title: Intercenter reproducibility of binary typing for Staphylococcus aureus.
abstract: The reproducibility of the binary typing (BT) protocol developed for epidemiological typing of Staphylococcus aureus was analyzed in a biphasic multicenter study. In a Dutch multicenter pilot study, 10 genetically unique isolates of methicillin-resistant S. aureus (MRSA) were characterized by the BT assay as presented by van Leeuwen et al. [J. Clin. Microbiol. 2001 39 (1) 328]. The BT assay, including a standardized DNA extraction protocol was performed in duplicate in eleven medical microbiology laboratories. Two different hybridization detection procedures were applied and a prelabeled DNA sample as process control was included. Only three laboratories accurately identified all strains. Divergence in technical procedures resulted in misinterpretation due to an increasing number of faint or absent hybridization signals in combination with high background staining. The binary type of the process control was determined correctly by all participating laboratories. The feasibility of the BT protocol was related directly to the skill of the laboratory personnel. On the basis of the national study, we concluded that the DNA extraction protocol needed modification to improve DNA yield and purity. Subsequently, seven European laboratories participated in an international study to determine the reproducibility of the modified BT protocol. Each center was asked to analyze 10 DNA samples previously extracted from 10 MRSA strains (phase 1) and, additionally, to analyze 10 MRSA strains, using the standardized or their in-house DNA isolation protocol (phase 2). A prelabeled DNA process control sample was included again. The binary types of all DNA samples were identified correctly by all but one laboratories. This latter laboratory diverged from the protocol by adding an excess of labeled DNA to the hybridization mixture, resulting in a high background and, therefore, noninterpretable BT results. All centers produced identical BT results for the process control. Five of the seven centers correctly identified the binary types of all 10 MRSA strains in phase 2 of the international study. Three of these centers used their in-house DNA extraction protocol. Divergence from the standard BT protocol in the remaining two centers resulted in no interpretable BT data for the 10 MRSA strains. The study demonstrated that each center that followed the BT protocol to the letter could generate reproducible results, irrespective whether or not an in-house DNA isolation protocol was used. The current BT protocol thus represents a simple method generating robust, reproducible genotype data for S. aureus strains.
authors.affiliation: Department of Medical Microbiology and Infectious Diseases, Erasmus MC, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands. vanleeuwen@bacl.azr.nl
authors.forename: Willem B
authors.initials: WB
authors.lastname: van Leeuwen
authors.forename: Sandor
authors.initials: S
authors.lastname: Snoeijers
authors.forename: Christel
authors.initials: C
authors.lastname: van der Werken-Libregts
authors.forename: Anita
authors.initials: A
authors.lastname: Tuip
authors.forename: Anneke
authors.initials: A
authors.lastname: van der Zee
authors.forename: Diane
authors.initials: D
authors.lastname: Egberink
authors.forename: Monique
authors.initials: M
authors.lastname: de Proost
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Bik
authors.forename: Bjorn
authors.initials: B
authors.lastname: Lunter
authors.forename: Jan
authors.initials: J
authors.lastname: Kluytmans
authors.forename: Etty
authors.initials: E
authors.lastname: Gits
authors.forename: Inge
authors.initials: I
authors.lastname: van Duyn
authors.forename: Max
authors.initials: M
authors.lastname: Heck
authors.forename: Kim
authors.initials: K
authors.lastname: van der Zwaluw
authors.forename: Wim
authors.initials: W
authors.lastname: Wannet
authors.forename: Gerda T
authors.initials: GT
authors.lastname: Noordhoek
authors.forename: Sije
authors.initials: S
authors.lastname: Mulder
authors.forename: Nicole
authors.initials: N
authors.lastname: Renders
authors.forename: Miranda
authors.initials: M
authors.lastname: Boers
authors.forename: Sebastiaan
authors.initials: S
authors.lastname: Zaat
authors.forename: Daniëlle
authors.initials: D
authors.lastname: van der Riet
authors.forename: Mirjam
authors.initials: M
authors.lastname: Kooistra
authors.forename: Adriaan
authors.initials: A
authors.lastname: Talens
authors.forename: Lenie
authors.initials: L
authors.lastname: Dijkshoorn
authors.forename: Tanny
authors.initials: T
authors.lastname: van der Reyden
authors.forename: Dick
authors.initials: D
authors.lastname: Veenendaal
authors.forename: Nancy
authors.initials: N
authors.lastname: Bakker
authors.forename: Barry
authors.initials: B
authors.lastname: Cookson
authors.forename: Alisson
authors.initials: A
authors.lastname: Lynch
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Witte
authors.forename: Christa
authors.initials: C
authors.lastname: Cuny
authors.forename: Dominique
authors.initials: D
authors.lastname: Blanc
authors.forename: Isabelle
authors.initials: I
authors.lastname: Vernez
authors.forename: Waleria
authors.initials: W
authors.lastname: Hryniewicz
authors.forename: Janusz
authors.initials: J
authors.lastname: Fiett
authors.forename: Marc
authors.initials: M
authors.lastname: Struelens
authors.forename: Ariane
authors.initials: A
authors.lastname: Deplano
authors.forename: Jim
authors.initials: J
authors.lastname: Landegent
authors.forename: Henri A
authors.initials: HA
authors.lastname: Verbrugh
authors.forename: Alex
authors.initials: A
authors.lastname: van Belkum
mesh_terms: D015373:Bacterial Typing Techniques / Q000379:methods / Q000592:standards*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005060:Europe; D016106:Methicillin Resistance; D009693:Nucleic Acid Hybridization / Q000379:methods*; D010865:Pilot Projects; D015203:Reproducibility of Results; D013211:Staphylococcus aureus / Q000145:classification* / Q000235:genetics
chemical_list: D004269:DNA, Bacterial