Characteristics of antitoxic and antibacterial immune response in nontoxic forms of oropharyngeal diphtheria

The enzyme immunoassay system (EIA) for differentiation of antibodies in therapeutic heterogeneous antitoxic serum and antibodies to Corynebacterium diphtheriae toxigenic strains in patients and carriers was developed. The use of EIA permitted the dynamic evaluation of the characteristics of humoral antitoxic and antibacterial immune response in 50 patients with the localized and disseminated forms of stomatopharyngeal diphtheria and 14 "healthy" carriers of toxigenic C. diphtheriae. As revealed in this study, the symptoms of the disease in patients with disseminated forms of stomatopharyngeal diphtheria developed in the presence of statistically significant low quantitative values of antitoxic and antibacterial antibodies to C. diphtheriae antigens. In the group of patients with the localized forms of the disease the initially low level of antitoxic antibodies was detected with the concentration of antibacterial antibodies remaining unchanged. During the period of convalescence the levels of antitoxic antibodies in both groups reached those of healthy persons. In case of localized forms of the disease the level of antibacterial antibodies decreased as compared with healthy persons, starting from the second week of the disease. The period of convalescence in the disseminated forms was characterized by the low concentration of antibacterial antibodies. Carrier state was formed in the presence of high levels of antitoxic antibodies and significantly low levels of antibacterial ones.     

Corynebacterium diphtheriae nontoxigenic strain carrying the gene of diphtheria toxin

Among 828 C. diphtheriae nontoxigenic cultures isolated in different region of Russia in 1994-2002, 114 cultures (13.8%) had the gene of diphtheria toxin (gene tox) and were thus called nontoxigenic tox-carrying (NTTC) strains. All NTTC strains were found to belong to biovar mitis and formed neither normal, nor "defective" diphtheria toxin. The most of NTTC strains (94%) belonged to ribotype "Moskva", not occurring among C. diphtheriae toxigenic strains. The incapacity of NNTC strains of forming diphtheria toxin was caused by mutation: the deletion of one nucleotide which led to the shift of the open reading frame and to the formation of the stop codon. The results of these studies are indicative of the fact that a sufficiently homogeneous and isolated group of C. diphtheriae nontoxigenic strains is spread in Russia. These strains carry the nonexpressing gene of diphtheria toxin and are of no epidemic importance in diphtheria infection.     

Pangenomic study of Corynebacterium diphtheriae that provides insights into the genomic diversity of pathogenic isolates from cases of classical diphtheria, endocarditis, and pneumonia

Corynebacterium diphtheriae is one of the most prominent human pathogens and the causative agent of the communicable disease diphtheria. The genomes of 12 strains isolated from patients with classical diphtheria, endocarditis, and pneumonia were completely sequenced and annotated. Including the genome of C. diphtheriae NCTC 13129, we herewith present a comprehensive comparative analysis of 13 strains and the first characterization of the pangenome of the species C. diphtheriae. Comparative genomics showed extensive synteny and revealed a core genome consisting of 1,632 conserved genes. The pangenome currently comprises 4,786 protein-coding regions and increases at an average of 65 unique genes per newly sequenced strain. Analysis of prophages carrying the diphtheria toxin gene tox revealed that the toxoid vaccine producer C. diphtheriae Park-Williams no. 8 has been lysogenized by two copies of the ω(tox)(+) phage, whereas C. diphtheriae 31A harbors a hitherto-unknown tox(+) corynephage. DNA binding sites of the tox-controlling regulator DtxR were detected by genome-wide motif searches. Comparative content analysis showed that the DtxR regulons exhibit marked differences due to gene gain, gene loss, partial gene deletion, and DtxR binding site depletion. Most predicted pathogenicity islands of C. diphtheriae revealed characteristics of horizontal gene transfer. The majority of these islands encode subunits of adhesive pili, which can play important roles in adhesion of C. diphtheriae to different host tissues. All sequenced isolates contain at least two pilus gene clusters. It appears that variation in the distributed genome is a common strategy of C. diphtheriae to establish differences in host-pathogen interactions.     

Characteristics of the antitoxic and antibacterial immune response in children with diphtheria

As found in this study, the development of the manifest forms of diphtheria occurred in the presence of lower levels of antitoxic antibodies, than bacterial carrier state. The level of antitoxic antibodies in all patients, irrespective of the time of their examination, was higher than that of antibacterial antibodies. In the dynamics of the disease a considerable increase in antitoxic and antibacterial antibodies was observed in localized and disseminated forms of diphtheria and, to a considerably lesser degree, in toxic and subtoxic forms of diphtheria, while not observed in the process of the formation of bacterial carrier state. The dynamics of a rise in the levels of antitoxic and antibacterial antibodies in the manifest forms of diphtheria infection made it possible to differentiate between the cases of toxigenic Corynebacterium diphtheriae carrier state and those of the disease.     

Pathogenicity of Corynebacterium diphtheriae circulating in Rostov-on-Don City and Rostov region during interepidemic period

Main pathogenic characteristics (toxin production, tox-gene detection, adhesiveness) of 59 strains of C. diphtheriae circulating in Rostov-on-Don city and Rostov region in 2004-2005 were studied. Study of toxigenicity of 15 tox+ C.di phtheriae strains showed full coincidence of Elek immunoprecipitation test and polymerase chain reaction (PCR). Presence of part of A-fragment of tox-gene was detected in 5 (11.4%) of 44 C. diphtheriae strains that were negative in Elek test. Hemagglutinating activity of toxin producing strains was intermediate (40%) or high (60%). Among non-toxigenic strains those with intermediate adhesiveness were predominated (45,5%), the intermediate or high adhesiveness was detected in strains positive in PCR. Obtained characteristics of C. diphtheriae can be useful for surveillance for diphtheria infection during interepidemic period.     

Diphtheria remains a threat to health in the developing world--an overview

Changes in the epidemiology of diphtheria are occurring worldwide. A large proportion of adults in many industrialized and developing countries are now susceptible to diphtheria. Vaccine-induced immunity wanes over time unless periodic booster is given or exposure to toxigenic Corynebacterium diphtheriae occurs. Immunity gap in adults coupled with large numbers of susceptible children creates the potential for new extensive epidemics. Epidemic emergencies may not be long in coming in countries experiencing rapid industrialization or undergoing sociopolitical instability where many of the factors thought to be important in producing epidemic such as mass population movements and difficult hygienic and economic conditions are present. The continuous circulation of toxigenic C. diphtheriae emphasizes the need to be aware of epidemiological features, clinical signs, and symptoms of diphtheria in vaccine era so that cases can be promptly diagnosed and treated, and further public health measures can be taken to contain this serious disease. This overview focused on worldwide data obtained from diphtheria with particular emphasis to main factors leading to recent epidemics, new clinical forms of C. diphtheriae infections, expression of virulence factors, other than toxin production, control strategies, and laboratory diagnosis procedures.     

Specificity of antibodies to diphtheria toxin subunits in children with various forms of diphtheria infections

In order to study the specificity of serum antibodies to separate subunits of diphtheria toxin, SDS-electrophoresis of diphtheria toxin preliminary disintegrated on the subunits via trypsin treatment was performed, followed by immunoblotting assay. 86 blood serum samples of children with diphtheria carriers of toxigenic and non-toxigenic strains of Corynebacterium diphtheriae as well as children with other infectious diseases similar to diphtheria in their clinical manifestation, and healthy ones immunized with DTP-vaccine were tested. A special computer program was written and applied for results processing and assumption. The data obtained showed that there were particular differences in frequency of predominating the antibodies to one or another subunit of diphtheria toxin among various groups of the children. We consider that the different specificity of antibodies of sick children and children-carriers is capable to predetermine the different course of infectious process.     

The diagnostic effectiveness of an immunoenzyme test system for determining diphtheria toxin antigens in the blood serum in a clinical laboratory trial

The data on the approbation of the diagnostic value of the enzyme immunoassay (EIA) system for the determination of diphtheria toxin in the blood sera of diphtheria patients and persons suspected for diphtheria are presented. The EIA system was prepared on the basis of F(ab)2 fractions of purified antidiphtheria antibodies. 240 serum samples from diphtheria and tonsillitis patients and from healthy persons were studied. Diphtheria toxin was determined in all patients with the toxic form of diphtheria and in 41.3% of patients with its localized forms. Blood was taken mainly of the first week of the disease. In healthy persons the results of EIA were negative. Thus, the trial of the assay system in a clinical laboratory showed its good diagnostic effectiveness. The use of this EIA system in medical practice is believed to be quite promising.     

Evidence that the regulation of diphtheria toxin production is directed at the level of transcription.

It has been known for several decades that iron inhibits the production of diphtheria toxin by Corynebacterium diphtheriae by preventing expression at maximal levels. We examined the inhibition kinetics of toxin production after the addition of either iron or rifampin to iron-limited cultures of C7 (betatox+). Iron-mediated inhibition of toxin production was found to be linear within the range of 16 nM to 16 micron. The inhibition kinetics following the addition of iron or rifampin was almost identical. [3H]RNA extracted from iron-limited toxigenic C. diphtheriae was found to hybridize to a greater extent to corynephage beta DNA than either [3H]RNA extracted from toxigenic C. diphtheriae before the onset of toxin production or [3H]RNA extracted from nonlysogenic, nontoxigenic C. diphtheriae.     

The adhesive activity of diphtheritic strains in relation to the characteristics of the infectious process they induce]

The degree of adhesiveness of 602 C. diphtheriae strains isolated from patients with different forms of diphtheria was studied on trypsinized sheep red blood cells (SRBC) used as an experimental model. The titer of bacterial suspension, i.e. its highest dilution ensuring the agglutination of 50% of SRBC, was assumed to be the index of adhesive activity. The toxigenic strains were more homogeneous with respect to the degree of their activity and proved to be moderately and highly adhesive, while among the nontoxigenic strains faintly and moderately adhesive ones prevailed. The degree of adhesiveness was not linked with the cultural biological strains variants, but depended on the form of C. diphtheriae infection. The toxigenic strains isolated from diphtheria patients were essentially more active than those isolated from carriers. Both toxigenic and nontoxigenic strains isolated in cases of prolonged carrier state (more than 4 weeks) did not differ in the degree of their adhesiveness and were essentially more active than the strains isolated from short-term carriers. The strains circulating in 10 closed groups with a high proportion of pronounced cases of carrier state (70.6% to 86.7%) were essentially more active than those circulating in 10 similar groups, but having a low proportion of pronounced cases of carrier state (6.7% to 23.8%). The conclusion was made that the degree of adhesiveness proved to be an important factor of C. diphtheriae pathogenicity, responsible for the formation of carrier state. Along with pathogenicity, this factor should be taken into consideration in the evaluation of the epidemiological importance of different sources of infection.     

Tissue Culture Method for Toxigenicity Testing of Corynebacterium diphtheriae

A method for toxigenicity testing of Corynebacterium diphtheriae in tissue cultures was developed. Results were obtained by comparing destruction of the monkey kidney or, preferably, rabbit kidney monolayer by 0.1 ml of the C. diphtheriae culture in Elek's broth containing 20% rabbit serum with the appearance after the addition of 0.2 ml of a mixture of the C. diphtheriae culture and diphtheria antitoxin. The mixture of C. diphtheriae broth culture and 10 antitoxin units per ml was incubated for 1 hr at room temperature before it was added to the tissue cultures which were then incubated as long as 5 days; most results, however, were read in 72 hr. Elek's broth medium was superior to heart infusion broth for toxin production by C. diphtheriae. Addition of 20% rabbit serum improved toxin production in either broth. Numerous toxigenic and atoxigenic C. diphtheriae cultures were tested for toxigenicity in primary rabbit and monkey kidney tissue cultures. If properly controlled, this in vitro method appeared to have an advantage over the in vitro agar gel method; its results were comparable with the rabbit intradermal test. With the wider use of tissue cultures in most laboratories, we believe that the tissue culture method for toxigenicity would be more economical and easier to perform than the animal intradermal method.     

Epiedmiologic significance of the distribution of corynephages in different collectives]

Corynephage distribution was studied in the nasopharyngeal washings of 252 persons infected with C. diphtheriae of gravis type, toxigenic (21 patients and 147 carriers) and non-toxigenic ones (84 carriers), and in 468 uninfected persons in collective bodies under different epidemic conditions. Corynephages were isolated from the nasopharyngeal washings only in persons infected with toxigenic C. diphtheriae--in 4 (of 21) patients, and in 21% (of 147) carriers. Phages tox+ (4--6.2%) were revealed only in carriers of toxigenic C. diphtheriae with numerous bacteria in the nasopharynx and in diphtheria patients. Carriers of nontoxigenic diphtheria bacilli can become infected with phage tox+ only together with the toxigenic strains (reinfection). The data obtained indicated that toxigenic and nontoxigenic C. diphtheriae strains were individual variants.     

Novelty of laboratory diagnosis for diphtheria

Selected elements of simplified, bacteriological diagnosis of diphtheria were presented. The procedure of Corynebacterium strains isolation from diphtheria suspected persons and performing of toxin testing of potentially toxigenic isolates: C. diphtheriae, C. ulcerans and C. pseudotuberculosis were shortened. The role of selective tellurite media was underlined but Loeffler medium was rejected. Columbia blood agar plate was utilized for preliminary culture. Biochemical tests and toxin testing were performed from this medium. Presented diphtheria diagnosis scheme may have practical application for the laboratory work in Poland.     

Characterization of production processes for tetanus and diphtheria anatoxins

Tetanus and diphtheria are diseases that still cause significant morbidity and mortality. Clostridium tetani produces the tetanus toxin, a 150-kDa protein. The diphtheria toxin is synthesized by Corynebacterium diphtheriae as a protein of 58 kDa. The objective of this study was to carry out a chemical characterization of the tetanus and diphtheria toxin forms in the several production process stages, and thus to establish an affordable alternative in vitro quality control to aggregate to the classical tests. The 150 kDa band of the tetanus toxin and approximately 58 kDa band of the diphtheria toxin were observed by electrophoresis similar as that described in the literature. The same band of 58 KDa was detected in Western blotting reactions. The results obtained for diphtheria toxin showed very similar protein profiles between distinct lots. For the tetanus toxin, the profiles of the initial stage showed some variability, but the ones of the following stages were similar. The similarity of the electrophoresis results indicated reproduction and consistency of the production processes in Butantan Institute and correlated with the yield and antigenic purity classical data. The establishment of alternative in vitro quality control tests can significantly contribute to achieve the consistency approach supported by WHO.     

Results of conducting an epidemiological survey of diphtheria in 21 territories of the RSFSR

As the result of epidemiological survey of diphtherial infection, carried out in conformity with the unified methodological recommendations in 21 regions of the RSFSR during 1980-1981, the expediency of such experiment was established. Immunity to diphtheria in children aged up to 14 years was high: children with negative Schick tests constituted 96.9-99.1%. No biological changes in Corynebacterium diphtheriae occurred during the term of observation. Toxigenic C. diphtheriae showed a high level of pathogenicity. The epidemiological survey contributed to a more thorough detection of diphtheria patients and carriers releasing toxigenic C. diphtheriae. The quality of clinical bacteriological diagnosis improved. In rare cases angina with the concomitant carriership of toxigenic C. diphtheriae could be diagnosed with the indispensable serological examination of the patients by Jensen's method.     

Cotranslational secretion of diphtheria toxin and alkaline phosphatase in vitro: involvement of membrane protein(s).

Crude messenger ribonucleic acid fractions isolated from Corynebacterium diphtheriae and Escherichia coli were translated in an E. coli in vitro protein-synthesizing system and yielded precursors of the secreted proteins diphtheria toxin and alkaline phosphatase, respectively. Addition of inverted E. coli inner membrane vesicles to the system during the initial stages of translation resulted in the intravesicular segregation of mature diphtheria toxin and alkaline phosphatase. Outer membrane vesicles or inner membrane vesicles whose cytoplasmic surfaces had been treated with pronase could not mediate transmembrane transfer of diphtheria toxin or alkaline phosphatase. However, inner membrane vesicles isolated from E. coli spheroplasts which had been treated with pronase and inner membrane vesicles complexed with ribosomes during pronase treatment were functional in transmembrane transfer. At temperatures below the phase transition of E. coli membranes, no intravesicular segregation of alkaline phosphatase or diphtheria toxin was observed. The precursor forms of each protein accumulated free from the vesicles. These results suggest that an inner membrane protein, exposed on the cytoplasmic surface, plays an integral role in secretion.     

Isolation and partial characterization of a corynebacteriophage beta, tox operator constitutive-like mutant lysogen of Corynebacterium diphtheriae.

We have isolated and partially characterized a beta-phage mutant lysogen of Corynebacterium diphtheriae, C7(betatoxct1+), which is partially insensitive to iron inhibition of diphtheria toxin production. tox expression by C7(betatoxct1+) was found to be partially constitutive. In the presence of concentrations of iron that almost completely inhibit the expression of diphtheria toxin by the wild type, C7(beta), the level of toxin production by C7(betatoxct1+) was found to be at least 25 times that of the parent. The purified tox gene product of C7(betatoxct1+) was immunologically and electrophoretically identical to, and equally as toxic as, diphtheria toxin purified from C7(beta). In addition, the partial N-terminal amino acid sequence was found to be identical to diphtheria toxin. This data strongly suggests that the mutation allowing for the constitutive expression of tox in C7(betatoxct1+) is outside of the structural gene. Furthermore, the constitutive expression of diphtheria toxin was found to be cis dominant in the double lysogen C7(betacrm45+/betatoxct1+). The data presented is consistent with the existence of a tox operator locus.