibacterial immunity and the development of supplementary means of specific prophylaxis is necessary.

Adolescent↗