Characterization of a Toxin A-Negative, Toxin B-Positive Variant Strain of Clostridium difficile

Bo-Moon Shin; Eun-Young Kuak

doi:10.3343/kjlm.2006.26.1.27

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Shin and Kuak: Characterization of a Toxin A-Negative, Toxin B-Positive Variant Strain of Clostridium difficile

Original Article

Korean J Leg Med 2006;26(1):27-31.

Published online: 10 February 2006

DOI: https://doi.org/10.3343/kjlm.2006.26.1.27

Characterization of a Toxin A-Negative, Toxin B-Positive Variant Strain of Clostridium difficile

Bo-Moon Shin, M.D., Eun-Young Kuak, M.T.

Department of Laboratory Medicine, Sanggye Paik Hospital, Inje University, Seoul, Korea

Received 17 October 2005 Revised 27 December 2005 Accepted 2 February 2006

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Clostridium difficile is one of the most important pathogens responsible for nosocomial diarrhea. Recently, we have frequently experienced culture positive, toxin A enzyme immunoassay negative strains. Therefore, we evaluated the strains with several PCR primer sets to characterize them.

Methods

A total of 351 stool specimens were examined for toxin A using enzyme linked fluorescent immunoassay (ELFA) and also cultured for C. difficile using cycloserine cefoxitine fructose agar incubated under anaerobic conditions. Spore stain and Vitek ANA identification card (BioMerieux, France) were used for identification of C. difficile. We amplified toxin A and toxin B genes in 81 isolates using primers NK1-NK2, NK3-NK2, NK9-NK11, and NK104-NK105.

Results

The concordance rate between ELFA and culture was 65.2% (229/351). PCR for the toxin A gene using NK1-NK2, NK3-NK2 and for the toxin B gene using NK104-NK105 showed almost the same results. However, toxin A gene PCR using NK9-NK11 showed that 45.7% (37/81) of the evaluated strains were toxin A (−)/ toxin B(+) variant strains; thus, the corrected sensitivity and specificity of the ELFA based on the PCR results for toxin A and B genes were 65.6% and 100%, respectively.

Conclusions

The low sensitivity of the ELFA results for toxin A was due to the toxin A(−)/toxin B(+) variants of C. difficile, suggesting that the prevalence of the variant strains could be higher in Korea than was expected.

Keywords: Clostridium difficile, Variant, Toxin A, toxin B, Enzyme immunoassay, PCR

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Fig. 1.

Agarose gel electrophoresis of PCR products of C. difficile strains. Primer pairs: NK1-NK2(A), NK3-NK2(B) and NK9-NK11(C) for toxin A genes, and NK104-NK105(D) for toxin B gene. The size marker used was a 100-bp ladder. (A) and (B) showed identical PCR results, but (C) disclosed different results in that only 2 cases (lane 2 & 7) showed 1200 bp bands and the other 5 cases showed 700 bp bands representing variant strains. (D) represented most of the C. difficile strains (except lane 3 and 9) that had toxin B genes.

Table 1.

Results of ELFA tests based on toxin A gene PCR using NK9-NK11primers

		C. difficile toxin A			Total
		positive	variant^*	negative	Total
ELFA	(+)	21 (65.6%)	1 (2.7%)	0	22 (27.2%)
	Equivocal	2 (6.3%)	1 (2.7%)	0	3 (3.7%)
	(−)	9 (28.1%)	35 (94.6%)	12 (100%)	56 (67.9%)
Total		32 (100%)	37 (100%)	12 (100%)	81 (100%)

^* C. difficile toxin A, variant was toxin A gene, showing atypical 700 bp in PCR using NK9-NK11 primers.

Abbreviation: ELFA, enzyme linked fluorescent assay.

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