Periodic Reporting for period 1 - DIFTERIA (Development of a simple and rapid assay for early diphtheria diagnosis.)
Periodo di rendicontazione: 2020-03-01 al 2022-02-28
Before the introduction of vaccine, diphtheria was a leading cause of childhood death around the world. With the evidenced rise in cases, it seems that diphtheria is a re-emerging old foe of humankind and could become a potential major global threat. Diphtheria is relatively easy to prevent, with vaccinations given to babies. Yet approximately 15% of the world’s infants do not have access to it. In a few low- and middle-income countries, the disease is still causing sporadic infections or outbreaks in unvaccinated and partially vaccinated communities. Increasing global travel is contributing to the spread of diphtheria to areas where it has not been seen for many years. Toxigenic Corynebacterium diphtheriae and C. ulcerans, the diphtheria agents, pose a serious risk for people with low level of immune protection, the number of which has increased in Europe due to low adult re-vaccination rate, vaccine hesitancy and COVID-19 pandemic’s backsliding on childhood vaccinations. Effective laboratory diagnostics makes it possible to indicate patients with diphtheria and carriers, thereby preventing the pathogen from capturing new territories. The current diphtheria diagnostic methodology is complex and time-consuming. Considering this, scientists of the EU-funded DIFTERIA project have raised the issue of a reliable diagnostic method to prepare for possible outbreaks. The Lateral Flow Immunoassay (LFIA) was developed, which detects the diphtheria toxin (DT) in just six hours of a liquid bacterial culture incubation. Implementation of this novel test will improve the accuracy of the diphtheria diagnosis in Europe and worldwide, enabling the timely initiation of treatment and preventing the spread of the pathogen in the community.
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.
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.
The approach developed by the participants of the DIFTERIA project makes the laboratory diagnosis of diphtheria standard, unified and reliable. This approach makes it possible to speed up diagnostics from 5-10 days to 24-30 hours, as well as simplify it, so it can be used not only in the reference laboratory, but also in any ordinary bacteriological laboratory in the world. Therefore, no need to transport specimens from patients with suspected diphtheria case to a national reference laboratory for disease diagnosis, but this can be done locally, allowing a timely clinical diagnosis of diphtheria. This will save lives and prevent complications, as well as stop the spread of the infection.
There are signals that the diphtheria pathogen is starting to adapt to our weapons. In frames of the DIFTERIA project we analyzed the genome of over 500 C. diphtheriae isolates taken from 16 countries and territories over the past 122 years. The diversity of their toxin gene, responsible for producing the deadly toxin, has recently increased. We identified 18 different variants of the tox gene, some of which could change the basic structure of the toxin (Fig. 2), which could make existing vaccines and treatments less effective. We also found here a strain of toxigenic diphtheria agent with a mutation in the toxin gene, which makes this strain "invisible" for diagnosis. So, the diagnostics, vaccine, and treatments that target the toxin, need to be appraised on a regular basis.
Within the last several years C. ulcerans was recognized as an emerging zoonotic pathogen. Diphtheria-like infections with toxigenic C. ulcerans have even outnumbered those caused by toxigenic C. diphtheriae in many industrialized countries. The most intriguing thing is that circulating in domestic (cats, dogs) and wild animals, the microbe accumulates mutations in the DT gene. In C. ulcerans toxin gene about 30 mutations were found compared with the C. diphtheriae tox gene. Thus, variant of C. ulcerans with a modified toxin may appear that will be able to penetrate through the immune barrier and cause diphtheria epidemics in people vaccinated against diphtheria. Such potentially dangerous strains will be indicated and studied in the next international project (Germany-Great Britain-India-Brazil), currently being prepared, and based on the DIFTERIA project results, with the aim of timely development of the improved diagnostics and remedies.
There are signals that the diphtheria pathogen is starting to adapt to our weapons. In frames of the DIFTERIA project we analyzed the genome of over 500 C. diphtheriae isolates taken from 16 countries and territories over the past 122 years. The diversity of their toxin gene, responsible for producing the deadly toxin, has recently increased. We identified 18 different variants of the tox gene, some of which could change the basic structure of the toxin (Fig. 2), which could make existing vaccines and treatments less effective. We also found here a strain of toxigenic diphtheria agent with a mutation in the toxin gene, which makes this strain "invisible" for diagnosis. So, the diagnostics, vaccine, and treatments that target the toxin, need to be appraised on a regular basis.
Within the last several years C. ulcerans was recognized as an emerging zoonotic pathogen. Diphtheria-like infections with toxigenic C. ulcerans have even outnumbered those caused by toxigenic C. diphtheriae in many industrialized countries. The most intriguing thing is that circulating in domestic (cats, dogs) and wild animals, the microbe accumulates mutations in the DT gene. In C. ulcerans toxin gene about 30 mutations were found compared with the C. diphtheriae tox gene. Thus, variant of C. ulcerans with a modified toxin may appear that will be able to penetrate through the immune barrier and cause diphtheria epidemics in people vaccinated against diphtheria. Such potentially dangerous strains will be indicated and studied in the next international project (Germany-Great Britain-India-Brazil), currently being prepared, and based on the DIFTERIA project results, with the aim of timely development of the improved diagnostics and remedies.