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J Clin Microbiol. Jan 2008; 46(1): 331–333.
Published online Nov 7, 2007. doi:  10.1128/JCM.01251-07
PMCID: PMC2224273

Analysis of Toxigenic Corynebacterium ulcerans Strains Revealing Potential for False-Negative Real-Time PCR Results[down-pointing small open triangle]

Abstract

Diphtheria surveillance depends on the rapid and reliable recognition of the toxin gene in Corynebacterium diphtheriae. Real-time PCR is a rapid tool to confirm the presence of the diphtheria toxin gene (tox) in an isolate or specimen. We report that some toxigenic Corynebacterium ulcerans strains show atypical results in a real-time PCR for tox.

Recent reports in the literature indicate that toxigenic Corynebacterium ulcerans strains are being isolated from patients with respiratory diphtheria-like illnesses in several countries (1, 2, 8-10, 15-21). We report the characterization of a strain (designated CD355) isolated from a throat membrane from a patient with suspected respiratory diphtheria in March 2005. Real-time PCR on DNA extracted from the strain showed atypical results (Fig. (Fig.1).1). However, phenotypic testing indicating that the organism isolated from the membrane was a toxigenic C. ulcerans isolate. In order to evaluate the potential extent of atypical or false-negative real-time PCR tests for the diphtheria toxin gene in C. ulcerans isolates, we further characterized all available human C. ulcerans strains (n = 19) stored in the CDC's Corynebacterium strain collection (Table (Table11).

FIG. 1.
Real-time PCR graph showing the detection of tox subunits A and B in typical and atypical strains of C. ulcerans.
TABLE 1.
Results for 20 C. ulcerans strains tested by real-time and conventional PCR and the Elek assay

Real-time PCR for the A and B subunits of tox was performed using primers and probes described previously by Mothershed et al. (12). Conventional PCR for the A and B subunits of tox was performed using primers and methods described previously by Nakao and Popovic (13). Conventional PCR was also performed using the real-time PCR primers. A modified Elek toxin-antitoxin precipitation test as described previously by Engler et al. (5) was used to test all strains for diphtheria toxin production using antitoxin produced by the Instituto Butantan, Sao Paulo, Brazil.

Sequencing and analysis as described previously by Gee et al. (6) were performed on the tox genes of 10 C. ulcerans strains. Sequencing primers included primers 107U21, 186U20, 354U21, 552U21, 1001U21, 1227U21, 1473U21, 605L21, 822L22, 1268L20, 1523L21, 1761L22, and 1929L18 (Table (Table2).2). The listed primers were originally described by Nakao et al. (14) and altered to include nucleotide changes observed in the diphtheria toxin gene from a previously reported C. ulcerans strain, A6361 (GenBank accession number AY141014). Primers 107U21 or 186U20 with 1929L18 were used to make a 1.8-kb amplicon that begins before the start of transcription and ends after the stop codon. Sequence data analysis was performed with the Genetics Computer Group Wisconsin package, version 10.2 (Accelrys, San Diego, CA). The tox sequences were aligned with a previously reported Corynebacterium diphtheriae diphtheria toxin gene (GenBank accession number K01722) (7, 11).

TABLE 2.
Primers used for sequencing of the tox gene

Real-time PCR, conventional PCR, and Elek results for the 20 study strains are presented in Table Table1.1. tox was not detected in eight Elek-negative strains by either PCR method. In one Elek-negative strain (CD022), tox was detected by conventional PCR but not by real-time PCR. tox was detected in four Elek-positive strains by both PCR methods. In the remaining seven Elek-positive strains, both subunits of tox were detected by conventional PCR with the conventional primers as well as with the real-time primers; however, these strains showed weak amplification of subunit A and no amplification of subunit B of tox by real-time PCR. Based on the results for the 20 study strains, positive and negative predictive values for real-time and conventional PCR for tox are presented in Table Table33.

TABLE 3.
Positive and negative predicative values for real-time and conventional PCR for the A and B subunits of the diphtheria toxin gene (tox) in 20 C. ulcerans isolates

Sequencing of the toxin gene identified several nucleotide differences in the seven atypical real-time PCR strains, specifically in the regions to where the primers and probes anneal. Three to four mismatches were seen in the probe and primer regions of real-time PCR for subunit A, and eight mismatches were observed in subunit B. In comparison, three to four mutations were seen in conventional PCR for subunit A, and no mismatches were observed for subunit B.

Mismatches were also found in nontoxigenic strain CD022. Six mismatches were seen in the probe and primer regions of the real-time PCR for subunit A, and nine mismatches were observed in subunit B. In comparison, five mismatches were found in conventional PCR for subunit A, and only one mismatch was observed in subunit B.

We have identified a toxigenic C. ulcerans strain that caused a respiratory diphtheria-like syndrome and compared it with 19 archival strains. We found that real-time PCR, using the primers and probes previously used to detect the A and B subunits of C. diphtheriae tox (17), was not a reliable method to determine the presence of tox in C. ulcerans. Seven of 11 (63%) toxigenic strains in this study would not have been identified as being toxigenic based solely on real-time PCR results. The fact that conventional PCR was able to work with both real-time and conventional PCR primers despite some sequencing mismatches indicates that annealing of the probe is much more vulnerable to mismatches than annealing of the primers. Therefore, real-time PCR may be less sensitive in identifying tox in C. ulcerans than conventional PCR if the probe happens to anneal to a relatively variable region.

PCR is a valuable and rapid method for detecting the presence of tox in a specimen from a suspected case of diphtheria. In many such cases, PCR on DNA extracted from a throat swab may be the only tool available to help diagnose the disease (12). However, the presence of tox does not always indicate toxin production, and isolates should be tested phenotypically for toxin production by the Elek test or Vero cell cytotoxicity assay (4). Strains of C. diphtheriae that carry tox but that do not express the toxin have been previously reported (3), and we report here, for the first time, a strain of C. ulcerans (CD022) that is not toxigenic but that contains detectable tox.

Physicians and clinical laboratorians should be aware that a diphtheria-like disease can be caused by C. ulcerans. Negative or unusual real-time PCR results should be verified by conventional PCR to verify the absence of tox in diphtheria-like illness caused by C. ulcerans, and the Elek test or Vero cell assay must be performed on isolates in a reference laboratory to test for the production of a functional toxin protein.

Lastly, it may be feasible to develop a real-time PCR that is more specific for C. ulcerans atypical tox sequences. However, the frequency of such strains circulating around the world is unknown. In cases where atypical real-time PCR results are obtained from a throat swab, it may be more practical to use conventional or real-time primers in conventional PCR to detect tox.

Acknowledgments

We acknowledge the contributions of Henrietta Harder and Timothy Jones of the Tennessee Department of Health to this project.

Footnotes

[down-pointing small open triangle]Published ahead of print on 7 November 2007.

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