title: Evaluation of an internally controlled real-time polymerase chain reaction assay targeting the groEL gene for the detection of Bartonella spp. DNA in patients with suspected cat-scratch disease.
abstract: Bartonella (B.) henselae is the causative agent of cat-scratch disease (CSD), which usually presents as a self-limiting lymphadenopathy. This study reports the development and evaluation of an internally controlled real-time polymerase chain reaction targeting the groEL gene for detection of Bartonella spp. DNA was extracted using the MagNA Pure system. The lower detection limit was 10-100 fg DNA and the in vitro sensitivity of the assay was not affected by duplexing with an internal control PCR. The real-time PCR assay detected DNA from all five B. henselae strains tested, and from B. birtlesii, B. vinsonii subsp. vinsonii, B. vinsonii subsp. arupensis and B. doshiae. The assay generated negative results with a selection of other bacteria, including several Mycobacterium spp., Streptococcus pyogenes and Staphylococcus aureus. Results of real-time PCR in clinical samples were compared with those of a conventional 16S rDNA-based PCR assay. During the period described in the Material and methods section, real-time PCR and conventional 16S PCR were performed on 73 clinical samples. Of these samples, 29 (40%) were found to give positive results and 44 (60%) gave negative results, both by real-time PCR and by conventional PCR, with a 100% agreement between the two tests. The PCR developed in this study is a rapid, sensitive, and simple method for the detection of Bartonella spp. in CSD and is suitable for implementation in the diagnostic laboratory.
authors.affiliation: Regional Laboratory of Public Health Haarlem, The Netherlands. bramdiederen@gmail.com
authors.forename: B M W
authors.initials: BM
authors.lastname: Diederen
authors.forename: M J
authors.initials: MJ
authors.lastname: Vermeulen
authors.forename: H
authors.initials: H
authors.lastname: Verbakel
authors.forename: A
authors.initials: A
authors.lastname: van der Zee
authors.forename: A
authors.initials: A
authors.lastname: Bergmans
authors.forename: M F
authors.initials: MF
authors.lastname: Peeters
mesh_terms: D000328:Adult; D001473:Bartonella / Q000235:genetics / Q000302:isolation & purification*; D002372:Cat-Scratch Disease / Q000175:diagnosis / Q000382:microbiology*; D018834:Chaperonin 60 / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D005260:Female; D006801:Humans; D008297:Male; D009161:Mycobacterium / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D013211:Staphylococcus aureus / Q000235:genetics; D013297:Streptococcus pyogenes / Q000235:genetics
chemical_list: D018834:Chaperonin 60; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S