Direct detection of vanA and vanB genes in clinical specimens for rapid identification of vancomycin resistant enterococci (VRE) using multiplex PCR

Surveillance for vancomycin resistant enterococci (VRE) by culture can be l abour intensive and lime consuming. We have developed a multiplex polymeras e chain reaction (MPCR) which can be performed directly on the clinical spe cimen. The assay allows sensitive detection of enterococci with vanA- and v anB- mediated resistance to vancomycin. DNA was purified from stool and rec tal specimens using the XTRAX(TM) DNA Extraction Kit (Gull Labs). Multiplex PCR amplified vanA and vanB targets were detected using a microtiter plate EIA. Two-hundred specimens were tested by routine culture and MPCR. Cultur e identified 44 VRE isolates and MPCR detected 38 of the 44 culture positiv es. Multiplex PCR detected three additional positive VRE specimens missed b y culture for a sensitivity and specificity of 86.4 and 98.1%, respectively . When the presence of PCR inhibitors was addressed in the six culture posi tive/MPCR negative specimens, four additional VRE positive specimens were d etected, Performing MPCR on the original specimens and on a 1:10 dilution o f all specimens to minimize the effect of inhibitors gave a sensitivity and specificity of 95.5 and 98.1%, respectively. Multiplex PCR with confirmati on by microtiter plate hybridization could be completed in 8 h compared wit h 24-48 h required for culture. (C) 1999 Academic Press.