Rapid identification of staphylococci and meticillin susceptibility testing directly from growth positive blood cultures by real-time polymerase chain reaction.
Cardiff Metropolitan University
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Staphylococci are the most commonly isolated organisms from blood cultures. The most important clinically being Staphylococcus aureus either meticillin sensitive or resistant. Of blood cultures containing staphylococci, 70% contain coagulase-negative staphylococci which may not require treatment. About 80% of these are Staphylococcus epidermidis. Currently if bacteria looking similar to staphylococci are seen in blood cultures, the clinicians are informed and a clinical judgement is made as to whether the bacteria are likely to be meticillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA) or other staphylococci. Species identification and definitive antibiotic susceptibility takes a further 48 hours, often resulting in patients being treated unnecessarily with expensive antibiotics in case the isolate is identified as MRSA. In some cases a decision not to treat may be made when in fact antibiotics are needed. The aim of this study was to identify the main types of staphylococci and meticillin susceptibility within hours, using real-time polymerase chain reaction (PCR), by targeting three genes, mecA,femA-SA and femA-SE. The species and susceptibility profiles of all the isolates were confirmed using the BD Phoenix automated system and conventional cultures. The results of this study identified the need for a DNA extraction method before the real-time PCR assay in order to remove potential inhibitors present in blood culture vials, from affecting the PCR. A method described by Louie et a1.,2002, was adapted and used to extract DNA from growth positive blood cultures. 100 growth positive blood cultures were tested by real-time PCR using SYBR green detection dye and TaqMan probes to identify target genes. Routine methods, BD Phoenix and real-time PCR results all correlated for all 39 S. aureus identification and susceptibility results. Amongst the 53 blood cultures containing coagulase negative staphylococci in pure culture, discrepancies were observed between methods in 7 cases using multiplex probe real-time PCR and SYBR green detection assays. Among the 5 mixed cultures there were 3 discrepant results. I Micrococcus species was correctly identified and two other growth positive blood cultures containing gram-positive cocci in clusters were subsequently no bacterial growth on culture. To conclude, this study used a simple and reliable method for DNA extraction with a multiplex or SYBR green dye detection real-time PCR assay for the rapid identification of staphylococcal species and meticillin susceptibility from growth positive blood cultures, within 2.5 hours. There were no major effors when identifying staphylococcal isolates and their meticillin susceptibility. This study identifies a method for same-day reporting of growth positive blood cultures which in turn will avoid unnecessary treatment and side-effects for some patients, allow correct treatment for those who do need treatment, avoid the need to treat all suspected S. aureus sepsis with a glycopeptide, allow earlier discharge of patients whose blood cultures are judged to contain skin contaminants, and avoid the need to recall patients with positive blood cultures which are later found to be harmless contaminants, saving time for both clinicians and clinical microbiologists with improved patient management.
MSc Biomedical Science
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