Human clinical isolates of Corynebacterium diphtheriae and Corynebacterium ulcerans collected in Canada from 1999 to 2003 but not fitting reporting criteria for cases of diphtheria.

Abstract

A 5-year collection of Corynebacterium diphtheriae and Corynebacterium ulcerans human clinical isolates yielded nine isolates from blood cultures of patients with invasive infections, stressing the importance of C. diphtheriae as a serious blood-borne pathogen. Seven percent of C. diphtheriae and 100% of C. ulcerans isolates produced diphtheria toxin, demonstrating that toxigenic corynebacteria continue to circulate.

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JOURNAL OF CLINICAL MICROBIOLOGY, July 2005, p. 3447–3449
0095-1137/05/$08.00?0doi:10.1128/JCM.43.7.3447–3449.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 43, No. 7
Human Clinical Isolates of Corynebacterium diphtheriae and
Corynebacterium ulcerans Collected in Canada from 1999
to 2003 but Not Fitting Reporting Criteria for Cases
of Diphtheria
Leanne M. DeWinter,1* Kathryn A. Bernard,1and Marc G. Romney2
National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada
R3E 3R2,1and Division of Medical Microbiology, Department of Pathology and Laboratory
Medicine, University of British Columbia, Vancouver, British Columbia, Canada2
Received 23 December 2004/Returned for modification 8 February 2005/Accepted 28 February 2005
A 5-year collection of Corynebacterium diphtheriae and Corynebacterium ulcerans human clinical isolates
yielded nine isolates from blood cultures of patients with invasive infections, stressing the importance of C.
diphtheriae as a serious blood-borne pathogen. Seven percent of C. diphtheriae and 100% of C. ulcerans isolates
produced diphtheria toxin, demonstrating that toxigenic corynebacteria continue to circulate.
Two serological studies of healthy adult Canadian blood
donors determined that roughly 20% had antibodies to diph-
theria toxin below the accepted protective threshold (12, 21).
When organized by age group, the 60-year and over age group
was the least protected group, with 41.8% and 36.3% below the
threshold in the two studies. Due to the fact that blood donors
comprise a relatively healthier group than the general popula-
tion, immunity among the general population would be ex-
pected to be lower (17). Furthermore, in industrialized coun-
tries, subpopulations of individuals refuse vaccination for
religious, philosophical, or other reasons and thus are without
protection against diphtheria and other vaccination-prevent-
able diseases. Therefore, outbreaks of diphtheria could occur
in susceptible, unvaccinated populations and among adults
whose antibody level has dropped below the protective thresh-
old.
Due to ongoing universal diphtheria vaccination programs in
Canada, there have been no or a few cases of diphtheria meet-
ing the criteria for notification per year since 1986 (4, 14).
Notifiable cases of diphtheria in Canada include those from
which Corynebacterium diphtheriae is isolated from an appro-
priate clinical specimen and those with a histopathological
diagnosis of diphtheria (1, 12, 15). Despite the low incidence of
notifiable diphtheria in Canada, numerous isolates of C. diph-
theriae or Corynebacterium ulcerans continue to be recovered
from patients seeking medical treatment for infections. The
majority of these isolations are not notifiable since they do not
meet case criteria for diphtheria.
The aim of this study was to characterize human clinical
isolates of C. diphtheriae and C. ulcerans that did not meet the
criteria for a notifiable case of diphtheria to determine whether
there is a potential reservoir of toxigenic organisms in Canada.
(The data were presented in part at the 8th European Lab-
oratory Working Group on Diphtheria/Diphtheria Surveil-
lance Network meeting in Copenhagen, Denmark, 16 to 18
June 2004.)
All isolates were human clinical isolates referred to the Na-
tional Microbiology Laboratory, Public Health Agency of Can-
ada, Winnipeg, between 1999 and 2003. A standard panel of
conventional biochemical tests which included fermentation of
sugars, amino acid decarboxylases, and other reactions was
performed as outlined previously (2). Production of diphtheria
toxin was assessed by the modified Elek test (7). Carriage of
the entire diphtheria toxin gene, tox, and the toxA fragment of
the gene was assessed by the PCR as previously described (6,
13). Isolates were assigned to biotypes based on biochemical
characteristics as described previously (8).
By conventional methods, C. diphtheriae isolates demon-
strated phenotypic characteristics consistent with those re-
ported in the literature (8), including fermentation of glucose,
maltose, and fructose but not xylose, mannitol, lactose, or
sucrose. All isolates were urease negative; citrate utilization
negative; esculin and bile esculin negative; lysine, arginine, and
ornithine decarboxylase negative; and Voges-Proskauer nega-
tive.
Six of 89 (7%) isolates of C. diphtheriae produced diphtheria
toxin and harbored the diphtheria toxin gene (Table 1). A
further 6 of 89 (7%) isolates carried the entire tox gene but did
not express it, and 2 of 89 (2%) isolates carried the toxA
fragment of the gene only. C. diphtheriae biotype mitis isolates
carried the full tox gene, whereas C. diphtheriae biotype belfanti
isolates tested positive for the toxA fragment only. Among C.
ulcerans isolates, three of three isolates (100%) produced diph-
theria toxin, which were also detected by PCR. Table 1 sum-
marizes the biotype/species and toxigenicity status of all iso-
lates between 1999 and 2003. By far the most commonly
received isolates were nontoxigenic C. diphtheriae biotype mi-
tis, 54/89 (61%) and C. diphtheriae biotype gravis 24/89 (27%).
Among the biotypes, there were more toxigenic C. diphtheriae
biotype gravis than C. diphtheriae biotype mitis, with rates of
4/28 (14%) and 2/56 (4%), respectively. Thus, over a 5-year
* Corresponding author. Mailing address: National Microbiology
Laboratory, Public Health Agency of Canada, 1015 Arlington St.,
Winnipeg, Manitoba, Canada R3E 3R2. Phone: (204) 789-2137. Fax:
(204) 789-5009. E-mail: leanne_dewinter@phac-aspc.gc.ca.
3447

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period, there was an overall rate of 10% (9/92) toxigenicity
among all isolates and a further 9% (8/92) of isolates carried
the toxin genes but did not express them. None of the nonno-
tifiable referrals were identified as C. pseudotuberculosis, C.
diphtheriae biotype intermedius, toxigenic C. diphtheriae bio-
type belfanti, or nontoxigenic C. ulcerans during this period.
The most common sources of C. diphtheriae and C. ulcerans
were nonsterile sites, including skin and wounds, representing
59% (54/92) of all isolates, while 17% (16/92) were isolated
from ears (Table 1). When examined by biotype, a different
picture emerges; 39% (11/28) of all C. diphtheriae biotype
gravis isolates and 9% (5/56) of C. diphtheriae biotype mitis
isolates came from ears. C. diphtheriae biotypes mitis and gravis
(all nontoxigenic) were isolated from throats at the low inci-
dence of 2% (1/56) among biotype mitis isolates and 7% (2/28)
among biotype gravis isolates. The total percentage of throat
isolates from all specimen sites was only 3% (3/92). Three of
four C. diphtheriae biotype belfanti isolates were from sinuses,
and they were the only biotype isolated from sinuses.
Nine of 92 (10%) C. diphtheriae isolates were isolated from
blood, including eight nontoxigenic biotype mitis isolates and
one toxigenic biotype gravis isolate. All nine isolates were iso-
lated from patients in the Vancouver area and were fairly
spaced out over a 5-year period. Four of the nine patients died
following the infection, though the patient who was infected
with the toxigenic C. diphtheriae biotype gravis survived. Un-
derlying medical and social conditions that appeared to con-
tribute to the development of bacteremia included intravenous
drug use, diabetes mellitus, homelessness, and skin coloniza-
tion with C. diphtheriae. Most, but not all, of the patients lived
in or frequented the impoverished skid row area of Vancouver,
which was associated with diphtheria outbreaks up to and
during the 1970s (3). This cluster of invasive isolates is consis-
tent with reports from around the world of nontoxigenic inva-
sive C. diphtheriae infections associated with known risk factors
(9–11, 16, 18) and highlights the importance of considering C.
diphtheriae a serious pathogen when isolated from blood.
There have been increasing reports of C. ulcerans human
infections in the literature in which toxigenic strains have been
responsible for classical pharyngeal and cutaneous diphtheria
as well as being associated with other infections (5, 19, 20).
Although only three isolates of C. ulcerans have been referred
to the national reference center between 1999 and 2003, all
three isolates were toxigenic. Disease caused by all diphtheria
toxin-producing species of Corynebacterium, including C.
pseudotuberculosis and C. ulcerans, must be considered in the
differential of causative agents of diphtheria. In the United
Kingdom, isolation of any toxigenic corynebacteria, including
C. pseudotuberculosis and C. ulcerans, requires notification of
local and national communicable disease control agencies (5).
Although universal vaccination has resulted in a very low
incidence of diphtheria in Canada, human clinical isolates
which harbor and produce the diphtheria toxin remain in cir-
culation. Between 1999 and 2003, 16% (14 of 89) of referred C.
diphtheriae isolates produced the diphtheria toxin or harbored
the diphtheria toxin gene without expressing it; 100% of re-
ferred C. ulcerans isolates produced the diphtheria toxin. The
continued circulation of toxigenic strains of C. diphtheriae and
C. ulcerans highlights the importance of continuing vaccination
programs against diphtheria.
We acknowledge Carol Shaw of the British Columbia Centre for
Disease Control for obtaining isolate information. We also thank De-
borah Wiebe and Emma Ongsansoy for excellent technical assistance.
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All isolates
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Biotype gravis
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site.
3448NOTESJ. CLIN. MICROBIOL.

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