Novel multiplex format of an extended multilocus variable-number-tandem-repeat analysis of Clostridium difficile correlates with tandem repeat sequence typing

• We successfully developed a multiplex MLVA format for subtyping of C. difficile.
• An excellent concordance between MLVA and TRST typing was found.
• We found loci potentially reducible in outbreak investigations of known TRST types.
• We identified variable loci providing discriminatory power to the MLVA scheme.

Subtyping of Clostridium difficile is crucial for outbreak investigations. An extended multilocus variable-number tandem-repeat analysis (eMLVA) of 14 variable number tandem repeat (VNTR) loci was validated in multiplex format compatible with a routine typing laboratory and showed excellent concordance with tandem repeat sequence typing (TRST) and high discriminatory power.