One-step sample preparation of positive blood cultures for the direct detection of methicillin-sensitive and -resistant Staphylococcus aureus and methicillin-resistant coagulase-negative staphylococci within one hour using the automated GenomEra CDX™ PCR system

• Published in: European journal of clinical microbiology & infectious diseases 2012-10, Vol.31 (10), p.2835-2842
• Main Authors: Hirvonen, J. J., von Lode, P., Nevalainen, M., Rantakokko-Jalava, K., Kaukoranta, S.-S.
• Format: Article
• Language: English
• Subjects: Automation; Bacterial diseases; Bacterial Proteins - analysis; Bacterial Typing Techniques - methods; Biological and medical sciences; Biomedical and Life Sciences; Biomedicine; Blood; Cell Survival; Charcoal; Coagulase; Culture media; Fluorometry - instrumentation; Fluorometry - methods; Hemoglobin; Human bacterial diseases; Humans; Indexing in process; Infectious diseases; Internal Medicine; Laboratories; Medical Microbiology; Medical sciences; Methicillin-Resistant Staphylococcus aureus - enzymology; Methicillin-Resistant Staphylococcus aureus - isolation & purification; Microbiology; Penicillin-Binding Proteins; Polymerase chain reaction; Polymerase Chain Reaction - instrumentation; Polymerase Chain Reaction - methods; Reagent Kits, Diagnostic - standards; Reproducibility of Results; Sample preparation; Sensitivity and Specificity; Staphylococcal Infections - blood; Staphylococcal Infections - diagnosis; Staphylococcal Infections - microbiology; Staphylococcal infections, streptococcal infections, pneumococcal infections; Staphylococcus aureus - enzymology; Staphylococcus aureus - genetics; Staphylococcus aureus - isolation & purification; Staphylococcus infections; Time Factors
• ISSN: 0934-9723; 1435-4373
• DOI: 10.1007/s10096-012-1637-3

A method for the rapid detection of methicillin-sensitive and -resistant Staphylococcus aureus (MSSA and MRSA, respectively) and methicillin-resistant coagulase-negative staphylococci (MRCoNS) with a straightforward sample preparation protocol of blood cultures using an automated homogeneous polymerase chain reaction (PCR) assay, the GenomEra™ MRSA/SA (Abacus Diagnostica Oy, Turku, Finland), is presented. In total, 316 BacT/Alert (bioMérieux, Marcy l’Etoile, France) and 433 BACTEC (Becton Dickinson, Sparks, MD, USA) blood culture bottles were analyzed, including 725 positive cultures containing Gram-positive cocci in clusters ( n  = 419) and other Gram stain forms ( n  = 361), as well as 24 signal- and growth-negative bottles. Detection sensitivities for MSSA, MRSA, and MRCoNS were 99.4 % (158/159), 100.0 % (9/9), and 99.3 % (132/133), respectively. One false-positive MRSA result was detected from a non-staphylococci-containing bottle, yielding a specificity of 99.8 %. The lowest detectable amount of viable cells in the blood culture sample was 4 × 10 4  CFU/mL. The results were available within one hour after microbial growth detection and the two-step, time-resolved fluorometric (TRF) measurement mode employed by the GenomEra CDX™ instrument showed no interference from blood, charcoal, or culture media. The method described lacks all sample purification steps and allows reliable and simplified pathogen detection also in clinical microbiology laboratory settings without specialized molecular microbiology competence.