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Eurosurveillance, Volume 7, Issue 1, 01 January 2002
Scientific review
Report on the Sixth International Meeting of the European Laboratory Working Group on Diphtheria, Brussels, Belgium

Citation style for this article: Lai S, Efstratiou A. Report on the Sixth International Meeting of the European Laboratory Working Group on Diphtheria, Brussels, Belgium. Euro Surveill. 2002;7(1):pii=350. Available online: http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=350

S. Lai, A. Efstratiou on behalf of the European Laboratory Working Group on Diphtheria.

WHO Collaborating Centre for Diphtheria and Streptococcal Infections, Respiratory and Systemic Infection Laboratory, PHLS Central Public Health Laboratory, London, United Kingdom.


In addition to the Eastern European resurgence of diphtheria during the last decade, there has also been an emergence of infections caused by non-toxigenic Corynebacterium diphtheriae and non-toxigenic, toxin gene bearing C. diphtheriae. Given that these strains may manifest as symptomatic infections of differing degrees of severity, their clinical and epidemiological significance need to be assessed. The persistence of toxigenic and non-toxigenic C. diphtheriae in circulation, together with genotypic and biotype variability means that innovative measures to vaccinate populations are pertinent. The most effective method of protecting the currently most vulnerable population group (adults) is to implement a booster dose of vaccine amongst the adult populations. Furthermore, in combination with an efficient surveillance system, effective antibiotic prophylaxis and an up-to-date vaccination programme, serological studies needs to be maintained to monitor the immunity status of the population.
 

Presentations at the Sixth International Meeting of the European Laboratory Working Group on Diphtheria (ELWGD), provided an overview on the microbiology and epidemiology of diphtheria in the Newly Independent States of the former Soviet Union (NIS) and an international updated review of progress in epidemiological, clinical and microbiological aspects.


Microbiological Surveillance of diphtheria

Two European Commission (EC) Fourth Framework Directorate General Research and Technological Development (DGRTD) programmes have contributed to the pan-European activities of the ELWGD (1); BioMed 2 BMH4-CT98.3793 and INCO-Copernicus IC15CT.98.0302. The objectives of both programmes are the microbiological surveillance of diphtheria, in Europe and Eastern Europe respectively. Within the remit of these programmes and the ELWGD, National Diphtheria Reference Centres have been established in Armenia, Austria, Belarus, Brazil, France, Georgia, Greece, Italy, Kazakhstan, Latvia and Turkey. This has facilitated concerted and networked efforts towards the standardisation and integration of methodologies for the laboratory diagnosis of diphtheria and epidemiological typing of the causative organism, C. diphtheriae. These initiatives have also led to the co-ordination of international External Quality Assessment (EQA) schemes to monitor and assess these activities and construction of the first international genotype database for C. diphtheriae. Awareness has been enhanced through training workshops and the recently launched website http://www.phls.co.uk/international/diphtheria/diphtheria.htm. Moreover, the establishment of the microbiological surveillance databases on diphtheria in London (UK) for reports in the Western European countries and in Smolensk (Russia) for Eastern European countries has provided a centralisation of data for continued monitoring of this disease. Further research to develop techniques in toxigenicity testing, and exploration into the genome sequence of this causative organism are providing sources of inspiration for other lines of enquiry regarding our understanding of its pathogenesis. This should lead to further development of novel diagnostics, therapeutics and prophylactics and ultimately, advance our understanding of the host-pathogen interaction to public health.

Epidemiology and microbiology of diphtheria in the NIS
Genotypic and phenotypic characteristics of diphtheria are not as homogeneous as was once thought. The predominant strain within the European Region has shifted from C. diphtheriae biotype mitis Toulouse-1 ribotype during the 1940-1970s to biotype gravis of "Sankt Petersburg" and "Rossiya" ribotypes (pers. comm. P. A. D. Grimont) in the more recent epidemic of the 1990s. Clinical manifestations of diphtheria caused by
C. diphtheriae var mitis have been frequently presented with croup amongst non-vaccinated children in Russia, whilst epidemiological data demonstrated that different immunised groups showed different severity levels of the disease; in particular, the adult population, the homeless, and the drug abusers.

The control of the diphtheria epidemic in most of the Baltic States and NIS countries is indicative of progress since the introduction of the improved public health strategy of prevention and control (2, 3). Diphtheria remains a major concern in Kyrgyzstan, Latvia, Tajikistan and Ukraine. Despite the improved situation observed in 1998 within Kyrgyzstan, Tajikistan and Ukraine, the diphtheria incidence rate is still higher than the WHO target for NIS countries, which is less than 1 case per 100 000 population (http://cisid.who.dk/dip/DipRO2.asp).

The situation is exasperated by incomplete microbiological surveillance of C. diphtheriae in some parts of the NIS. Funding provided by the United Nations Children's Fund (UNICEF), International Federation of Red Cross and Red Crescent Societies (IFRC), the WHO and other donors (1) for WHO/PHLS Diphtheria Laboratory Kits has ceased. This has resulted with some countries experiencing inadequate supplies of reagents for microbiological diagnosis, and forced to rely solely upon clinical diagnosis of diphtheria cases.

Emergence of non-toxigenic C. diphtheriae
Non-toxigenic C. diphtheriae in England and Wales has been emerging as a pathogen associated with recurrent and sometimes severe pharyngitis (4). Sporadic cases of endocarditis caused by non-toxigenic C. diphtheriae biotype gravis have been reported between 1990-1991 in New South Wales and a smaller cluster in Victoria, Australia (5, 6).

In the latter half of the 1990s, a similar trend towards non-toxigenic C. diphtheriae was also reported from Belarus, the Republic of Georgia, Moldova, and St. Petersburg. Studies at the St. Petersburg Pasteur Institute showed their strains to be predominantly biotype mitis (62%) and genotypically diverse on the basis of ribotyping. Meanwhile, among the UK referrals to the PHLS Streptococcus and Diphtheria Reference Unit (SDRU) the predominant biotype is gravis (7, 8).

The phenotypic and genotypic characterisation of non-toxigenic strains of C. diphtheriae carrying the tox-gene was first described by Groman et al. in 1983 (9). These non-toxigenic tox-bearing C. diphtheriae (NTTB) strains currently represent 20-30% of the non-toxigenic C. diphtheriae biotype mitis from the Russian Federation showing an indistinguishable biotype, ribotype and other biological properties (10). Amongst the Belarussian strains circulating during 1996-1997, 58% were phenotypically non-toxigenic, though more than half of these strains possessed the diphtheria toxin gene. Twenty of 105 (19.0%) strains were isolated from patients with pharyngitis and seven of 76 were isolated from close contacts (9.2%).

The emergence of non-toxigenic and NTTB strains poses several questions; is there selection against toxigenic strains of C. diphtheriae as postulated by Clarridge et al. (11), what is the pathogenicity of these non-toxigenic C. diphtheriae and NTTB strains, and what is the incidence of infections caused by these organisms globally?

Clinical and public health impact of infections caused by C. diphtheriae
Diphtheria affects both children and adults, which can manifest in different degrees of severity, from asymptomatic carriers to more severe and complicated forms of the disease. This in turn is dependent upon two main factors, the characteristics of the bacterium and the immune status of the host. Unfortunately, there are still extensive groups of people at risk of infection through close contact and poor hygiene and living conditions, namely, people in lower socio-economic groups; the homeless and alcohol users. In addition, groups who are predisposed to infection are usually children with chronic respiratory diseases and non-vaccinated groups (mainly due to mistaken grounds for contraindications to immunisation).

Diphtheria cases and carriers in Diyarbakir in the Southeast region of Turkey revealed that despite expanded vaccine coverage to 81% in 1998 for the primary immunisation series in Turkey, the number of diphtheria cases is still high (12). This has been the result of a number of contributing factors associated with urbanisation, poor socio-economic condition and regions with low immunisation coverage.

Microbiological aspects of infections caused by other corynebacteria

Infection caused by C. ulcerans
In the UK, contact tracing of infections caused by C. ulcerans was not recommended prior to 1999, as there were no reports of person-to-person transmission. However, the increasing frequency of isolation of toxigenic C. ulcerans, the severity of infection, and its infectivity has led to subsequent changes in the UK guidelines to recommend contact tracing of infections caused by toxigenic strains of C. ulcerans towards the control of diphtheria (7).

Infection associated with other corynebacteria
The National Microbiology Laboratory of Health Canada (NMLHC), Winnipeg, Canada undertakes detailed polyphasic studies for the identification of Corynebacterium species. Results from conventional biochemical characterisation, cellular fatty acid composition studies and 16S rRNA sequence analysis have revealed the association of the newly described Corynebacterium species; Corynebacterium imitans (13) and C. durum (14) isolated from blood culture with diphtheria-like symptoms among Canadian and international isolate referrals. Such findings suggest that microbiologists need to be aware of other Corynebacterium spp. that may induce virulent manifestations.

Laboratory diagnosis of diphtheria
A limited number of key tests for the differential diagnosis of diphtheria are often relied upon in laboratories, with limited resources.

At the Brazilian Public Health Laboratory, an efficient screening approach has been developed. The algorithm for the laboratory diagnosis of
C. diphtheriae relies upon the elimination of other corynebacteria by incorporating the test for porphyrin production on King B Medium using the double sugar-urease test for glucose, maltose and urease activity in combination with a test for toxin production using a radial immunodiffusion method (15).

Developments in toxigenicity testing
Recently, a novel and rapid immunochromatographic strip (ICS) test for the detection of diphtheria toxin from bacterial cultures and clinical specimens was developed by the Public Health Laboratory Service (PHLS, UK) in collaboration with Program for Appropriate Technology in Health (PATH, USA) (16). Results from field studies using strains from the UK, Ukraine and Latvia showed complete congruence with conventional and modified Elek tests. The unparalleled sensitivity (0.5ng/ml) of the test, the availability of results within three hours (from the selection of colonies from the culture plate) and its correlation with other phenotypic tests emphasise the potential importance of the ICS test in the primary routine screening procedure for suspect colonies.

Advances in quantitative Polymerase Chain Reaction have led to the development of the TaqMan® PCR assay for rapid detection of diphtheria toxin. This assay was designed and evaluated by the Diphtheria Reference Laboratory at the Centres for Disease Control and Prevention, Atlanta and employs the direct sequence detection of the toxin gene in real-time quantitative PCR from clinical specimens (17). Preliminary investigations show benefits which include a sensitivity that is ten fold greater than the standard conventional PCR detection of the tox gene, obviates post-amplification handling, enables a high throughput (96 well plate format) and easy quantification of amplified products. Further evaluation will place the TaqMan® PCR format as an invaluable alternative tool to the standard PCR detection of the tox gene directly from clinical material. However, the initial capital cost will restrict its application to central reference laboratories.

Quality assessment of laboratory diagnosis of diphtheria
Annual distributions of the EQA schemes since 1996 have been instrumental towards maintaining awareness and laboratory capabilities within specialised areas of microbiology. The results have indicated the proficiency of laboratories in the diagnosis of diphtheria and toxigenicity testing (18). Moreover, it has highlighted the need for at least, yearly distributions of specimens for quality assurance within each laboratory.

Molecular characterisation of diphtheria
Much remains to be learned about the changing molecular epidemiology of this resurgent disease. Ribotyping has been successfully used for molecular subtyping in identification and monitoring of the evolution of a clonal group.

Within the remit of the the ELWGD, ribotyping has been adopted as
the gold standard and forms the basis of a genotype database for C. diphtheriae (19). The nomenclature for C. diphtheriae ribotypes is currently being established, based on geographic location.

Serological aspects of diphtheria immunity
The continuing epidemics in the Baltic States were associated with low immunisation coverage rates in some areas, the lack of immunity among adults and variable antibody responses upon administering different doses of vaccine. In Italy, a population immunity study against diphtheria, concluded that the prevalence of 45-49 years female (1086) and 50-54 years males (974) among the susceptible population was indicative of the proportionate correlation between a non-protected population and increasing age group (20). A similar trend was identified among the adult population (>50 years) in Greece and Israel. Although it was not possible to generalise the situation in every country, the high primary vaccine coverage in Israel, Italy, Finland, France, Netherlands, UK and Sweden provided satisfactory protection (antitoxin level =0.1 IU/ml) among children and young adults (approx.21 years) (21). Thus, the noticeable decreasing antibody levels among populations over the age of 21 years in these countries supports the recommendation of implementing a routine booster dose of diphtheria-tetanus (Td) vaccine every ten years. This was reinforced by the study undertaken within the remit of the DGXII European Commission funded programme, European Sero-Epidemiological Network (ESEN) (22, 23). Furthermore, the benefit of such a regime was evident when antibody responses among a group of adults immunised with a booster dose of Td vaccine showed a significant (14 fold) increase in antitoxin levels (24).

Developments and the future of diphtheria
Finally, the completion of the genome sequence of C. diphtheriae offers new strategies for studying the unique host-pathogen interactions, thus providing a novel tool for diagnosis of infection and intervention (25).

So far, the collaborative C. diphtheriae sequencing project between the Sanger Centre (http://www.sanger.ac.uk) with the PHLS Respiratory and Systemic Infection Laboratory (RSIL) as a leading collaborator is near completion. Preliminary in silico analysis of the genome has indicated an apparent capability for the production of fimbriae through the discovery of sortase-like proteins; representing important host attachment factors involved in diphtheria pathogenicity (26).

Acknowledgements

The authors gratefully acknowledge the contribution of all participants of the Sixth International ELWGD; the European Commission DGRTD, Dr Ludovica Serafini; Dr Colette Roure and the WHO Regional Office for Europe; by no means least, the partners from the two EC-funded diphtheria programmes, BioMed 2 BMH4.CT98. 3793 and INCO-Copernicus IC15.CT98.0302 as listed:

EC Project Coordinator: Androulla Efstratiou
EC Project Scientist: Sandra Lai
EC Project Secretary: Jocelyne Seyve


References

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