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.
TABLE 1.
tox-positive Corynebacterium sp. clinical isolates tested in this study
| Strain | Species | Yr of isolation | Source/sex/age (yr)b | Elek test result | tox PCR resulta | Real-time PCR result | Reference |
|---|---|---|---|---|---|---|---|
| A6361 | C. ulcerans | 2000 | Human/M/40 | Negative | Positive | Positive | 11 |
| A2911 | C. ulcerans | 2001 | Human/M/77 | Positive | Positive | Positive | 11 |
| 43 | C. diphtheriae subsp. mitis | 2001 | Human/ND/ND | Positive | Positive | Positive | 10 |
| 52 | C. diphtheriae subsp. mitis | 2002 | Human/M/5 | Positive | Positive | Positive | 10 |
| 53 | C. diphtheriae subsp. mitis | 2002 | Human/M/10 | Positive | Positive | Positive | 10 |
| X959 | C. ulcerans | 2004 | Human/W/53 | Positive | Positive | Positive | 9 |
| 89 | C. diphtheriae subsp. mitis | 2005 | Human/M/4 | Positive | Positive | Positive | |
| 90 | C. ulcerans | 2005 | Human/W/35 | ND | Positive | Positive | |
| 102 | C. ulcerans | 2006 | Human/ND/ND | Positive | Positive | Positive | |
| 107 | C. ulcerans | 2007 | Cat/ND/ND | Positive | Positive | Positive | |
| 109 | C. ulcerans | 2007 | Human/W/ND | Positive | Positive | Positive | |
| 110 | C. ulcerans | 2007 | Human/W/ND | Positive | Positive | Positive | |
| 111 | C. diphtheriae subsp. mitis | 2007 | Laboratory trial DIPNET | Negative | Positive | Positive | |
| 112 | C. diphtheriae subsp. gravis | 2007 | Laboratory trial DIPNET | Positive | Positive | Positive | |
| 114 | C. ulcerans | 2007 | Laboratory trial DIPNET | Positive | Positive | Positive | |
| 115 | C. diphtheriae | 2007 | Laboratory trial DIPNET | Negative | Positive | Positive | |
| 117 | C. diphtheriae subsp. gravis | 2007 | Laboratory trial DIPNET | Positive | Positive | Positive | |
| 118 | C. diphtheriae subsp. gravis | 2007 | Laboratory trial DIPNET | Positive | Positive | Positive | |
| 126 | C. ulcerans | 2007 | Human/W/56 | Positive | Positive | Positive |
The tox PCR was performed as described previously (2).
M, man; W, woman; ND, not determined; DIPNET, Diphtheria Surveillance Network.
TABLE 2.
tox-negative Corynebacterium sp. clinical isolates used for validation of the real-time PCR
| Species | No. of isolates | Elek test result | tox PCR resulta | Real-time PCR result |
|---|---|---|---|---|
| C. diphtheriae subsp. gravis | 14 | Negative | Negative | Negative |
| C. diphtheriae subsp. mitis | 29 | Negative | Negative | Negative |
| C. diphtheriae subsp. belfanti | 11 | Negative | Negative | Negative |
| C. diphtheriae subsp. intermedius | 5 | Negative | Negative | Negative |
| C. amycolatum | 3 | Negative | Negative | Negative |
| C. striatum | 2 | Negative | Negative | Negative |
| C. pseudodiphtheriticum | 2 | Negative | Negative | Negative |
| C. propinquum | 2 | Negative | Negative | Negative |
| C. ulcerans | 1 | Negative | Negative | Negative |
| C. coyleae | 1 | Negative | Negative | Negative |
The tox PCR was performed as described previously (2).
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).
Acknowledgments
We thank Wolfgang Schmidt, Angela Huber, Henrike Skala, and Birgit Groβ for the cultivation and microbiological characterization of the corynebacteria.
Footnotes
Published ahead of print on 11 June 2008.
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