Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Shrestha, N. K. Articles by Procop, G. W. Search for Related Content PubMed PubMed Citation Articles by Shrestha, N. K. Articles by Procop, G. W.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, December 2005, p. 6144-6146, Vol. 43, No. 12
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.12.6144-6146.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Evaluation of the LightCycler Staphylococcus M^GRADE Kits on Positive Blood Cultures That Contained Gram-Positive Cocci in Clusters

Nabin K. Shrestha,^1* Marion J. Tuohy,² Ravindran A. Padmanabhan,¹ Gerri S. Hall,² and Gary W. Procop²

Department of Infectious Diseases,¹ Section of Clinical Microbiology, The Cleveland Clinic Foundation, Cleveland, Ohio²

Received 10 August 2005/ Returned for modification 8 September 2005/ Accepted 5 October 2005

Top Abstract Text References

 
We evaluated the Roche LightCycler Staphylococcus M^GRADE kits to differentiate between Staphylococcus aureus and coagulase-negative staphylococci in blood cultures growing clusters of gram-positive cocci. Testing 100 bottles (36 containing S. aureus), the assay was 100% sensitive and 98.44% specific for S. aureus and 100% sensitive and specific for coagulase-negative staphylococci.

Top Abstract Text References

 
Gram-positive cocci in clusters (GPCC) noted on Gram staining of blood cultures are usually either Staphylococcus aureus or coagulase-negative staphylococci (CoNS). While the latter may be considered contaminants, the former is usually considered a true pathogen. It takes an additional day and supplementary testing to differentiate these two organisms by conventional methods. Multiple studies have reported rapid and accurate detection of S. aureus in blood culture bottles growing GPCC, which speak to the feasibility and interest in this approach (1-3, 5, 6, 9, 10). Accurate detection of the presence of all staphylococci in blood culture bottles and simultaneous identification of S. aureus among them using real-time PCR have also been demonstrated (8). This approach identifies CoNS by actually detecting its presence rather than by assuming its presence in the absence of S. aureus in cultures growing GPCC. A limitation to the use of real-time PCR for detection of staphylococci in a clinical laboratory is the expertise necessary to conduct real-time PCR and control for all the variables that might give rise to inconsistencies in the reaction over a period of time. The availability of a standardized kit would ease the process to the point where staphylococcal detection in blood cultures by real-time PCR may be seriously considered for practical application in clinical microbiology laboratories. The purpose of this study was to evaluate the LightCycler Staphylococcus M^GRADE kits for the detection and identification of S. aureus and CoNS in blood culture bottles growing GPCC.

Aerobic and anaerobic FAN-containing blood culture bottles that gave a positive signal in the BacT/ALERT blood culture system (bioMérieux, Inc., Durham, NC) and revealed the presence of GPCC were studied. A 0.5-ml aliquot of fluid was immediately removed for PCR from the positive blood culture bottle after brief manual agitation. Identification of the bacterium proceeded according to the standard laboratory protocol that included inoculation onto 5% sheep blood agar, incubation at 37°C, and identification based on colony morphology and coagulase test results. The aliquot removed for PCR was processed to remove the charcoal and DNA was prepared by a differential centrifugation and lysis buffer treatment as previously described (9). Charcoal was removed by centrifuging the aliquot at 850 x g for 2 min and discarding the pellet. The supernatant was centrifuged at 11,500 x g for 5 min. The resulting pellet was resuspended in 200 µl of a lysis buffer (7) containing 1% Triton X-100, 0.5% Tween 20, 10 mM Tris-HCl (pH 8.0), and 1 mM EDTA and incubated in a screw-cap reaction tube at 100°C for 10 min. The mixture was again centrifuged at 850 x g for 2 min, and the supernatant was removed and stored at –20°C for future testing.

Real-time PCR was performed in the LightCycler instrument (Roche Diagnostics, Indianapolis, IN) using the following LightCycler reagents (Roche): FastStart DNA master hybridization probes, Staphylococcus primer/hybridization probes, and the Staphylococcus template set. With these kits, a portion of the ITS region (internal transcribed spacer between the 16S and 23S genes) of Staphylococcus spp. are amplified and S. aureus and CoNS are detected and differentiated by melting curve analysis using fluorescence resonance energy transfer probes. In addition, PCR inhibition is monitored by the inclusion of a probe for the recovery template.

Each 20-µl PCR mixture consisted of 8.4 µl of PCR H₂O, 1.6 µl of 25 mM MgCl₂ (final concentration of 3 mM), 2.0 µl of primer/hybridization probe mix, 1.0 µl of the recovery template diluted 1:10, 2.0 µl of Fast Start mix and 5 µl of DNA target. The LightCycler reaction protocol was as follows: denaturation and Taq polymerase activation (95°C for 10 min), 45 cycles of PCR (1 cycle consists of 95°C for 10 s, 50°C for 15 s, and 72°C for 10 s), a melting phase (95°C for 60 s, and 40°C to 80°C in 60 s), and a cooling phase at 40° for 30 s. Positive amplification curves indicated the presence of Staphylococcus spp. Melting curve analysis was performed with channel F2 (LightCycler Red 640 probe) for differentiating between S. aureus and CoNS and with channel F3 (LightCycler Red 705 probe) for detecting the recovery template (internal control for PCR inhibition). Interpretation of melting temperatures was done according to the package insert for the Staphylococcus primer/hybridization probe M^GRADE kit.

The specimens were also analyzed by another real-time PCR assay that had been previously described, targeting the sa442 DNA fragment of S. aureus, to serve as an independent confirmation of the presence of S. aureus (9).

One hundred positive bottles analyzed in this manner contained 36 S. aureus isolates, 63 CoNS isolates, and 1 Micrococcus species. Positive and negative controls reacted appropriately. The LightCycler Staphylococcus M^GRADE kits detected the presence of staphylococci in all 99 specimens that contained the microorganism. The specimen containing Micrococcus returned a negative result. All bottles containing S. aureus were correctly identified. The LightCycler Staphylococcus M^GRADE kits revealed the presence of CoNS in all of the 63 positive blood culture bottles that contained this organism. However, one of these bottles that grew only CoNS also tested positive for the presence of S. aureus by the LightCycler Staphylococcus M^GRADE kits by postamplification melting curve analysis. The result of the S. aureus-specific sa442 assay on this specimen was negative. Interestingly, this specimen came from a patient on hemodialysis; patients requiring hemodialysis are known to be at risk for S. aureus infection. The possibility that the LightCycler Staphylococcus M^GRADE kit PCR detected DNA from nonviable S. aureus and that the discrepancy between the two PCR assays represents differences in sensitivity remains. Although the sa442 assay was previously shown to have a sensitivity of 100% (9), it may really be less sensitive than the Staphylococcus M^GRADE kit PCR when examining small bacterial loads because the sa442 DNA fragment exists as a single copy and the ITS region is present in multiple copies, but this remains speculative. This result was categorized as a false positive for the LightCycler Staphylococcus M^GRADE kit. The mean crossing thresholds (standard deviations shown in parentheses) were 30.13 (3.58) and 29.07 (4.84) cycles for S. aureus and CoNS, respectively; Fig. 1 shows their distributions graphically. The mean melting temperatures (standard deviations shown in parentheses) were 60.68°C (0.34) and 51.41°C (1.16), respectively, with Fig. 2 demonstrating that the two were clearly distinguishable. Therefore, in this analysis, we found the LightCycler Staphylococcus M^GRADE kit to be 100% sensitive and specific for the detection of the Staphylococcus genus, 100% sensitive and specific for the detection of the presence of CoNS, and 100% sensitive and 98.44% specific for the detection of S. aureus in positive blood culture bottles that contained GPCC.

View larger version (11K): [in this window] [in a new window]

FIG. 1. Box plot comparing crossing thresholds for S. aureus (SA) and CoNS. *, data point that lies more than 1.5 times the interquartile range from the end of the box.

View larger version (10K): [in this window] [in a new window]

FIG. 2. Box plot comparing melting temperatures for S. aureus (SA) and CoNS. *, data point that lies more than 1.5 times the interquartile range from the end of the box.

It would be ideal if we could use PCR to identify microorganisms in blood culture bottles as soon as they give a positive signal. A limitation of many real-time PCR assays is that they allow for accurate identification of a selected species or genus of microorganism. Multiplex PCR using multiple-species-specific probes are one approach for the simultaneous detection of multiple microorganisms (13). However, the complexities of these assays, which arise mainly from the interactions between the various oligonucleotide primers and probes make this approach less practical in many laboratories. Other approaches to rapid identification of bacteria in blood cultures have been reported (11). Although PCR-single-strand conformation polymorphism (PCR-SSCP) analysis has been shown to be able to detect a wide variety of microorganisms, multiple organisms may have the same SSCP pattern, and mixtures of organisms will confuse the interpretation of results. A more practical approach would be to identify specific important microorganisms by targeted real-time PCR in positive blood culture bottles. As a genus, staphylococci are the commonest cause of positive blood cultures, and of these staphylococci, S. aureus is almost always a significant pathogen (12). Thus, laboratories may be interested in targeting this genus, and specifically S. aureus, for rapid identification in blood culture systems, and the LightCycler Staphylococcus M^GRADE kit is one commercially available system that is available to laboratories that contemplate doing so, especially those that already have a LightCycler in place. The question that arises is if there is any utility in knowing the identity of the microorganism 1 day in advance while susceptibility test results are still pending. The assay could make a difference where single cultures are positive. If the microorganism were identified as S. aureus, the culture result would be taken seriously with further workup initiated immediately, rather than hesitating to proceed with immediate further workup because of the possibility that a single CoNS may be a contaminant. In our laboratory we are currently using fluorescence in situ hybridization with peptide nucleic acid probes for the identification of S. aureus as soon as blood culture bottles turn positive (4). By this strategy we identify gram-positive cocci in blood culture bottles as being S. aureus 1 day earlier than would have been possible by conventional methods. The use of the LightCycler Staphylococcus M^GRADE kits would be an equally effective alternative for achieving the same end. In order to provide same-day results, this would need planning on batching of specimens with cutoff times specified in a manner that would suit the workflow of the laboratory planning to use the test. It would be more effective if combined with real-time PCR detection of methicillin resistance. The cost of the assay, excluding capital costs, using the kits described in this study would be about $25 per sample, of which the three reagent kits account for $16.43, assuming a batch size of 20 with a positive and negative control in every batch and a 15% repeat rate. Institutions will have to decide if the added cost is worth the knowledge of the identity of the staphylococcus 1 day earlier than would be available by conventional testing. Real-time PCR identification of S. aureus and methicillin resistance in signal-positive blood culture bottles targeting the femA and mecA genes, respectively, has been implemented in the routine work of a lab (6). This signifies an interest in early identification of S. aureus and the presence of methicillin resistance in positive blood cultures. We conclude that S. aureus and CoNS can be accurately detected and identified using the LightCycler Staphylococcus M^GRADE kits in positive blood cultures that contain GPCC. The availability of such kits makes the application of real-time PCR for detection of staphylococci in positive blood culture bottles practical. The use of PCR assays augments our diagnostic capabilities when used in conjunction with automated blood culture systems.

 
* Corresponding author. Mailing address: Department of Infectious Diseases, The Cleveland Clinic Foundation, 9500 Euclid Avenue, S-32, Cleveland, OH 44195. Phone: (216) 444-1687. Fax: (216) 445-9446. E-mail: shrestn{at}ccf.org.

Top Abstract Text References

 

1
1. Carroll, K. C., R. B. Leonard, P. L. Newcomb-Gayman, and D. R. Hillyard. 1996. Rapid detection of the staphylococcal mecA gene from BACTEC blood culture bottles by the polymerase chain reaction. Am. J. Clin. Pathol. 106:600-605.[Medline]
2
2. Lem, P., J. Spiegelman, B. Toye, and K. Ramotar. 2001. Direct detection of mecA, nuc and 16S rRNA genes in BacT/Alert blood culture bottles. Diagn. Microbiol. Infect. Dis. 41:165-168.[CrossRef][Medline]
3
3. Louie, L., J. Goodfellow, P. Mathieu, A. Glatt, M. Louie, and A. E. Simor. 2002. Rapid detection of methicillin-resistant staphylococci from blood culture bottles by using a multiplex PCR assay. J. Clin. Microbiol. 40:2786-2790.[Abstract/Free Full Text]
4
4. Oliveira, K., G. W. Procop, D. Wilson, J. Coull, and H. Stender. 2002. Rapid identification of Staphylococcus aureus directly from blood cultures by fluorescence in situ hybridization with peptide nucleic acid probes. J. Clin. Microbiol. 40:247-251.[Abstract/Free Full Text]
5
5. Palomares, C., M. J. Torres, A. Torres, J. Aznar, and J. C. Palomares. 2003. Rapid detection and identification of Staphylococcus aureus from blood culture specimens using real-time fluorescence PCR. Diagn. Microbiol. Infect. Dis. 45:183-189.[Medline]
6
6. Paule, S. M., A. C. Pasquariello, R. B. Thomson, Jr., K. L. Kaul, and L. R. Peterson. 2005. Real-time PCR can rapidly detect methicillin-susceptible and methicillin-resistant Staphylococcus aureus directly from positive blood culture bottles. Am. J. Clin. Pathol. 124:404-407.[CrossRef][Medline]
7
7. Reischl, U., H. J. Linde, M. Metz, B. Leppmeier, and N. Lehn. 2000. Rapid identification of methicillin-resistant Staphylococcus aureus and simultaneous species confirmation using real-time fluorescence PCR. J. Clin. Microbiol. 38:2429-2433.[Abstract/Free Full Text]
8
8. Sakai, H., G. W. Procop, N. Kobayashi, D. Togawa, D. A. Wilson, L. Borden, V. Krebs, and T. W. Bauer. 2004. Simultaneous detection of Staphylococcus aureus and coagulase-negative staphylococci in positive blood cultures by real-time PCR with two fluorescence resonance energy transfer probe sets. J. Clin. Microbiol. 42:5739-5744.[Abstract/Free Full Text]
9
9. Shrestha, N. K., M. J. Tuohy, G. S. Hall, C. M. Isada, and G. W. Procop. 2002. Rapid identification of Staphylococcus aureus and the mecA gene from BacT/ALERT blood culture bottles by using the LightCycler system. J. Clin. Microbiol. 40:2659-2661.[Abstract/Free Full Text]
10
10. Tan, T. Y., S. Corden, R. Barnes, and B. Cookson. 2001. Rapid identification of methicillin-resistant Staphylococcus aureus from positive blood cultures by real-time fluorescence PCR. J. Clin. Microbiol. 39:4529-4531.[Abstract/Free Full Text]
11
11. Turenne, C. Y., E. Witwicki, D. J. Hoban, J. A. Karlowsky, and A. M. Kabani. 2000. Rapid identification of bacteria from positive blood cultures by fluorescence-based PCR-single-strand conformation polymorphism analysis of the 16S rRNA gene. J. Clin. Microbiol. 38:513-520.[Abstract/Free Full Text]
12
12. Weinstein, M. P., M. L. Towns, S. M. Quartey, S. Mirrett, L. G. Reimer, G. Parmigiani, and L. B. Reller. 1997. The clinical significance of positive blood cultures in the 1990s: a prospective comprehensive evaluation of the microbiology, epidemiology, and outcome of bacteremia and fungemia in adults. Clin. Infect. Dis. 24:584-602.[Medline]
13
13. Wellinghausen, N., B. Wirths, A. R. Franz, L. Karolyi, R. Marre, and U. Reischl. 2004. Algorithm for the identification of bacterial pathogens in positive blood cultures by real-time LightCycler polymerase chain reaction (PCR) with sequence-specific probes. Diagn. Microbiol. Infect. Dis. 48:229-241.[CrossRef][Medline]

---

Journal of Clinical Microbiology, December 2005, p. 6144-6146, Vol. 43, No. 12
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.12.6144-6146.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Shrestha, N. K. Articles by Procop, G. W. Search for Related Content PubMed PubMed Citation Articles by Shrestha, N. K. Articles by Procop, G. W.