title: Improved protocol for rapid identification of certain spa types using high resolution melting curve analysis.
abstract: Methicillin-resistant Staphylococcus aureus is one of the most significant pathogens associated with health care. For efficient surveillance, control and outbreak investigation, S. aureus typing is essential. A high resolution melting curve analysis was developed and evaluated for rapid identification of the most frequent spa types found in an Austrian hospital consortium covering 2,435 beds. Among 557 methicillin-resistant Staphylococcus aureus isolates 38 different spa types were identified by sequence analysis of the hypervariable region X of the protein A gene (spa). Identification of spa types through their characteristic high resolution melting curve profiles was considerably improved by double spiking with genomic DNA from spa type t030 and spa type t003 and allowed unambiguous and fast identification of the ten most frequent spa types t001 (58%), t003 (12%), t190 (9%), t041 (5%), t022 (2%), t032 (2%), t008 (2%), t002 (1%), t5712 (1%) and t2203 (1%), representing 93% of all isolates within this hospital consortium. The performance of the assay was evaluated by testing samples with unknown spa types from the daily routine and by testing three different high resolution melting curve analysis real-time PCR instruments. The ten most frequent spa types were identified from all samples and on all instruments with 100% specificity and 100% sensitivity. Compared to classical spa typing by sequence analysis, this gene scanning assay is faster, cheaper and can be performed in a single closed tube assay format. Therefore it is an optimal screening tool to detect the most frequent endemic spa types and to exclude non-endemic spa types within a hospital.
authors.affiliation: University of Applied Sciences Wiener Neustadt, Department of Biomedical Analytics, Wiener Neustadt, Austria; Austrian Agency for Health and Food Safety, Institute of Medical Microbiology and Hygiene, Vienna, Austria.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Mayerhofer
authors.affiliation: Austrian Agency for Health and Food Safety, Institute of Medical Microbiology and Hygiene, Vienna, Austria.
authors.forename: Anna
authors.initials: A
authors.lastname: Stöger
authors.affiliation: Austrian Agency for Health and Food Safety, Institute of Medical Microbiology and Hygiene, Vienna, Austria.
authors.forename: Ariane T
authors.initials: AT
authors.lastname: Pietzka
authors.affiliation: Austrian Agency for Health and Food Safety, Institute of Medical Microbiology and Hygiene, Vienna, Austria.
authors.forename: Haizpea Lasa
authors.initials: HL
authors.lastname: Fernandez
authors.affiliation: Austrian Agency for Health and Food Safety, Institute of Medical Microbiology and Hygiene, Vienna, Austria; University of Natural Resources and Life Sciences, Department of Biotechnology, Institute of Applied Microbiology, Vienna, Austria.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Prewein
authors.affiliation: KABEG, Klinikum Klagenfurt, Institute of Laboratory Diagnostics and Microbiology, Klagenfurt, Austria.
authors.forename: Sieglinde
authors.initials: S
authors.lastname: Sorschag
authors.affiliation: University of Natural Resources and Life Sciences, Department of Biotechnology, Institute of Applied Microbiology, Vienna, Austria.
authors.forename: Renate
authors.initials: R
authors.lastname: Kunert
authors.affiliation: Austrian Agency for Health and Food Safety, Institute of Medical Microbiology and Hygiene, Vienna, Austria.
authors.forename: Franz
authors.initials: F
authors.lastname: Allerberger
authors.affiliation: Austrian Agency for Health and Food Safety, Institute of Medical Microbiology and Hygiene, Vienna, Austria; University of Natural Resources and Life Sciences, Department of Biotechnology, Institute of Applied Microbiology, Vienna, Austria.
authors.forename: Werner
authors.initials: W
authors.lastname: Ruppitsch
mesh_terms: D015373:Bacterial Typing Techniques / Q000379:methods*; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D055624:Methicillin-Resistant Staphylococcus aureus / Q000145:classification* / Q000235:genetics; D009691:Nucleic Acid Denaturation; D013203:Staphylococcal Infections / Q000382:microbiology; D013205:Staphylococcal Protein A / Q000235:genetics*
chemical_list: D004269:DNA, Bacterial; D013205:Staphylococcal Protein A