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An immunochromatographic strip (ICS) test was developed for the detection of diphtheria toxin by using an equine polyclonal antibody as the capture antibody and colloidal gold-labeled monoclonal antibodies specific for fragment A of the diphtheria toxin molecule as the detection antibody. The ICS test has been fully optimized for the detection of toxin from bacterial cultures; the limit of detection was approximately 0.5 ng of diphtheria toxin per ml within 10 min. In a comparative study with 915 pure clinical isolates of Corynebacterium spp., the results of the ICS test were in complete agreement with those of the conventional Elek test. The ICS test was also evaluated for its ability to detect toxigenicity from clinical specimens (throat swabs) in two field studies conducted within areas of the former USSR where diphtheria is epidemic. Eight hundred fifty throat swabs were examined by conventional culture and by use of directly inoculated broth cultures for the ICS test. The results showed 99% concordance (848 of 850 specimens), and the sensitivity and specificity of the ICS test were 98% (95% confidence interval, 91 to 99%) and 99% (95% confidence interval, 99 to 100%), respectively.

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The biochemical test of the reduction of nitrates to nitrites made it possible to identify 5.2% of strains belonging to biovar belfanti among 135 C. diphtheriae strains, initially classified within biovar mitis. Out of 7 identified C. diphtheriae belfanti strains, 2 toxigenic strains were isolated from multiple foci diphtheria. According to the results of the polymerase chain reaction, 1 out of 5 non-toxigenic strains had tox gene. All C. diphtheriae belfanti strains were found to have pronounced capacity for adhesion to sheep and human red blood cells. At the stage of the extinction of diphtheria epidemic the practical identification of C. diphtheriae belfanti strains is necessary, as increased adhesion in combination with toxigenic properties may probably promote for bacteria of this biovar to take the leading role at the period of sporadic morbidity.

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Molecular subtyping of Corynebacterium diphtheriae identified significant genetic diversity within the species and led to the identification of a unique clonal group that emerged in Russia in 1990 at the beginning of the current epidemic. Strains of this group belong to a distinct electrophoretic type complex and are of ribotypes D1 and D4. Identification of the group allowed for precise monitoring of the epidemic's progression and for rapid detection of cases imported to other countries. The evolution of this clonal group was monitored, and changes were identified. Molecular analysis revealed that no amino acid substitutions have occurred in the diphtheria toxin gene of the epidemic clone strains, reaffirming the use of the current vaccine as the single most effective preventive measure. Application of molecular subtyping methods and continuous monitoring of the spread of these clones has made it possible to distinguish rapidly between epidemic, endemic, and imported cases, allowing for implementation of timely and adequate preventive measures and providing reassurance that no secondary spread resulted from importations.

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Epidemic diphtheria spread to Ukraine in 1991, where it peaked in 1995 with >5,000 reported cases. To refine epidemic control strategies, immunogenicity of a tetanus-diphtheria toxoids vaccine (Td) containing 2 limits of flocculation (Lf) diphtheria toxoid was evaluated. During a mass vaccination campaign, adults at a clinic in Odessa received one dose of Td. At enrollment, 57.2% of 341 study participants had levels of diphtheria antitoxin (DAT) >/=0.1 IU/mL. Thirty and 180 days after receiving one dose of Td, 91.5% and 84.5% of the participants, respectively, had DAT levels >/=0.1 IU/mL. However, among 40- to 49-year-old participants, only 78.8% and 73.8% had DAT levels >/=0.1 IU/mL at 30 and 180 days, respectively. This study suggests that one dose of 2 Lf diphtheria toxoid is highly effective in raising DAT to protective levels in most adults; however, the study also shows that certain age groups, particularly persons 40-49 and, to a lesser degree, 30-39 years old may require additional doses or a complete three-dose primary vaccination series for optimal protection against diphtheria.

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Despite the success of mass immunization in many countries, diphtheria continues to play a major role as a potentially lethal resurgent infectious disease. Early, accurate diagnosis is imperative since delay in specific therapy may result in death. The microbiologic diagnosis of the disease, the identification of contacts and carriers, and the appropriate clinical management of these patients are therefore crucial. The epidemiology of diseases caused by Corynebacterium diphtheriae has changed dramatically over the decades, a situation that is highlighted by the resurgence of infections in the European region. These factors have strengthened the need for laboratories to screen for C. diphtheriae. Many modified and new methodologies are now used widely within laboratories for diphtheria diagnosis. Recent developments have focused upon methods for detection of the lethal and potent exotoxin produced by the causative organism, C. diphtheriae; this detection is the definitive test for the microbiologic diagnosis of diphtheria.

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The detection of toxigenicity among Corynebacterium diphtheriae and Corynebacterium ulcerans strains is the most important test for the microbiological diagnosis of diphtheria. Difficulties with current methods, in particular the Elek test, are well documented. We therefore describe a modified Elek test which provides an accurate result after only 16 h of incubation, in contrast to 48 h for the conventional test.

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The largest diphtheria outbreak in the developed world since the 1960s is in progress in the Russian Federation. Seventy-two Corynebacterium diphtheriae strains from throughout Russia and the Ukraine, selected for temporal and geographic diversity, and 6 reference and control strains were assayed by DNA direct sequencing, and DNA sequences of their diphtheria toxin gene, tox, and the regulatory dtxR gene, were compared to those of the Park-Williams no. 8 strain (PW8). Twenty-eight C. diphtheriae strains had entire tox sequences identical to that of the PW8 strain. Among the remaining 40 strains which were toxigenic, 4 point mutations were detected in the tox gene, one within the A and three within the B subunit gene. All four were silent mutations, indicating that diphtheria toxin is highly conserved at the amino acid sequence level; therefore, changes in the efficacy of the current vaccines would be unlikely to occur. Within the open reading frame of the regulatory dtxR gene, 35 point mutations were detected. Only 15 strains had entire dtxR sequences identical to that of the PW8 strain. Nine amino acid substitutions were found in the carboxyl half of dtxR: 22 and 25 strains differed from the PW8 strain in one and two amino acids, respectively. Given that naturally occurring variations of dtxR might be associated with increased diphtheria toxin production, studies to investigate the association of these point mutations and amino acid substitutions with quantified toxin production in the strains causing the current epidemic are under way.

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Diphtheria toxin (tox) and its regulatory element (dtxR) from 72 Corynebacterium diphtheriae strains isolated in Russia and Ukraine before and during the current diphtheria epidemic were studied by PCR-single-strand conformation polymorphism analysis (PCR-SSCP). Twelve sets of primers were constructed (eight for tox and four for dtxR), and three regions within tox and all four regions of dtxR showed significant variations in the number and/or sizes of the amplicons. Two to four different SSCP patterns were identified in each of the variable regions; subsequently, tox and dtxR could be classified into 6 and 12 different types, respectively. The great majority of epidemic strains from both Russia and Ukraine had tox types 3 and 4, and only in a single preepidemic strain isolated in Russia were all eight tox regions identical to those of C. diphtheriae Park-Williams No. 8 (tox type 1). Epidemic strains from Ukraine can easily be identified by dtxR type 5, while the majority of the Russian epidemic strains have dtxR of types 2 and 8. No differences in the tox regions between mitis and gravis biotype strains were observed. However, dtxR types 2, 5, and 8 were identified only in the gravis biotype, and dtxR type 1 was characteristic for the mitis biotype strains. PCR-SSCP is a simple and rapid method for the identification of variable tox and dtxR regions that allows for the clear association of tox and dtxR types with strains of distinct temporal and/or geographic origins.

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The method for the determination of the toxigenicity of C. diphtheriae by means of discs impregnated with diphtheria antitoxin is proposed. The discs can be prepared long before use and stored for a year. The determination of toxigenicity with these discs is not inferior in its sensitivity to the routine procedure of the immunoprecipitation test. The disc method decreases the consumption of unavailable diphtheria antitoxin by 10 times, that of culture media by 2-2.5 times: besides, it is less labor consuming.

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