title: Advantages and limitations of ribosomal RNA PCR and DNA sequencing for identification of bacteria in cardiac valves of danish patients.
abstract: Studies on the value of culture-independent molecular identification of bacteria in cardiac valves are mostly restricted to comparing agreement of identification to what is obtained by culture to the number of identified bacteria in culture-negative cases. However, evaluation of the usefulness of direct molecular identification should also address weaknesses, their relevance in the given setting, and possible improvements. In this study cardiac valves from 56 Danish patients referred for surgery for infective endocarditis were analysed by microscopy and culture as well as by PCR targeting part of the bacterial 16S rRNA gene followed by DNA sequencing of the PCR product. PCR and DNA sequencing identified significant bacteria in 49 samples from 43 patients, including five out of 13 culture-negative cases. No rare, exotic, or intracellular bacteria were identified. There was a general agreement between bacterial identity obtained by ribosomal PCR and DNA sequencing from the valves and bacterial isolates from blood culture. However, DNA sequencing of the 16S rRNA gene did not discriminate well among non-haemolytic streptococci, especially within the Streptococcus mitis group. Ribosomal PCR with subsequent DNA sequencing is an efficient and reliable method of identifying the cause of IE, but exact species identification of some of the most common causes, i.e. non-haemolytic streptococci, may be improved with other molecular methods.
authors.affiliation: Microbiological Surveillance & Research, Statens Serum Institut, Copenhagen, Denmark ; Department of Clinical Microbiology, Odense University Hospital, Odense, Denmark.
authors.forename: Michael
authors.initials: M
authors.lastname: Kemp
authors.affiliation: Department of Clinical Microbiology, Herlev University hospital, Copenhagen, Denmark.
authors.forename: Jette
authors.initials: J
authors.lastname: Bangsborg
authors.affiliation: Department of Clinical Microbiology, Herlev University hospital, Copenhagen, Denmark.
authors.forename: Anne
authors.initials: A
authors.lastname: Kjerulf
authors.affiliation: The Emergency Department, Holbaek University Hospital, Holbaek, Denmark.
authors.forename: Thomas Andersen
authors.initials: TA
authors.lastname: Schmidt
authors.affiliation: Department of Cardiothoracic Surgery, Gentofte University Hospital, Copenhagen, Denmark.
authors.forename: John
authors.initials: J
authors.lastname: Christensen
authors.affiliation: Microbiological Surveillance & Research, Statens Serum Institut, Copenhagen, Denmark.
authors.forename: Akhmadjon 6
authors.initials: A6
authors.lastname: Irmukhamedov
authors.affiliation: Department of Cardiology, Gentofte University Hospital, Copenhagen, Denmark.
authors.forename: Niels Eske
authors.initials: NE
authors.lastname: Bruun
authors.affiliation: Microbiological Surveillance & Research, Statens Serum Institut, Copenhagen, Denmark ; Department of Clinical Microbiology, Slagelse Hospital, Slagelse, Denmark.
authors.forename: Rimtas
authors.initials: R
authors.lastname: Dargis
authors.affiliation: Microbiological Surveillance & Research, Statens Serum Institut, Copenhagen, Denmark.
authors.forename: Keld
authors.initials: K
authors.lastname: Andresen
authors.affiliation: Microbiological Surveillance & Research, Statens Serum Institut, Copenhagen, Denmark ; Department of Clinical Microbiology, Slagelse Hospital, Slagelse, Denmark.
authors.forename: Jens Jørgen
authors.initials: JJ
authors.lastname: Christensen
keywords: 16S rRNA gene; BLAST; NCBI database; Streptococcus mitis group; culture independent identification; viridans streptococci.