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Introduction
The epidemic of diphtheria in the Newly Independent States (NIS) began
in the Russian Federation in 1990 and affected all the NIS countries
by the end of 1994. The emergence of this epidemic resulted in the need
for the development of modern laboratory techniques for diphtheria diagnosis
and analysis. At the initiative of the World Health Organization Regional
Office for Europe, the European Laboratory Working Group on Diphtheria
(ELWGD) was formed in July 1993 as a result of the epidemic situation
in the NIS. In 2001, the network became `The Diphtheria Surveillance
Network (DIPNET)', and included both the epidemiological and microbiological
aspects of diphtheria and other infections caused by potentially toxigenic
corynebacteria. The Eighth International meeting of the European Laboratory
Working Group on Diphtheria (ELWGD) and the Diphtheria Surveillance
Network (DIPNET) was held and co-organised with the WHO Regional Office
for Europe, Copenhagen, Denmark, in June 2004. Following are the main
issues discussed and all they all highlight the importance of improving
surveillance systems and carrying out epidemiological studies to sustain
diphtheria control.
Current state of diphtheria in the European Region
In the last fifty years, the incidence of diphtheria in western Europe
has declined dramatically. However, in 1990 a diphtheria epidemic occurred
in the Newly Independent States (NIS) of the former USSR. The epidemic
began in the Russian Federation in 1990 and affected all the NIS countries
by the end of 1994. At the peak of the epidemic in 1995, 50 425 cases
were reported in the NIS, compared with 24 cases in other countries;
the NIS accounted for 88% of cases reported worldwide. Diphtheria control
measures were implemented in the Russian Federation in 1992, and mass
immunisation campaigns were set up in all the Newly Independent States
(NIS), achieving a high coverage rate (³ 80% in all age groups)
relatively quickly. As a result of the action taken, the incidence of
diphtheria in the Russian Federation and in the NIS began to decrease.
Between 1990 and 2001, over 160 000 cases were reported in the region
with over 4000 deaths. In 2002, 1189 cases were reported from the WHO
European region: 95% of the cases were from the Russian Federation and
the NIS. In 2003, a total of 896 cases were reported from the WHO European
region and 99% (892) were from the Russian Federation and the NIS; the
four remaining cases were reported from Turkey (n=1) and the UK (n=3)
(FIGURE)[1,2].
Many of the western, central and eastern European countries now report
none or very few cases of diphtheria each year, including importated
cases. Since 2003, excellent progress in the control of diphtheria has
been achieved and the incidence has remained very low in most of the
NIS. However, in a few countries, such as Georgia, Latvia, Ukraine and
the Russian Federation, the situation still appears to be problematic
[2].
Sustaining diphtheria control is still a high priority for the WHO
European Region and can only be achieved effectively by maintaining
high population immunity in all age groups together with a good epidemiological
and microbiological surveillance system with reliable laboratory diagnosis,
for timely detection, investigation and management of cases and contacts
[4, 5].
Clinical, epidemiological and microbiological aspects of infection
caused by C. diphtheriae and C. ulcerans
Diphtheria is rare in western Europe and this makes it difficult to
establish a standardised surveillance system. The policy for screening
of throat swabs varies from country to country and only five of 19 countries
routinely screen throat swabs for corynebacteria [6]. If throat swabs
are not screened routinely, this could result in cases being diagnosed
late. Clinicians providing insufficient information to laboratories
along with mild or atypical clinical presentations in vaccinated patients
may also lead to a delayed diagnosis. In England and Wales, between
1986 and 2003, only 14 of 90 (16%) cases of toxigenic C. diphtheriae
infection presented with classical diphtheria; 84% of cases had milder
infections such as sore throat. Mild infections can only be detected
by screening throat swabs and if routine screening ceases, more than
80% of the infections will probably not be detected. This could result
in inappropriate treatment of cases, higher fatality ratios and secondary
cases and increase risk of outbreaks [7, 8].
Most cases of diphtheria result from infection with toxin producing
strains of C. diphtheriae. However, strains of C. ulcerans
found more commonly in cattle than other animals, can carry the same
bacteriophage that codes for the toxin produced by toxigenic strains
of C. diphtheriae. Human C. ulcerans infections are usually
acquired through contact with animals or by eating or drinking unpasteurised
dairy products [9, 10, 11]. However, such risk factors have not been
identified for some cases of classical diphtheria caused by C. ulcerans,
which suggests that there may be other routes of infection [12]. In
2001, a case of diphtheria-like illness in a Japanese woman caused by
toxigenic C. ulcerans was documented. The patient had no direct
contact with dairy livestock or unpasteurised dairy products, but a
week before illness onset, the patient had been scratched by a cat,
which had rhinorrhea [13]. Toxigenic C. ulcerans has also been
isolated in the UK from domestic cats with bilateral nasal discharge
[14, 15] and recently C. ulcerans was isolated from a 47-year-old
French woman with severe sore throat and dyspnea who had close contact
with an infected dog. Molecular typing confirmed that the human isolate
and the dog isolate had indistinguishable ribotypes.
Molecular and genetic characterisation of Corynebacterium diphtheriae
Data on the analysis of the complete genome sequence of Corynebacterium
diphtheriae NCTC 13129 has been reported [16]. The genome sequence
data can be obtained from the GeneDB website (http://www.genedb.org).
The genome sequence data will permit the discovery of novel virulence
factors and factors responsible for colonising the host. Sequencing
the genome of a non-toxigenic C. diphtheriae strain and also
a C. ulcerans strain in future would give further insight into
specific virulence mechanisms associated with these organisms and therefore
may help to clarify the role of these organisms as emerging pathogens.
The international nomenclature for Corynebacterium diphtheriae
ribotypes has now been established and a database of all recognised
ribotypes has been built and requires regular updating [17]. Ribotyping
is an effective and a discriminatory typing method, which can be used
to study the global epidemiology of Corynebacterium diphtheriae.
It is still the most recognised and straightforward method for typing
Corynebacterium diphtheriae isolates and the ribotype database
should facilitate global communication between typing laboratories [17].
A study which analysed 302 toxigenic C. diphtheriae isolated
between 2001-2003 and 974 non-toxigenic C. diphtheriae isolated
between 1996-2003 from Russia, showed that among the toxigenic strains,
the biotype gravis was most common and amongst the non-toxigenic strains,
biotype mitis was most common [18]. Among the non-toxigenic strains,
164 were non-toxigenic tox-bearing strains (NTTB) (these strains possess
the tox gene, however, they do not express toxin phenotypically).
Ribotyping strains isolated between 2001-2003 revealed 12 ribotypes
amongst the toxigenic strains and nine ribotypes amongst the non-toxigenic
strains. The predominant ribotypes amongst the toxigenic strains were
St. Petersburg, Rossija, Otchakov, Cluj, Londinium and Schwarzenberg.
The majority of the NTTB strains were ribotype Moskva, however recently,
three new ribotypes (provisionally named as NTTB1, NTTB2 and NTTB3)
have been documented amongst the NTTB strains isolated from Moscow [18].
The role of NTTB strains is still uncertain in the epidemiology of
diphtheria. The isolation rate of NTTB strains varied from year to year.
To establish mutations in the tox gene, NTTB strains were analysed
by peptide nucleic acid (PNA)- mediated PCR clamping. Deletion of one
guanine of four between positions 52-55 leading to a DNA open reading
frame shift, and a nucleotide substitution in position 60 (adenine to
guanine), which did not result in an amino acid substitution were revealed
in all strains. These results were confirmed by direct sequencing of
the tox gene. The epidemiological significance of NTTB strains
and reasons for these particular mutations are currently been investigated
[19].
Diphtheria immunity: strategies and sero-epidemiological studies
The European Sero-Epidemiological Network (ESEN-2) [20], based on the
original ESEN project was established in 2001 [21], and the network
undertook an evaluation of several diphtheria antibody test kits.
A panel of 150 human serum samples were tested by eight participating
laboratories. The Vero cell toxin neutralisation assay (VCA) is the
only assay that measures functional antibodies and is therefore used
as the reference in vitro assay. Comparison of the results obtained
from the different laboratories revealed a high correlation between
the VCA results (R2 > 0.9). Comparison of the VCA results
with results obtained from other assays such as the double-antigen delayed
time-resolved fluorescence (DA-DELFIA), double antigen enzyme-linked
immunosorbent assay (DA-ELISA), toxin binding inhibition test (ToBI),
passive haemagglutination assay (PHA) and two commercially available
enzyme-linked immunosorbent assay (ELISA) kits revealed that there is
good correlation between the VCA and the DA-DELFIA, DA-ELISA, ToBI and
the PHA assays (R2 > 0.8). There was poor correlation
between the two ELISA kits and the VCA (R2< or = 0.6).
Therefore, these ELISA kits, even though cheaper and simpler to use
than neutralisation tests, lack sensitivity for serum samples containing
low levels of antitoxin and are not recommended for use [22, 23].
However, a new enzyme immunoassay (EIA) with an improved correlation
to the Vero cell assay (VCA), which is available commercially from Binding
Site Ltd, United Kingdom, was tested and compared with the VCA. Thirty-four
serum samples from the Respiratory and Systemic Infection Laboratory,
HPA, Colindale, UK were tested using the EIA and the results were compared
with those obtained by the VCA. Linear regression analysis showed excellent
correlation between the assays (R2 = 0.974). Using WHO guidelines of
0.01-0.1 IU/mL as minimum protective level, and >0.1 IU/mL as protective,
only 2 of 34 samples gave discordant results. However, both samples
had VCA results within one doubling dilution of the EIA result. The
EIA assay measuring range was 0.004 - 3.0 IU/mL. Intra-assay percentage
coefficient of variation was found to be between 5.8% and 2.7% by testing
0.06, 0.71 and 2.6 IU/mL samples 16 times. Assay linearity was assessed
at serum dilutions of 1:100 - 1:128000 using three positive samples.
Comparison of the achieved and expected values by linear regression
gave values of (R2 = 0.998, 1.000 and 0.999
respectively. As the two assays produce very similar results, the newly
developed EIA could be a possible alternative to the VCA. Use of an
EIA assay offers significant advantages in terms of cost, speed, ease
of use and adaptability to automation than the kits used previously
[24].
Studies performed on immunity to diphtheria in various countries such
as, Russia, Kazakhstan, Latvia, Turkey and Brazil have shown that in
spite of mass immunisation programmes, there are still many adults who
have inadequate immunity levels and are susceptible to diphtheria. The
age group with the lowest levels of immunity varies from country to
country and probably depends on the year that childhood immunisation
programme was implemented on a routine basis [25, 26]. Immunity induced
by childhood immunisation usually wanes and if adults do not receive
booster doses of diphtheria toxoid, they become susceptible to the disease
[25, 27, 28].
Conclusions
Diphtheria made a dramatic return in eastern Europe and remains a serious
disease throughout many countries of the world. The eastern European
epidemic has clearly shown that diphtheria will always return whenever
immunity levels decrease and highlights the importance of childhood
vaccination, maintenance of immunity in adults, and the role of socioeconomic
conditions in the spread of diphtheria. Also, with increasing international
travel and the emergence of epidemic clones, the existence of diphtheria
anywhere in the world poses a threat to the unimmunised and those persons
with low levels of immunity. These problems further highlight the importance
of microbiological and epidemiological surveillance and the use of new
molecular methodologies. The changing epidemiology of the disease poses
a threat and ongoing efforts to further enhance our understanding of
this disease must continue.
Further information on the ELWGD/DIPNET can be found at:
http://www.hpa.org.uk/hpa/inter/elwgd_menu.htm
* DIPNET collaborators: Armenia, S Gabrielyan; Austria, R Bauer, R
Strauss; Azerbaijan, R Mammadbayova; Belarus, L Titov; Belgium, D Pierard;
Brazil, A Mattos Guraldi; Canada, K Bernard; Cyprus, D Bagatzouni; Czech
Republic, B Kriz; Denmark, P Andersen, J Christensen, Estonia, U Joks;
Finland, J Vuopio-Varkila; France, P Grimont; Georgia, T Gomelauri;
Germany, A Sing; Greece; A Pangalis India, N Sharma; Israel, E Marva;
Italy, S Salmaso, C Von Hunolstein; Kazakhstan, V Kim; Kyrghzia, G Djumalieva;
Latvia, I Selga, I Velicko; Moldova, P Galetchi; Romania, M Damian,
A Diaconescu; Russian Federation, R Kozlov, I Mazurova, G Tseneva, Tajikistan,
M Boltaeva, Turkey, E Akbas, S Tumay; UK, A Efstratiou, N Crowcroft;
Ukraine, T Glushkevich; USA, T Popovic; Uzbekistan, K Iskhakova; WHO
EURO, N Emiroglu.
The authors gratefully acknowledge the contribution of all participants
of the Eighth International Meeting of the ELWGD and DIPNET. We also
thank the INTAS Programme 01-2289 for support and the WHO Regional Office
for Europe for hosting the meeting in Copenhagen.
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