It is well acknowledged that the earlier diphtheria is diagnosed, the more favorable the outcome of the disease can be expected and the earlier the epidemiological surveillance service can be warned. Because of the likelihood of diphtheria outbreaks in Europe due to the significant number of refugees from diphtheria-endemic regions of Asia and Africa, and from war-torn Ukraine, and therefore the need for rapid and simultaneous examination of a large number of clinical samples, an important task is to speed up the procedure for detecting the diphtheria agent in clinical material, as well as to simplify the diagnostic methodology so that it can be implemented in ordinary bacteriological laboratories.
Main element in the diphtheria diagnostic scheme is a detection of the bacterial toxin, the major virulence factor in pathogenic corynebacteria. The gold standard method for detecting the production of DT by corynebacteria, reaction of immunoprecipitation in agar or Elek test, a sophisticated technology requiring time, the availability of proven reagents and special knowledge, is performed mainly by specialized reference laboratories. In order to accelerate and simplify the diagnostic technology, we designed and evaluated a monoclonal antibodies-based ELISA and LFIA, that make it possible to quickly and accurately detect DT in a liquid culture of suspicious corynebacteria (Fig. 1). The LFIA was designed with the help of the DIFTERIA project host organization Senova Immunoassay Systems GmbH, Weimar, Germany.
Validation of the ELISA and LFIA was performed on a large strains collection: 416 strains of diphtheria and non-diphtheria corynebacteria. LFIA was also tested on 510 human pharyngeal specimens supplemented with a varied concentration of 17 test strains of corynebacteria (diphtheria simulated specimens). The LFIA validation was performed at the National Conciliary Laboratory on Diphtheria, Bavarian Health and Food Safety Authority, Oberschleißheim, Germany (the project Secondment organisation). As a result of the project, a new diphtheria diagnostic approach was developed: clinical sample from a diphtheria patient is plated onto selective Hoyle medium, after 18-24 hours of incubation suspicious black bacterial colonies are inoculated on the LFIA bouillon and in 6 hours of culturing the LFIA is performed. The “classical” stage of isolation and identification of pure culture was omitted. So, the result of the new methodology is registered in 24-30 hours, i.e. on the 2-nd day after clinical sample plating. We have also improved the Elek test, which is currently used to diagnose diphtheria in all reference laboratories over the world.
The DIFTERIA project results were reported at 6 international conferences (in 2021 and 2022), an article was published at the ‘Nature Communications’ (2021), one of the world’s leading scientific journals, 1 article is accepted to ‘Infection’ journal and 2 more articles are submitted for open-access publication to ‘Diagnostics” journal (2022). All the diphtheria researchers were informed about the results of this project via the WHO Diphtheria Working Group network which was created by us 30 years ago (in 1992) at the times of diphtheria epidemics in Russia, Ukraine and other former USSR countries.
