| DIPNET will continue a collaborative and coordinated
approach to support countries to improve diphtheria surveillance for early
detection of cases and contacts by accurate microbiological and epidemiological
surveillance (3,4,5). The network of national and international laboratories
not only microbiologists, but also epidemiologists from most of the EU
member states and accession countries. Communication between laboratories
and public health authorities within and between countries enhances the
capacity of an EU member state to respond rapidly to this public health
Participants in this meeting came from 39 different countries including
12 EU member states, six accession countries, 12 NIS countries, and Brazil,
Canada, India, Israel, Norway, Switzerland, Turkey, and the United States
(US). This report summarises the presentations given at this meeting which
provided an interesting overview of the surveillance of diphtheria within
the Russian Federation and NIS as well as the EU member states and accession
countries, a review of the clinical and microbiological aspects of infection
caused by corynebacteria, and an update on recent studies on the molecular
and genetic characteristics of Corynebacterium diphtheriae and
Microbiological surveillance of diphtheria in the NIS of the former
The recent epidemic of diphtheria in the NIS began in the Russian Federation
in 1990 and affected all 12 NIS by the end of 1994 (7). In 1990, the number
of reported diphtheria cases in the NIS was 1 436. However, in 1993 the
number of cases dramatically increased to 19 604, then to 47 869 in 1994,
and finally reached a peak of 50 434 in 1995. The prominent strains during
this epidemic were C. diphtheriae var gravis ribotypes Sankt-Petersburg
and Rossija. At the beginning of the epidemic, the case fatality rate
was very high and was probably due to inadequate supplies of antitoxin,
improper case management, and delayed treatment. Diphtheria control efforts,
coordinated by the World Health Organization (WHO), began in the Russian
Federation in 1992 and included mass immunisation campaigns which covered
most of the adult population (8). Further control efforts and mass immunization
campaigns in the other NIS followed. In recent years, the number of cases
reported to the European Region of WHO have fallen to approximately 1
500 in 1999 and 2000.
Excellent progress has been achieved in the control of diphtheria in Armenia,
Azerbaijan, Belarus, Estonia, Kazakhstan, Lithuania, Republic of Moldova,
Turkmenistan, and Uzbekistan, where the incidence of the disease has reached
low levels. However, the number of cases are higher in Georgia, Kyrghyzstan,
Latvia, the Russian Federation, Tajikistan and the Ukraine (9,10). Some
regions appear to have higher incidence rates than others: during recent
years, for example, there has been an increase in the number of diphtheria
cases reported in the Smolensk region of the Russian Federation. In 2001,
30 cases were reported in Smolensk, giving an incidence of 2.66 per 100
000 population, while the incidence in the Russian Federation as a whole
was only 0.57 per 100 000 population. Seventy three per cent of the cases
were adults, and two adult patients died. A dramatic increase in carriage
rates of toxigenic C. diphtheriae strains was also observed. When
there is an increase in the incidence of diphtheria, additional anti-epidemic
measures may be needed to increase the coverage rates in all age groups.
Beginning in 1998, an annual increase of diphtheria incidence was also
observed in the St Petersburg region, with an incidence of 2.6 per 100
000, mainly in adult patients. Since 2000, most of the diphtheria cases
have been caused by toxigenic C. diphtheriae var mitis ribotype
Otchakov. Screening and serological investigations amongst different social
and professional risk groups have recently been performed in this region.
A total of 472 sera from individuals aged between 20 to 69 years were
studied, and 35% were susceptible to diphtheria. Fifty two per cent of
individuals aged between 40 and 59 years had only ever been given one
booster vaccination. Among those who had been given two boosters, the
proportion of susceptibles was only 25%. For effective control of the
epidemic in this district, immunisation of adults with two boosters may
be required. Immunisation of the booster vaccine to the adult population
has begun in this region, but it seems that ribotype Otchakov may present
a new public health threat in the future.
Epidemiology of diphtheria in EU member states and accession countries
Although the risk of resurgence of diphtheria in EU member state countries
is relatively low, the capacity to control and prevent diphtheria must
be maintained due to close links with other higher incidence countries.
The ability to recognise an imported case of diphtheria rapidly and to
control any potential spread of the disease within the community is a
public health priority (3). Laboratory facilities, clinical expertise,
and surveillance all need to be maintained. Additionally, travellers to
countries where the disease is endemic need to be protected, so adequate
supplies of antitoxin and toxoid vaccine must be available when required.
Many of the EU member states and accession countries see either no cases
or very few cases of diphtheria each year. However, the WHO Collaborating
Centre for Diphtheria at the Respiratory and Systemic Infection Laboratory
of the Health Protection Agency in the United Kingdom (UK) identified
36 isolates of toxigenic corynebacteria between the beginning of 1997
and the end of 2002 from throat swabs and other samples types referred
for toxigenicity testing, 58% (21/36) of which were identified as C.
ulcerans. Infection with C. ulcerans in humans is 'traditionally'
associated with consumption of raw dairy products or contact with farm
animals; however, no such risk factors were identified for about half
of the cases.
In April 1993, the first imported case of diphtheria for nearly thirty
years was identified in Finland and was found to be caused by toxigenic
C. diphtheriae var gravis. Since then, a total of fifteen isolations of
C. diphtheriae have been reported, the majority produced toxin
(13/15) and were var gravis (10/15). Twelve of the cases had epidemiological
links to Russia and were caused by toxigenic C. diphtheriae. Based on
ribotype analysis, most of the var gravis isolates were indistinguishable
from the the Russian epidemic clone group Sankt-Peterburg/Rossija (11,12).
Despite extensive traffic across the border, the small number of diphtheria
cases identified in Finland in recent years suggests that the overall
immunity of the population is relatively good (13).
During 2002, the first case of diphtheria since 1989 was identified in
France in a female Chinese immigrant who presented with dysphasia and
fever. She had lived in France for the last two years and was probably
unimmunised. Toxigenic C. diphtheriae var mitis was isolated from
a throat swab taken from this patient which revealed a new ribotype pattern
Lariboisière. Also in 2002, the first case of diphtheria in Italy
since 1998 was seen in a 14 year old boy from Friuli, northern Italy,
who presented with fever, sore throat, lymphadenopathy and pseudomembrane.
Toxigenic C. ulcerans was isolated from a throat swab, but the source
of the infection was not ascertained.
Currently, Latvia has the highest incidence rate of diphtheria of any
of the EU member states or accession countries. Between September 1993
and 2001, 1 288 cases of diphtheria were reported, including 96 deaths.
Diphtheria incidence has peaked twice during this period, first between
1994 and 1996 and then between 1999 and the present time. During the first
peak, more than 71% diphtheria cases were reported amongst adults, most
of whom were unvaccinated, and especially high morbidity levels was seen
in those aged between 30 and 59 years (14). This is consistent with the
usual epidemiology of the disease in a population that has high levels
of childhood vaccination, but incomplete adult coverage. During the last
increase in incidence, however, the proportion aged between 30 and 59
years decreased by 19%, and 97% had been vaccinated against diphtheria
in childhood. The reasons for this last resurgence are still not completely
Clinical and microbiological aspects of infection caused by potentially
toxigenic and other corynebacteria
A large seroepidemiological study of 558 injured patients aged between
18 and 70 years was conducted at an outpatient clinic of a trauma department
in Vienna, Austria. Twenty seven per cent of the subjects were susceptible
to diphtheria, 27% had basic protection, and 46% were fully protected
(15). Multiple linear regression analysis revealed that age and gender
both had a significant independent influence on diphtheria immunity level.
In another study carried out in northern Greece on sera from 429 healthy
children and adults aged between 0 and 80 years, immunity rates against
diphtheria were found to be protective in high proportions of individuals
up to 20 years of age, after which time immunity levels declined sharply.
Another seroepidemiological study in Slovenia also showed a high level
of protective immunity in children and young adults and, again, there
was evidence of a fall in the level of protective antibodies in adults.
Between 1998 and 2000, antibiotic susceptibility tests were carried out
on 148 isolates of C. diphtheriae from diphtheria patients and carriers
in the Moscow, Murmansk, Omsk, Kaliningrad, Vladimir and Krasnodar regions
of Russia. Sixteen of these isolates (11%), all of which had been characterised
as the epidemic clonal group of toxigenic C. diphtheriae var gravis
ribotype Sankt-Peterburg/Rossija, were found to be resistant to
macrolide and lincosamide antibiotics such as erythromycin, azythromycin,
lincomycin, and clindamycin (11). No antibiotic-resistant strains were
found among isolates that circulated in Russia between the early 1980s
The completion of the genome sequence of C. diphtheriae offers new strategies
for studying host-pathogen interactions. An investigation was described
that was recently undertaken in Brazil to examine the interactions leading
up to and involving the adherence process of C. diphtheriae to human epithelial
cells, the primary step made in the colonisation and invasion of the host
(16,17). Thirteen strains were tested and displayed varying degrees of
attachment to HEp-2 cell monolayers with two distinct adherence patterns:
the predominant type called localised (LA), and diffuse (DA) (18). The
LA pattern was mainly observed among the glass adherent/sucrose fermenting
strains (19). Bacterial adherence to HEp-2 cells was inhibited in varied
degrees by carbohydrate moieties. Initial indications point to these carbohydrate
components as putative adhesins of C. diphtheriae to human epithelial
Molecular and genetic characteristics of corynebacteria
Ribotyping, a universal molecular typing method for bacteria based upon
rRNA gene restriction pattern determination, has previously been recognised
as the best marker for the molecular typing of C. diphtheriae isolates.
The endonucleases chosen for the standard methodology were BstEII (first
choice) and PvuII (second choice), because when these enzymes are used,
ribotypes often share common bands, which make pattern comparison easier.
International exchange of typing data can be carried out and there is
now a database of all C. diphtheriae ribotypes with a standard
ribotype nomenclature to facilitate communication between laboratories.
The recommended software for interpretation of C. diphtheriae ribotypes
is Taxotron 2000® (Taxolab, Institut Pasteur, Paris, France) for interpolation
of fragment sizes (20). To date, 86 distinct ribotypes with the endonuclease
BstEII have been chosen for the ribotype database, and each ribotype pattern
has been represented by a reference strain possessing a unique geographical
name, producing a stable and reproducible ribotype pattern (21). These
strains have now also been submitted to automated ribotyping using the
RiboPrinter® Microbial Characterization System (Qualicon, Wilmington,
DE, USA) using both endonucleases (22). Identification and verification
of new and existing genetically defined clones of C. diphtheriae with
the potential to cause epidemics can be made.
In 2002, a toxigenic strain of C. diphtheriae var mitis was isolated
from a throat swab of a boy aged 11 years from a religious community in
Salford, north west England (23). A toxigenic strain of C. diphtheriae
var mitis was also isolated from a family member aged 3 years in Jerusalem
and the two isolates were found to be indistinguishable from their ribotype
patterns. This is an excellent example of how ribotyping can be used in
'tracking' the international spread of this disease. Disease transmission
does not necessarily occur as an isolated event in one country but may
have implications for spread to other countries (3).
In Belarus, the majority of toxigenic strains in the last few years have
been biotype var gravis and their ribotypes were mainly Sankt-Petersburg,
Rossija, and Lyon ribotypes. Many of the strains circulating
in Belarus, however, have been non-toxigenic tox-bearing (NTTB) strains
of C. diphtheriae var mitis that are phenotypically non-toxigenic but
carry the tox-gene, and these have been mainly represented by the ribotype
Moskva, while the other non-toxigenic strains were mainly ribotype Cluj
(11,21). Ribotyping has successfully been used for molecular subtyping
and the identification and monitoring of clonal groups (24).
As previously mentioned, human infection with C. ulcerans is usually
acquired through contact with animals or by ingestion of unpasteurised
dairy products, although risk factors are often undetermined (3). In the
UK, the frequency and severity of C. ulcerans infections appear
to be increasing. Between 1986 and 2001 a total of 51 C. ulcerans isolates,
84% of which were toxigenic strains, were identified. Ribotyping results
of toxigenic C. ulcerans isolated from three cats with bilateral nasal
discharge have shown that the profiles produced are so far indistinguishable
from human isolates (25). The isolation of toxigenic C. ulcerans from
domestic cats, in addition to the possibility of person to person spread,
highlights the importance of further research into the role of C. ulcerans
in the epidemiology of diphtheria (15).
The complete genome of the C. diphtheriae NCTC 13129 strain, an
isolate of the epidemic clone (ribotype Sankt-Petersburg), circulating
within eastern Europe during the 1990s, has been sequenced at the Pathogen
Sequencing Unit at the Sanger Institute. A whole-genome shotgun technique
was used and the final sequence was assembled from 66 099 sequencing reads.
Analysis of the sequence was carried out using the software tool Artemis
and the Artemis Comparison Tool (ACT) was also used to compare this genome
with other bacterial genomes, both at the DNA or protein level. The genome
sequence data is available at http://www.sanger.ac.uk/Projects/C_diphtheriae/.
The completion of this genome sequence has enabled the development of
further studies into the molecular and genetic characteristics of corynebacteria.
A comprehensive study of NTTB-strains, all of biotype C. diphtheriae var
mitis, was recently carried out in Moscow (26,27,28). The lack of diphtheria
toxin production by these strains was explained by a mutation a single
base deletion at nucleotides 52-55 - that was the result of a frameshift
that subsequently produced a loss of the tox-gene open reading frame and
the truncation of the tox protein. However, some researchers have discovered
toxigenic properties in these NTTB strains and argued that toxin production
can be easily restored after passage through dialysis cellophane on iron-deficient
At the University of Colorado in the US, an impressive molecular analysis
was carried out on the regulation and function of the diphtheria toxin
repressor (DtxR) of C. diphtheriae (27). The conclusion to this
study was that DtxR represses siderophore biosynthesis more stringently
than diphtheria toxin production, and that that DtxR was not essential
for the viability of C. diphtheriae, although those strains with
mutations within the dtxR gene did exhibit enhanced susceptibility to
oxidative stress (29).
Further research and exploration into the genome sequence of C. diphtheriae
are providing sources of inspiration and new understanding of the pathogenicity
of toxigenic, NTTB, and non-toxigenic strains. The numbers of participants
in this international conference highlighted the expansion of this specialised
area of microbiology, with attendees several continents. An international
surveillance network within Europe is essential for the control of a resurging,
potentially fatal, vaccine-preventable infectious disease such as diphtheria.
Diphtheria is an excellent example of a situation where public health
services are expected to deliver a timely and coordinated response to
a public health threat such as an imported case that may originate outside
Europe, but which has the potential to spread within that country.
The authors gratefully acknowledge the contribution of all participants
of the Seventh International Meeting of the ELWGD, the European Commission
(EU DG SANCO Agreement No. S12.324473 (2001 CVG4-012)), the Public Health
Laboratory Service, the Department of Traumatology at the University of
Vienna, the Federal Ministry of Social Security and Generations in Vienna,
the Vienna Medical Academy, the WHO Regional Office for Europe, and the
The Programme and Abstracts
Book for the meeting are accessible
in pdf format.