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Correspondence |
Faculdade de Ciências Médicas, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil
Correspondence
Ana L. Mattos-Guaraldi
(guaraldi{at}pesquisador.cnpq.br)
The importance of identifying Corynebacterium diphtheriae can be appreciated since diphtheria remains endemic in many countries, alongside the risk of epidemic outbreaks and the existence of a large number of non-immunized people worldwide (Galazka & Robertson, 1995; Damasco et al., 2005). The features of infections caused by C. diphtheriae have changed over the decades, and are most clearly emphasized by the emergence of non-toxigenic strains causing atypical diseases, such as endocarditis, septic arthritis or infection in unusual anatomical sites (Mattos-Guaraldi & Formiga, 1998; Efstratiou & George, 1999; Reacher et al., 2000; Mattos-Guaraldi et al., 2003). Rapid and reliable methods are needed for identifying a C. diphtheriae infection, thus aiding in appropriate and timely patient management, and improved monitoring of cases.
The use of multiplex PCR is becoming increasingly important in the diagnosis of infectious diseases, and could be a simple and fast alternative procedure for identification of C. diphtheriae, including for screening for non-toxin-gene- and toxin-gene-bearing strains (Cianciotto & Groman, 1997). In this study, a multiplex PCR assay was developed as a rapid and simple method for the identification of C. diphtheriae, and for the differentiation between non-toxigenic and toxigenic strains among non-pigmented Gram-positive rods.
The study was undertaken with 84 C. diphtheriae strains from the culture collection of the Diphtheria Laboratory, Universidade do Estado do Rio de Janeiro, Brazil, as follows: 74 strains of C. diphtheriae subsp. mitis, 5 of C. diphtheriae subsp. gravis and 5 of C. diphtheriae subsp. belfanti; 33 of the strains were toxigenic and 51 were non-toxigenic; 40 of the strains were non-sucrose fermenting and 44 were sucrose fermenting. These C. diphtheriae strains were isolated from skin infections (n=34), blood-stream infections (n=4) and respiratory tract infections (n=46). This study also included three non-toxigenic Corynebacterium ulcerans, in addition to five non-toxigenic Corynebacterium pseudotuberculosis clinical strains kindly provided by Vasco Azevedo, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil. C. diphtheriae reference strains from the American Type Culture Collection (ATCC), Rockville, MD, USA, and from Centers for Disease Control and Prevention (CDC), Atlanta, GA, USA, were also used in experiments: non-toxigenic C. diphtheriae subsp. mitis ATCC 27010, toxigenic C. diphtheriae subsp. mitis ATCC 27012, CDC E8392 and Park Williams 8, C. diphtheriae subsp. intermedius CDC D7920, C. diphtheriae subsp. gravis CDC E6651. Micro-organisms were identified by conventional biochemical methods (Mattos-Guaraldi & Formiga, 1998; Funke & Bernard, 2003), DNase test (Pimenta et al., 2008b) and by the API Coryne system (bioMérieux) in accordance with the manufacturer's instructions.
Cytotoxicity of C. diphtheriae strains, including reference strains, was also evaluated by PCR using a primer pair targeted to a portion of fragment A of the tox gene (PCR-DTA, 258 bp), as well as by the ‘gold-standard’ Vero cells cytotoxicity assay (Efstratiou et al., 1998). The multiplex PCR assay was performed using three primer pairs corresponding to dtxR gene (DtxR1F and DtxR1R, 258 bp) and fragments of portions A (Dipht 2F and Dipht 4R, 719 bp) and B (Dipht 6F and Dipht 7R, 534 bp) of the tox gene (Nakao et al., 1996). Bacterial DNA was extracted by boiling a suspension composed of a loopful of freshly cultured bacteria in 500 µl sterile water for 10 min. The PCR was performed in a 25 µl volume containing: 1x Taq polymerase buffer, 1.5 mM MgCl2, 200 µM dNTPs, 4 µM each of the primers, 1.25 units Taq DNA polymerase (all reagents from Gibco-BRL) and 2 µl template DNA. Thermo cycling was performed using 35 cycles of 94 °C for 1 min, 55 °C for 1 min and 72 °C for 1.5 min. The reaction was concluded with a final extension step of 72 °C for 10 min.
Results of the multiplex PCR of C. diphtheriae strains are shown in Fig. 1
. Despite differences in the sites of isolation, biotypes and subspecies, all 84 C. diphtheriae clinical and 6 control strains tested exhibited at least one amplification product with the expected size of 258 bp, corresponding to the dtxR gene. A recent investigation indicated the use of the dtxR gene as a species-specific marker for C. diphtheriae (Pimenta et al., 2008a). In that work, results of the PCR-dtxR were 100 % positive for non-toxigenic and toxigenic C. diphtheriae strains. Conversely, the PCR-dtxR did not show an amplification signal from clinical isolates identified as non-diphtherial Corynebacterium species. In the present study, all three C. ulcerans and five C. pseudotuberculosis strains tested were negative for PCR-dtxR. PCR-dtxR completely correlated with the standard biochemical and commercial identification for all C. diphtheriae strains tested, indicating the feasibility of this reaction. In the present study, a complete agreement between results of multiplex PCR and PCR-dtxR was observed for all C. diphtheriae strains tested.
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The identification of C. diphtheriae should be initiated without delay following the isolation of any suspicious colonies. Phenotypic identification procedures tend to be technically demanding, or lacking in sensitivity, or else have limited evaluations. In general, these methods require at least 2 days from the selection of a suspicious organism. Important advantages of multiplex PCR over conventional biochemical procedures are its rapidity, ease of performance, the large number of strains that can be simultaneously tested for and the fact that interpretation of a PCR multiplex assay is simple. Identification by PCR proved to be reliable, and could be extremely important because it allows rapid application of control procedures and treatment of diphtheria or C. diphtheriae systemic infections. The multiplex PCR system may be incorporated into routine analyses in clinical diagnosis laboratories and/or epidemiological research.
ACKNOWLEDGEMENTS
This work was supported by CNPq, CAPES, FAPERJ, SR-2/UERJ and the Programa de Núcleo de Excelência (PRONEX) of the Brazilian Ministry of Science and Technology.
References
Cianciotto, N. P. & Groman, N. B. (1997). Characterization of bacteriophages from tox-containing, nontoxigenic isolates of Corynebacterium diphtheriae. Microb Pathog 22, 343–351.[CrossRef][Medline]
Damasco, P. V., Pimenta, F. P., Filardy, A. A., Brito, S. M., Andrade, A. F. B., Lopes, G. S., Hirata, R., Jr & Mattos-Guaraldi, A. L. (2005). Prevalence of IgG diphththeria antitoxin in blood donors in Rio de Janeiro. Epidemiol Infect 133, 911–914.[CrossRef][Medline]
Efstratiou, A. & George, R. C. (1999). Laboratory guidelines for the diagnosis of infections caused by Corynebacterium diphtheriae and C. ulcerans. Commun Dis Public Health 2, 250–257.[Medline]
Efstratiou, A., Engler, K. H., Dawes, C. S. & Sesardic, D. (1998). Comparison of phenotypic and genotypic methods for detection of diphtheria toxin among isolates of pathogenic corynebacteria. J Clin Microbiol 36, 3173–3177.
Funke, G. & Bernard, A. K. (2003). Coryneform Gram-positive rods. In Manual of Clinical Microbiology, pp. 472–501. Edited by P. R. Murray, E. J. Baron, M. A. Pfaller, J. H. Jorgensen & R. H. Yolken. Washington, DC: American Society for Microbiology.
Galazka, A. M. & Robertson, S. E. (1995). Diphtheria: changing patterns in the developing world and the industrialized world. Eur J Epidemiol 11, 107–117.[CrossRef][Medline]
Mattos-Guaraldi, A. L. & Formiga, L. C. D. (1998). Bacteriological properties of a sucrose-fermenting Corynebacterium diphtheriae strain isolated from a case of endocarditis. Curr Microbiol 37, 156–158.[CrossRef][Medline]
Mattos-Guaraldi, A. L., Moreira, L. O., Damasco, P. V. & Hirata, R., Jr (2003). Diphtheria remains a threat to health in the developing world: an overview. Mem Inst Oswaldo Cruz 98, 987–993.[Medline]
Nakao, H., Pruckler, J. M., Mazurova, I. K., Narvskaia, O. V., Glushkevich, T., Marijevski, V. F., Kravetz, A. N., Fields, B. S., Wachsmuth, I. K. & Popovic, T. (1996). Heterogeneity of diphtheria toxin gene, tox, and its regulatory element, dtxR, in Corynebacterium diphtheriae strains causing epidemic diphtheria in Russia and the Ukraine. J Clin Microbiol 34, 1711–1716.[Abstract]
Pimenta, F. P., Matias, G. A. M., Pereira, G. A., Camello, T. C. F., Alves, G. B., Rosa, A. C. P., Hirata, R., Jr & Mattos-Guaraldi, A. L. (2008a). A PCR for dtxR gene: application to diagnosis of non-toxigenic and toxigenic Corynebacterium diphtheriae. Mol Cell Probes 22, 189–192.[CrossRef][Medline]
Pimenta, F. P., Souza, M. C., Pereira, G. A., Hirata, R., Jr, Camello, T. C. & Mattos-Guaraldi, A. L. (2008b). DNase test as a novel approach for the routine screening of Corynebacterium diphtheriae. Lett Appl Microbiol 46, 307–311.[CrossRef][Medline]
Reacher, M., Ramsay, M., White, J., de Zoysa, A., Efstratiou, A., Mann, G., Mackay, A. & George, R. C. (2000). Nontoxigenic Corynebacterium diphtheriae: an emerging pathogen in England and Wales? Emerg Infect Dis 6, 640–645.[Medline]
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