Accuracy and Potential Usefulness of Triplex Real-Time PCR for Improving Antibiotic Treatment of Patients with Blood Cultures Showing Clustered Gram-Positive Cocci on Direct Smears (original) (raw)
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Journal of clinical …, 1999
Isolates of Staphylococcus aureus with decreased susceptibilities to glycopeptide antimicrobial agents, such as vancomycin and teicoplanin, have emerged in the United States and elsewhere. Commercially prepared brain heart infusion agar (BHIA) supplemented with 6 g of vancomycin per ml was shown in a previous study to detect glycopeptide-intermediate S. aureus (GISA) with high sensitivity and specificity; however, this medium, when prepared in-house, occasionally showed growth of vancomycin-susceptible control organisms. This limitation could significantly impact laboratories that prepare media in-house, particularly if they wished to conduct large surveillance studies for GISA. Therefore, a pilot study to detect GISA was performed with vancomycin-containing Mueller-Hinton agar (MHA) prepared in-house in place of commercially prepared BHIA. MHA was selected for this study because this medium is widely available and well standardized. The results of the pilot study showed that supplementation of MHA with 5 g of vancomycin per ml was both a sensitive and a specific method for screening for GISA isolates. This method was used to screen for GISA among 630 clinical isolates of methicillin-resistant S. aureus collected during 1997 from 33 U.S. hospitals. Although 14 S. aureus isolates grew on the screening agar, all were vancomycin susceptible (MICs were <1 g/ml) by broth microdilution testing. Population analyses of five isolates revealed two with a subpopulation for which vancomycin MICs were 8 g/ml. In summary, the MHA screen plate containing 5 g of vancomycin per ml prepared in-house provides a sensitive and cost-effective method for large-scale screening for GISA for which vancomycin MICs are 8 g/ml. However, confirmation of isolates as vancomycin resistant is critical. This study suggests that GISA was not a widespread problem in the United States in 1997.
Journal of Clinical Microbiology, 2007
The rapid detection of Staphylococcus aureus bacteremia and a swift determination of methicillin susceptibility has serious clinical implications affecting patient mortality. This study evaluated the StaphSR assay (BD GeneOhm, San Diego, CA), a real-time PCR assay, for the identification and differentiation of methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) from 300 positive blood cultures. The BD GeneOhm StaphSR assay was performed and interpreted according to the manufacturer's recommendations. Positive blood cultures (containing predominantly gram-positive cocci in clusters) were subcultured on 5% sheep blood agar plates. After 18 to 24 h of incubation, isolates morphologically consistent with S. aureus were presumptively identified by latex agglutination (Staphaurex Plus; Remel, Lenexa, KS). Susceptibility testing was initially performed with the Phoenix automated microbiology system (BD Diagnostics, Sparks, MD). Additional susceptibility testing of samples with discrepant results was done using BBL oxacillin screen agar (BD Diagnostics, Sparks, MD), oxacillin and cefoxitin Etests (AB Biodisk, Solna, Sweden) on Mueller-Hinton agar, an immunoassay for penicillin binding protein 2 (Denka Seiken Co., Tokyo, Japan), and mecA PCR. The sensitivity, specificity, and positive and negative predictive values of the BD GeneOhm StaphSR assay for MSSA detection were 98.9, 96.7, 93.6, and 99.5%, respectively. For the detection of MRSA, the BD GeneOhm StaphSR assay was 100% sensitive and 98.4% specific; positive and negative predictive values for MRSA detection were 92.6 and 100%, respectively. Inhibition was seen with only one sample, and the issue was resolved upon retesting. The BD GeneOhm StaphSR assay appears to be a valuable diagnostic tool for quickly differentiating bacteremia caused by MSSA and MRSA from that caused by other gram-positive cocci. Staphylococcus aureus is a significant cause of communityacquired and nosocomial infections. Antimicrobial resistance to methicillin is on the rise (11, 40), complicating patient management (32, 39). S. aureus bacteremia increases one's risk of death (19, 39-41), and most studies have shown an increase in mortality among patients infected with methicillin-resistant S. aureus (MRSA) compared to those infected with methicillinsusceptible S. aureus (MSSA) (39, 40). In a meta-analysis published by Cosgrove et al., the authors reported a significant increase in mortality associated with MRSA bacteremia compared to that associated with bloodstream infections caused by MSSA (9). The increase in mortality remained after adjustment for confounding variables (9). Traditionally, the detection of bloodstream infections caused by S. aureus requires at least a day for culture and another day for identification and susceptibility testing. Nucleic acid amplification methods can provide same-day results once the blood cultures are positive for microbial growth; however, the majority of presently described assays are not designed to differentiate between MSSA and MRSA. Additionally, those nucleic acid amplification procedures that can differentiate between
Journal of Clinical Microbiology, 2007
Methicillin-resistant Staphylococcus aureus (MRSA) isolates (n ؍ 3,189) from 2,990 patients were investigated by agar screening and by the Etest macromethod for reduced susceptibility to glycopeptide. No vancomycin-resistant S. aureus or glycopeptide-intermediate S. aureus (GISA) isolates were detected, but 178 isolates were confirmed as hetero-GISA (hGISA) by vancomycin population analysis profile (vPAP)-area under the curve (AUC) ratio determination and/or teicoplanin PAP (tPAP) methods. Of 139 isolates detected using the recommended Etest macromethod cutoff values of >8 mg/liter for both vancomycin and teicoplanin or >12 mg/liter for teicoplanin alone, 73 were confirmed as hGISA by vPAP-AUC, 95 were confirmed as hGISA by tPAP, and 108 were confirmed as hGISA by both methods. An Etest macromethod cutoff value of 8 mg/liter for teicoplanin alone detected a further 70 hGISA (17 were confirmed by vPAP-AUC and 70 were confirmed by tPAP). Agar screening utilizing brain heart infusion (BHI) agar containing 6 mg of vancomycin/liter (BHIV6) and Mueller-Hinton (MH) agar containing 8 mg of teicoplanin/liter (MHT8) failed to detect hGISA. MH agar containing 5 mg of teicoplanin/liter (MHT5) and BHI containing 5 mg of teicoplanin/liter (BHIT5) were evaluated using 10-l volumes of three inoculum concentrations (with densities equivalent to 0.5 and 2.0 McFarland turbidity standards and stationary-phase BHI broth subcultures [MHT5 0.5 , MHT5 2.0 , MHT5 S , BHIT5 0.5 , BHIT5 2.0 , and BHIT5 S ]). The sensitivity of all methods except MHT5 0.5 and MHT5 2.0 was 100%. The specificity ranged from 4 to 82%. BHIT5 0.5 yielded the best performance, with a specificity of 84% for detecting isolates with teicoplanin Etest macromethod values of >8 mg/liter. Screening on BHIT5 0.5 is useful where screen-positive isolates are investigated with the Etest macromethod and confirmed by vPAP-AUC and tPAP. The prevalence of hGISA among patients with blood culture isolates recovered in Irish hospitals between 1999 and 2003 was 2.6%, whereas the prevalence among patients with isolates from all specimen sites collected during a 2-week survey in 1999 was 12%. The prevalence in one hospital decreased from 5.3% in 2003 to 1.5% in 2004.
Journal of Antimicrobial Chemotherapy, 2005
Objectives: Glycopeptides are the drugs of choice to treat infections due to methicillin-resistant Staphylococcus aureus, but since 1995, glycopeptide-intermediate S. aureus (GISA) and heterogeneous GISA (hGISA) have been reported worldwide. Detection of reduced susceptibility to glycopeptides in S. aureus is very difficult in a routine clinical laboratory. The aim of this study was to investigate the prevalence of hGISA/GISA strains using a three-step approach during a 1 year period.
BMC Infectious Diseases, 2009
Coagulase-negative staphylococci (CoNS) are a major cause of nosocomial blood stream infection, especially in critically ill and haematology patients. CoNS are usually multidrug-resistant and glycopeptide antibiotics have been to date considered the drugs of choice for treatment. The aim of this study was to characterize CoNS with reduced susceptibility to glycopeptides causing blood stream infection (BSI) in critically ill and haematology patients at the University Hospital Tor Vergata, Rome, Italy, in 2007. Hospital microbiology records for transplant haematology and ICU were reviewed to identify CoNS with elevated MICs for glycopeptides, and isolates were matched to clinical records to determine whether the isolates caused a BSI. The isolates were tested for susceptibility to new drugs daptomicin and tigecycline and the genetic relationship was assessed using f-AFLP. Of a total of 17,418 blood cultures, 1,609 were positive for CoNS and of these, 87 (5.4%) displayed reduced susceptibility to glycopeptides. Clinical review revealed that in 13 cases (7 in haematology and 6 in ICU), CoNS with reduced susceptibility to glycopeptides were responsible for a BSI. Staphylococcus epidermidis was the causative organism in 11 instances and Staphylococcus haemolyticus in 2. The incidence of oxacillin resistance was high (77%), although all isolates remained susceptible to linezolid, daptomycin and tigecycline. Fingerprinting of CoNS identified one clonal relationship between two isolates. Multi-resistant CoNS with reduced susceptibility to glycopeptides, although still relatively infrequent in our hospital, are emerging pathogens of clinical concern. Surveillance by antibiotyping with attention to multi-resistant profile, and warning to clinicians, is necessary.
Diagnostic Microbiology and Infectious Disease, 2010
We describe here a 1-step, triplex real-time polymerase chain reaction (PCR) assay for the detection and identification of staphylococci directly from signal-positive blood culture bottles containing Gram-positive cocci in clusters (GPCC). The triplex assay targeted and detected tuf, nuc, and mecA genes in a single tube and had a detection limit of 10 5 CFU/mL for each gene target. A total of 341 GPCC-positive blood culture bottles were collected between November 12, 2008, and August 11, 2009. Among them, 230 methicillin-resistant coagulase-negative staphylococci (CoNS), 54 methicillin-susceptible CoNS, 22 methicillin-resistant Staphylococcus aureus, 22 methicillin-susceptible S. aureus, and 13 nonstaphylococci species were identified by conventional methods. The results obtained by triplex assay were in agreement with those of conventional methods for tuf (99.7%), nuc (100.0%), and mecA (99.1%), respectively. The triplex assay was found to have sensitivities of 99.7%, 100%, and 99.2% and specificities of 100%, 100%, and 98.7%, respectively, for the tuf, nuc, and mecA gene targets. The triplex real-time PCR assay accurately detects and identifies staphylococci directly from positive blood cultures without nucleic acid extraction prior to amplification.
Journal of Microbiological Methods, 2004
Detection of methicillin-resistant Staphylococcus aureus (MRSA) isolates exhibiting intermediate susceptibility to glycopeptides (GISA) is challenging for clinical microbiology laboratories. We compared three different screening assays for evaluating trends in decreased glycopeptide susceptibility during two periods. Ninety four and ninety five consecutive MRSA blood isolates from 189 bacteremic patients collected during periods A (1989-1994) and B (1999-2001), respectively, were screened in parallel for vancomycin or teicoplanin susceptibility by glycopeptide-containing brain-heart infusion agar (BHIA) tests, Etest MICs performed at a standard (0.5 McFarland) or high (2.0 McFarland) inoculum on Mueller-Hinton (MHA) or BHIA, respectively. Any MRSA isolate yielding < 50 CFU (representing < 10 À 6 of the plated inoculum) on either BHIA containing 2 mg/l of vancomycin (V2-BHIA) or 5 mg/l of teicoplanin (T5-BHIA) was considered as fully susceptible to vancomycin or teicoplanin, respectively. The proportion of MRSA isolates yielding>50 CFU on either V2-BHIA or T5-BHIA significantly (P < 0.01) increased from 7/94 (7.4 %) or 8/94 (8.5%) in period A to 16/95 (16.8 %) or 14/95 (14.7%) in period B, respectively. Vancomycin Etests MICs on MHA were of lower sensitivity (< 30% and < 65%), but high specificity (100% and 99%) on periods A and B isolates, respectively, compared to those on V2-BHIA. Vancomycin Etests MICs on BHIA were of a higher sensitivity (57% and 81%) but lower specificity (91% and 65%) compared to those on V2-BHIA, on periods A and B isolates, respectively, reflecting an unexpectedly high number of false positive isolates on period B isolates (28/95). Screening of decreased susceptibility to vancomycin or teicoplanin on V2-BHIA or T5-BHIA, respectively, may represent simple low-cost alternatives to Etest MICs, minimising the risk of missing potential GISA isolates.