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