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Journal of Clinical Microbiology, August 2008, p. 2822-2823, Vol. 46, No. 8
0095-1137/08/$08.00+0     doi:10.1128/JCM.01010-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

Detection of Toxigenic Corynebacterium diphtheriae and Corynebacterium ulcerans Strains by a Novel Real-Time PCR

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Diphtheria is caused by both toxigenic Corynebacterium diphtheriae and Corynebacterium ulcerans strains harboring lysogenic, tox-bearing beta-corynephages. While C. diphtheriae has caused most diphtheria cases for decades, the emerging pathogen C. ulcerans is becoming the major cause for diphtheria in many industrialized countries. Among the 25 Diphtheria Surveillance Network (DIPNET) members, Germany reported the most C. ulcerans-associated diphtheria patients after the United Kingdom and France (7).

In C. diphtheriae, tox encodes diphtheria toxin (DT), causing the systemic diphtheria symptoms. Recently, we described nucleotide and amino acid sequence differences between the C. diphtheriae and C. ulcerans DTs (11). tox and DT in C. ulcerans are much more heterogenous than in C. diphtheriae (6), since the 11 published C. ulcerans tox and DT sequences fall into five different groups (4, 8, 9, 11).

The differences between C. diphtheriae tox and C. ulcerans tox allow their differentiation by PCR (11), and a conventional C. diphtheriae tox PCR used by many laboratories worldwide for the identification of toxigenic corynebacteria amplifies both C. diphtheriae and C. ulcerans tox (2). Disturbingly, Tiwari et al. reported that a real-time C. diphtheriae tox PCR (5) used at the CDC caused atypical or false-negative results for two C. ulcerans diphtheria patients (12). Similarly, Cassiday et al. very recently published a study on the failure of their real-time PCR in detecting C. ulcerans tox (1). Since a reliable real-time PCR for detecting toxigenic corynebacteria is urgently needed, we developed and evaluated a novel 5'-nuclease real-time PCR detecting both C. diphtheriae and C. ulcerans tox.

The forward primer (RTDT_fw, TTA TCA AAA GGT TCG GTG ATG GTG), reverse primer (RTDT_rev2, AAT CTC AAG TTC TAC GCT TAA C), and probe (RTDT_So, 6FAM-CGC GTG TAG TGC TCA GCC TTC CCT-BBQ, where 6FAM is 6-carboxyfluorescein and BBQ is BlackBerry Quencher) are based on all tox sequences currently available at GenBank—six for C. ulcerans, three for C. diphtheriae, and five for DT-encoding prophages. The Mx3000P cycler (Stratagene) program consisted of a single denaturation step (15 min, 95°C) and 45 cycles of 94°C for 1 min and 60°C for 1 min, according to the QuantiTect No Rox (Qiagen) protocol.

Nineteen tox-bearing and 70 DT-negative C. diphtheriae and C. ulcerans clinical isolates obtained from patients with suspected respiratory or cutaneous diphtheria and collected by the German Consiliary Laboratory on Diphtheria since 1999 (Tables 1 and 2) were analyzed. Species identification was achieved by using API Coryne (bioMérieux) (9, 11) and by 16S rRNA and rpoB sequencing (3). Toxigenicity was tested by the tox PCR (2) and by a modified Elek test (11, 13). All 19 tox-bearing strains yielded positive results, and all 70 tox-negative strains yielded negative results, thus showing 100% sensitivity and specificity for the real-time PCR. All amplicons were verified by sequencing. DNA dilution revealed a detection limit of 100 fg/reaction, equivalent to 37 genome molecules. In comparison to the tox PCR (2), our method is 25-fold more sensitive. As expected, our C. ulcerans tox-specific PCR (11) was positive only in all tox-bearing C. ulcerans and not in tox-bearing C. diphtheriae strains.

View this table: [in this window] [in a new window]

TABLE 1. tox-positive Corynebacterium sp. clinical isolates tested in this study

View this table: [in this window] [in a new window]

TABLE 2. tox-negative Corynebacterium sp. clinical isolates used for validation of the real-time PCR

Since our real-time PCR based on all thus-far-published C. diphtheriae and C. ulcerans tox genes was reliable in rapidly detecting tox, it may serve as a screening method for the presence of C. diphtheriae or C. ulcerans tox in the management of a patient suspected of having diphtheria. However, physicians and microbiologists should be aware that an Elek test must be performed on tox-positive isolates for the testing of DT production. As with C. diphtheriae, the existence of tox-positive, Elek test-negative C. ulcerans strains has been described by us (11) and recently confirmed by others (1).

 
We thank Wolfgang Schmidt, Angela Huber, Henrike Skala, and Birgit Groβ for the cultivation and microbiological characterization of the corynebacteria.

 
Published ahead of print on 11 June 2008.

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1. Cassiday, P. K., L. C. Pawloski, T. Tiwari, G. N. Sanden, and P. P. Wilkins. 2008. Analysis of toxigenic Corynebacterium ulcerans strains revealing potential for false-negative real-time PCR results. J. Clin. Microbiol. 46:331-333.[Abstract/Free Full Text]
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2. Hauser, D., M. R. Popoff, M. Kiredjian, P. Boquet, and F. Bimet. 1993. Polymerase chain reaction assay for diagnosis of potentially toxinogenic Corynebacterium diphtheriae strains: correlation with ADP-ribosylation activity assay. J. Clin. Microbiol. 31:2720-2723.[Abstract/Free Full Text]
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3. Khamis, A., D. Raoult, and B. La Scola. 2004. rpoB gene sequencing for identification of Corynebacterium species. J. Clin. Microbiol. 42:3925-3931.[Abstract/Free Full Text]
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4. Lartigue, M.-F., X. Monnet, A. Le Flèche, P. A. D. Grimont, J.-J. Benet, A. Durrbach, M. Fabre, and P. Nordmann. 2005. Corynebacterium ulcerans in an immunocompromised patient with diphtheria and her dog. J. Clin. Microbiol. 43:999-1001.[Abstract/Free Full Text]
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5. Mothershed, E. A., P. K. Cassiday, K. Pierson, L. W. Mayer, and T. Popovic. 2002. Development of a real-time fluorescence PCR assay for rapid detection of the diphtheria toxin gene. J. Clin. Microbiol. 40:4713-4719.[Abstract/Free Full Text]
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6. Nakao, H., I. K. Mazurova, T. Glushkevich, and T. Popovic. 1997. Analysis of heterogeneity of Corynebacterium diphtheriae toxin gene, tox, and its regulatory element, dtxR, by direct sequencing. Res. Microbiol. 148:45-54.[Medline]
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7. Neal, S., and A. Efstratiou. 2007. DIPNET—establishment of a dedicated surveillance network for diphtheria in Europe. Euro Surveill. 12:E9-E10.[Medline]
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8. Seto, Y., T. Komiya, M. Iwaki, T. Kohda, M. Mukamoto, M. Takahashi, and S. Kozaki. 2008. Properties of corynephage attachment site and molecular epidemiology of Corynebacterium ulcerans isolated from humans and animals in Japan. Jpn. J. Infect. Dis. 61:116-122.[Medline]
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9. Sing, A., S. Bierschenk, and J. Heesemann. 2005. Classical diphtheria caused by Corynebacterium ulcerans in Germany: amino acid sequence differences between diphtheria toxins from Corynebacterium diphtheriae and C. ulcerans. Clin. Infect. Dis. 40:325-326.[CrossRef][Medline]
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10. Sing, A., and J. Heesemann. 2005. Imported cutaneous diphtheria, Germany, 1997-2003. Emerg. Infect. Dis. 11:343-344.[Medline]
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11. Sing, A., M. Hogardt, S. Bierschenk, and J. Heesemann. 2003. Detection of differences in the nucleotide and amino acid sequences of diphtheria toxin from Corynebacterium diphtheriae and Corynebacterium ulcerans causing extrapharyngeal infections. J. Clin. Microbiol. 41:4848-4851.[Abstract/Free Full Text]
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12. Tiwari, T. S., A. Golaz, D. T. Yu, K. R. Ehresmann, T. F. Jones, H. E. Hill, P. K. Cassiday, L. C. Pawloski, J. S. Moran, T. Popovic, and M. Wharton. 2008. Investigations of 2 cases of diphtheria-like illness due to toxigenic Corynebacterium ulcerans. Clin. Infect. Dis. 46:395-401.[CrossRef][Medline]
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13. Wellinghausen, N., A. Sing, W. V. Kern, S. Perner, R. Marre, and J. Rentschler. 2002. A fatal case of necrotizing sinusitis due to toxigenic Corynebacterium ulcerans. Int. J. Med. Microbiol. 292:59-63.[CrossRef][Medline]

						Regina Schuhegger Marion Lindermayer Rudolf Kugler National Consiliary Laboratory for Diphtheria Veterinärstraβe 2 85764 Oberschleissheim, Germany Jürgen Heesemann Max von Pettenkofer-Institut Ludwig-Maximilians-Universität München Pettenkoferstraβe 9a 80336 Münich, Germany Ulrich Busch Andreas Sing* Bavarian Health and Food Safety Authority Veterinärstraβe 2 85764 Oberschleissheim, Germany
						* Phone: 49-89-31560-814 Fax: 49-89-31560-458 E-mail: andreas.sing{at}lgl.bayern.de

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Journal of Clinical Microbiology, August 2008, p. 2822-2823, Vol. 46, No. 8
0095-1137/08/$08.00+0     doi:10.1128/JCM.01010-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Schuhegger, R. Articles by Sing, A. Search for Related Content PubMed PubMed Citation Articles by Schuhegger, R. Articles by Sing, A.