STAPHYLOCOCCUS QUICK-FISH BC TEST COMPARED WITH TUBE COAGULASE TEST IN POSITIVE BLOOD CULTURES:

Transcription

1 JCM Accepts, published online ahead of print on 24 October 2012 J. Clin. Microbiol. doi: /jcm Copyright 2012, American Society for Microbiology. All Rights Reserved STAPHYLOCOCCUS QUICK-FISH BC TEST COMPARED WITH TUBE COAGULASE TEST IN POSITIVE BLOOD CULTURES: EVALUATION AND APPLICATION IN A CLINICAL ROUTINE SETTING. 4 5 E. Carretto 1#, M. Bardaro 1, G. Russello 1, M. Mirra 1, C. Zuelli 1, D. Barbarini Clinical Microbiology Laboratory IRCCS Arcispedale Santa Maria Nuova, AO Reggio Emilia Italy 2 Virology and Microbiology Laboratory Fondazione IRCCS Policlinico San Matteo, Pavia - Italy KEYWORDS: blood cultures, coagulase negative staphylococci, Staphylococcus aureus, PNA-FISH, Quick-FISH, tube coagulase test RUNNING TITLE: DTCT and Staph Quick-FISH test and their clinical use # CORRESPONDING AUTHOR: Edoardo Carretto Clinical Microbiology Laboratory IRCCS Arcispedale Santa Maria Nuova Viale Risorgimento, Reggio Emilia, Italy - Phone (+39) ; fax (+39) ; edoardo.carretto@asmn.re.it 23 1

2 ABSTRACT Many studies demonstrate that a delayed proper therapy in bloodstream infections caused by Staphylococcus aureus increase the mortality rates, emphasizing the need to shorten the turn-around time for positive blood cultures. Different techniques are currently available, from phenotypic methods to more complex test such as MALDI-TOF, RT-PCR and PNA-FISH. This study evaluates the performance of the Staphylococcus QuickFISH BC test (QFT), a novel FISH methodology, compared with the direct tube coagulase test (DTCT) on blood cultures positive, at Gram stain, for Gram-positive cocci in clusters. A total of 173 blood cultures collected from 128 different patients were analyzed using the DTCT test, evaluated both after 4 and 24 hours, and the QFT. 179 isolates were identified using the Vitek2 system. Thirty-five out of 35 Staphylococcus aureus were correctly identified by the QFT (sensitivity = 100%), with a specificity of 100% (no green fluorescence detected for strains different from SA). The DTCT test was positive after 4h in 28 out of the 35 samples (sensitivity = 80%) and the DTCT after 24h in 31 out of the 35 samples (sensitivity = 88.57%). Among the remaining 144 isolates, one was then identified as Corynebacterium striatum and two as Micrococcus luteus. QFT identified 139 out of the 141 coagulase negative staphylococci (sensitivity = 98,58%), showing also a specificity of 100% (no fluorescent red signals detected for strains different from CNS). The paper discuss also the implementation process of this methodology in our setting, with particular emphasis to the workflow and the cost-effectiveness. 2

3 INTRODUCTION Staphylococci are the microorganisms most frequently isolated from blood cultures. Bloodstream infections caused by Staphylococcus aureus (SA-BSI) are severe infections, with an incidence that varies greatly around the world: from 19.7 episodes/ person-years in a Canadian study (10), to more than 50 episodes/ person-years (8), with risk factors known to be the advancing age, male gender, community onset, comorbidities (such as alcoholism, immunosuppression, cirrhosis, malignancies, chronic renal failure and frequent heath care contacts (20). Different studies demonstrate that a delayed appropriate therapy in SA-BSIs is related to an increase in the mortality rates, prolonging also the hospital stay and increasing costs (14, 15). On the other hand, coagulase negative staphylococci (CNS) are the microorganisms most frequently isolated in clinical microbiology laboratories, but are often contaminants that raise the laboratory working load. Their treatment leads to antibiotic abuse increasing also microbial resistance. Moreover, the misinterpretation of the CNS role causes side effects, underestimation of the true pathogens and extra costs (19). To date, different techniques are available to shorten the turn-around time (TAT) for positive blood cultures: e.g. MALDI-TOF, RT-PCR, traditional techniques and PNA-FISH. The MALDI-TOF approach seems promising, although the proper preparatory methods are still to be established and some variability in sensitivity is reported (different from Gram positive and Gram 3

4 negative) (11, 17, 22). A real-time PCR approach is available (Gene X-pert MRSA/SA, Cepheid, USA); the method is really easy to perform and seems to have an exquisite sensitivity (21), but its main disadvantage is that it is too expensive, especially for settings managing a large amount of samples. The direct tube coagulase test (DTCT), performed from positive blood cultures, is used for the discrimination between S. aureus and CNS strains; this test is cheap, easy to perform and possess high specificity but a great inconsistency in sensitivity, ranging from 60% to 100% (4, 16). For different microrganisms, including staphylococci, the peptide nucleic acid fluorescence in situ hybridization test (PNA-FISH) was extensively studied as in-house method (12, 13), and is now commercially available in clinical practice, allowing for identification of these microorganisms directly from positive blood culture bottles (6, 7). In our Institution we use the PNA-FISH, AdvanDx, USA, to presumptively identify yeasts and Gram positive cocci in pairs or chains. Recently, a modification of the technique was developed (QuickFISH BC, AdvanDx, USA) (QFT). This modified technique is currently (September 2012) available only for staphylococci and it allows for distinction between SA and CNS with a simple and short procedure. The time for processing has been reduced with faster fixation and hybridization phases (15 minutes), as well as removal of a wash step The pathogen detection requires a maximum of 20 minutes, allowing the clinical microbiologist to report the obtained ID together with initial Gram stain. This technique has been recently validated (4). This study evaluates the performance of this new FISH methodology, comparing it 4

5 with the DTCT, evaluated after 4h (DTCT4) and 24h (DTCT24) on different sets of blood cultures showing at Gram stain, Gram-positive cocci in clusters (GPCC), collected in a three-months period in the IRCCS Arcispedale Santa Maria Nuova of Reggio Emilia, Italy, in order to assess the usefulness in clinical practice and implementation in routine laboratory workflow MATERIALS AND METHODS Samples selection - Blood cultures were analyzed using the Bactec system (Becton Dickinson, USA) using the aerobic and anaerobic Bactec PLUS/F medium. During the study period (15th Oct th Dec 2011), all the blood cultures showing GPCC at Gram stain (alone or with other forms, such as Gram negative bacilli or Gram positive cocci in pairs or chains) were considered eligible. If a set from a patient (aerobic + anaerobic bottles) was positive, a single bottle was evaluated (the first one which resulted positive). If different sets from the same patient revealed GPCC, a single bottle for any set was analyzed, with a maximum of three analysis for a single patient. DTCT4, DTCT24, QFT were performed on all the samples included in the study. The samples were then cultured using standard techniques, as in CLSI document (3). The identification of the microorganisms grown from cultures was performed using the Vitek2 system (BioMérieux, Marcy l'etoile, France), according to manufacturer's recommendation. Supplementary identification tests were performed when needed. On few strains where identification was 5

6 not unambiguous even after supplementary tests, we used automated ribotyping (Qualicon DuPont, USA) to achieve the proper speciation (2). Direct tube coagulase test Two or three drops (50 μl) of a positive blood culture broth were added in a tube containing 0.5 ml of rabbit plasma with EDTA (BBL, Cockeysville, MD) and incubated at 35 C. The tubes were read the first time after 4 hours and then after 24 h to evaluate DTCT4 or DTCT24, respectively. Any clotting was interpreted as indicative of S. aureus (16). Staphylococcus QuickFISH BC test - The QFT was performed according to the procedure of the manufacturer. The procedure started immediately after the detection of GPCC at Gram stain in positive blood cultures. Drops of blood culture sample ( μl) were added into an AdvanDx Filter vial. A filter plunger was then inserted into the vial, pushed down to remove the resin beads. Ten μl of sample were then transferred into the center of the sample area of an AdvanDx QuickFISH slide, previously placed on a workstation heated at 55 C ± 1 C. Samples were then fixed with two different solutions (QuickFix-1 + QuickFix-2 in different times). After smear fixation, one drop of Staphylococcus PNA Blue was placed into the center of a coverslip, and then one drop of Staphylococcus PNA Yellow was added directly on top of the first one. PNA Blue and PNA Yellow was mixed until they produce a uniform green color. The coverslip was then applied to the slide placed on the workstation heated at 55 C ± 1 C. This hybridization phase lasted 15 minutes. The slides were examined using a fluorescence microscope with a 100x oil objective. S. aureus was 6

7 identified as bright green fluorescent cocci in multiple fields of view, whereas CNS was identified as multiple red fluorescent cocci in multiple fields of view. The QFT slides have built-in positive and negative controls that were read together with the sample. QFT slides were read independently by different test operators and blinded to the final results RESULTS During the study period, a total of 173 blood cultures collected from 128 different patients were positive for GPCC. In thirteen bottles the Gram stain revealed a mixed culture: we recognized 7 GPCC together with Gram negative bacilli and 6 GPCC mixed with Gram positive cocci in chains. QFT and DTCT were performed on all 173 samples. As shown in table 1, 179 isolates were identified from the selected blood cultures. Six bottles yielded two different staphylococcal strains: in one case, we isolated SA + S. epidermidis and in five cases we documented mixed cultures with S. epidermidis and S. hominis. Thirty-five out of the 179 isolates were identified as SA, one isolate was a Corynebacterium striatum strain which was misidentified at the Gram stain, 2 were identified as Micrococcus luteus and 141 strains were CNS, with S. epidermidis being the species most represented. In 18 out of the 141 CNS Vitek2 was unable to provide an unequivocal species identification for the CNS and supplementary phenotypical tests were needed to achieve the proper identification. In five cases even these tests did not provide a definitive result and we analyzed these strains using automated 7

8 ribotyping. This technique allowed us to correctly identify 2 Staphylococcus hominis strains,1 Staphylococcus simulans, 1 Staphylococcus capitis whereas one strain was not identified even by this method (the highest similarity index was 0.64 with S. epidermidis DUP-20388). Considering the performance of DTCT test, among the 35 S. aureus, 28 were positive after 4 h incubation (sensitivity for DTCT4 = 80% - 28/35), 3 were negative after 4h but positive after 24h (raising sensitivity for DTCT24 to 88.57% - 31/35) whereas four bottles remained negative after 24h. These tests were negative, both after 4 hours and after 24 hours, for all CNS. A sample containing both SA and S. epidermidis resulted positive both for DTCT4 and DTCT24 and was excluded for computational evaluation of the sensitivity and specificity of the method for CNS. Thus, regarding CNS, DTCT test showed a sensitivity and specificity of 100%. As regards the QFT, the images obtained were brilliant and easily interpretable (figure 1). No discrepancies between the QFT results and the phenotypic identification for SA were detected (sensitivity and specificity for SA = 100%). Among the 144 GPCC different from SA, QFT showed a red fluorescence for 139 samples. The five samples that did not produce fluorescence were 2 Micrococcus luteus, 1 Corynebacterium striatum, 1 Staphylococcus simulans and the staphylococcal strain we did not manage to identify. The overall sensitivity of the QFT was 97,14% and increased to 98,58% if Micrococcaceae were excluded from computation (table 2). The specificity was 100%, since no fluorescent red signals were detected in samples yielding strains different from CNS. 8

9 DISCUSSION QuickFISH (AdvanDx, USA) is a modification of traditional FISH techniques, using a simpler and faster protocol. This innovation is now FDA cleared and marked for CE-IVD and available for clinical routine use. It has to be expected that the manufacturer will broad the number of pathogens this new technology can detect. Our study represents an early clinical laboratory evaluation of the first product available on this platform after the validation paper of Deck et al. (4). This molecular procedure is based on the PNA-FISH methodology with shortened fixation and hybridization phases and elimination of the washing steps. In our experience the new QuickFISH appeared quick, reliable and easy to perform and allowed us to reduce TAT compared with DTCT and traditional PNA-FISH. Using this method, the microbiologist is able to provide the clinicians with the results of both Gram stain and the presumptive identification of a Staphylococcus aureus in half an hour from the bottle positivity. The performance of the QFT in distinguishing SA and CNS appeared very good. The five strains which gave a negative result by QFT were 1 Corynebacterium striatum (erroneously considered at Gram stain as GPCC), 2 Micrococcus luteus, 1 Staphylococcus simulans (which is known not to be targeted by the probes used for the QFT) and 1 staphylococcal strain that we were unable to identify at species level even using ribotyping techniques. We assume it could be a species hardly ever isolated in clinical practice, even because its 9

10 fingerprint is not included in the Riboprinter database, which showed good performances on clinical CNS (2). According to these considerations, the sensitivity of the method in CNS detection is 97.14%, but if we exclude for the computation the two Micrococcus lutes strains, the performance of the QFT increases to 98.58% and appears even more impressive. The data is in overall agreement with the sensitivity revealed in the validation study (4). An excellent agreement between QFT and a standard laboratory technique such as DTCT was demonstrated. As regards DTCT, a wide range of sensitivity is reported in different times and in different papers. Our data are in range with those published in the majority of the studies (4, 5, 9, 18). It has to be noted that among the four strains which gave a negative results after 24 hours, in three cases the test repeated the day after provided a positive result, confirming in our experience some kind of intrinsic variability of the method. Thus, our experience confirms that although DTCT is cheaper, its sensitivity was lower compared to QFT. Furthermore it should be considered that the DTCT value is poor if the test is performed on the blood cultures that automated instruments reveal positive in the afternoon, because the results, even after the 4h analysis, are available only after the closing time of a laboratory that has a workflow of 12h/day. To date, different techniques are available to shorten the TAT for positive blood cultures (MALDI-TOF, RT-PCR, traditional techniques, PNA-FISH). In a worldwide period of economical troubles and funding shortage, the microbiologist should made an effort to evaluate the best pharmacoeconomic 10

11 approach in providing clinicians with preliminary results able to correctly address the empiric therapy. These approaches cannot be easily standardized because of the high variability of the settings: the laboratory settings (opening time, mixed laboratories, number of cultures processed, automation level etc.) and the type of the Hospital (number of acute-care beds, patients typology, numbers of ICUs or transplant units etc.) should be taken in account carefully. In general, MALDI-TOF seems a promising tool to allow the presumptive identification of strains directly from blood cultures, but strong validation studies are required to focus the limitations of this technique (if any) and its proper use (11, 17, 22). A previous paper of Brown and Paladino focused the cost-effectiveness of RT- PCR for MRSA in reducing mortality rates while being less costly than empiric therapy (1), whereas the work of Hermsen et al. pointed out that DTCT4 and DTCT24 were more cost-effective than RT-PCR and PNA-FISH, respectively (7). However, it has to be emphasized that the prices of the different tests may vary in different Institution. Our Clinical Microbiology Laboratory supports a tertiary hospital (1000 acute care beds), three small hospitals (both for acute or long-term care beds) and some healthcare facilities (with long-term care beds). Medical wards are the most represented. More than blood culture sets are performed each year (13.42% positives) and the laboratory is opened 12h/day. MALDI-TOF is not still available, whereas Gene X-pert MRSA/SA, (Cepheid, USA) is available and used. In our setting, the cost of PNA-FISH is two-thirds compared with the cost 11

12 of RT-PCR, whereas DTCT appear unequivocally the cheapest test. Currently, the model used in our setting for blood cultures positive for GPCC is based on DTCT if the positivity is detected in the morning, followed by a RT-PCR for samples positive to the DTCT at 4h, in order to confirm the SA identification and evaluate the resistance to methicillin. If a blood culture is positive in the afternoon, it could be more cost-effective to use the QuickFISH to presumptively identify SA, and on these samples perform the RT-PCR. Considering the future price of QFT as the same of PNA-FISH, the possibility to use the QFT, that demonstrated a very good sensitivity and specificity on SA with the advantage of being extremely rapid, will allow to work on GPCC positive samples until the late afternoon also for laboratories that have a 12 hours/day workflow. Finally, it has to be emphasized that the hands-on time of the QFT test is less than 5 minutes; the technique is easy and the slides can be prepared even by technicians after a short training period. The introduction of the method in the laboratory routine has therefore a low implication in modifying the usual workflow. In conclusion, the QuickFISH technology appears robust and reliable, with the major advantage of providing results in half an hour. It could be expected that this technique will be used in routine for all blood cultures to detect all the different pathogens currently detected by traditional PNA-FISH, allowing major advantages in reporting preliminary identification together with the Gram stain. The results presented in this study confirm the very good 12

13 performance of the test on staphylococci and encourage the use of this methodology instead of more traditional FISH AKNOWLEDGMENTS None of the Authors has any financial interest in AdvanDx, Inc. or received financial supports to perform this study. Kits and disposables were kindly provided by AdvanDx A/S, Denmark. A special thank is due to Henrik Aspe, Steen Hesthaven and Lidija Horvat, for their helpful support throughout the study. This work was partially supported by grants from the Scientific Committee of the IRCCS Arcispedale Santa Maria Nuova, Reggio Emilia, to Edoardo Carretto REFERENCES Brown, J., and J. A. Paladino Impact of rapid methicillin-resistant Staphylococcus aureus polymerase chain reaction testing on mortality and cost effectiveness in hospitalized patients with bacteraemia: a decision model. Pharmacoeconomics 28: Carretto, E., D. Barbarini, I. Couto, D. De Vitis, P. Marone, J. Verhoef, H. De Lencastre, and S. Brisse Identification of coagulase-negative 13

14 staphylococci other than Staphylococcus epidermidis by automated ribotyping. Clin Microbiol Infect 11: CLSI Principles and procedures for blood cultures. Approved guideline. Wayne, PA CLSI Document:M47-A Deck, M. K., E. S. Anderson, R. J. Buckner, G. Colasante, J. M. Coull, B. Crystal, P. Della Latta, M. Fuchs, D. Fuller, W. Harris, K. Hazen, L. L. Klimas, D. Lindao, M. C. Meltzer, M. Morgan, J. Shepard, S. Stevens, F. Wu, and M. J. Fiandaca Multicenter evaluation of the Staphylococcus QuickFISH method for simultaneous identification of Staphylococcus aureus and coagulase-negative staphylococci directly from blood culture bottles in less than 30 minutes. J Clin Microbiol 50: Goldstein, J., and J. W. Roberts Microtube coagulase test for detection of coagulase-positive staphylococci. J Clin Microbiol 15: Hensley, D. M., R. Tapia, and Y. Encina An evaluation of the AdvanDx Staphylococcus aureus/cns PNA FISH assay. Clin Lab Sci 22:

15 Hermsen, E. D., S. S. Shull, D. G. Klepser, P. C. Iwen, A. Armbrust, J. Garrett, A. G. Freifeld, and M. E. Rupp Pharmacoeconomic analysis of microbiologic techniques for differentiating staphylococci directly from blood culture bottles. J Clin Microbiol 46: Klevens, R. M., M. A. Morrison, J. Nadle, S. Petit, K. Gershman, S. Ray, L. H. Harrison, R. Lynfield, G. Dumyati, J. M. Townes, A. S. Craig, E. R. Zell, G. E. Fosheim, L. K. McDougal, R. B. Carey, S. K. Fridkin, and M. I. Active Bacterial Core surveillance Invasive methicillinresistant Staphylococcus aureus infections in the United States. JAMA 298: Lagace-Wiens, P. R., M. J. Alfa, K. Manickam, and J. A. Karlowsky Thermostable DNase is superior to tube coagulase for direct detection of Staphylococcus aureus in positive blood cultures. J Clin Microbiol 45: Laupland, K. B., T. Ross, and D. B. Gregson Staphylococcus aureus bloodstream infections: risk factors, outcomes, and the influence of methicillin resistance in Calgary, Canada, J Infect Dis 198:

16 Meex, C., F. Neuville, J. Descy, P. Huynen, M. P. Hayette, P. De Mol, and P. Melin Direct identification of bacteria from positive anaerobic BacT/Alert(R) blood cultures by MALDI-TOF MS: MALDI Sepsityper(R) kit (Bruker) versus in-house saponin method for bacterial extraction. J Med Microbiol Jul 26. [Epub ahead of print] Oliveira, K., S. M. Brecher, A. Durbin, D. S. Shapiro, D. R. Schwartz, P. C. De Girolami, J. Dakos, G. W. Procop, D. Wilson, C. S. Hanna, G. Haase, H. Peltroche-Llacsahuanga, K. C. Chapin, M. C. Musgnug, M. H. Levi, C. Shoemaker, and H. Stender Direct identification of Staphylococcus aureus from positive blood culture bottles. J Clin Microbiol 41: Oliveira, K., G. W. Procop, D. Wilson, J. Coull, and H. Stender Rapid identification of Staphylococcus aureus directly from blood cultures by fluorescence in situ hybridization with peptide nucleic acid probes. J Clin Microbiol 40: Paul, M., G. Kariv, E. Goldberg, M. Raskin, H. Shaked, R. Hazzan, Z. Samra, D. Paghis, J. Bishara, and L. Leibovici Importance of appropriate empirical antibiotic therapy for methicillin-resistant Staphylococcus aureus bacteraemia. J Antimicrob Chemother 65:

17 Paul, M., V. Shani, E. Muchtar, G. Kariv, E. Robenshtok, and L. Leibovici Systematic review and meta-analysis of the efficacy of appropriate empiric antibiotic therapy for sepsis. Antimicrob Agents Chemother 54: Qian, Q., K. Eichelberger, and J. E. Kirby Rapid identification of Staphylococcus aureus in blood cultures by use of the direct tube coagulase test. J Clin Microbiol 45: Saffert, R. T., S. A. Cunningham, J. Mandrekar, and R. Patel Comparison of three preparatory methods for detection of bacteremia by MALDI-TOF mass spectrometry. Diagn Microbiol Infect Dis 73: Sturm, P. D., D. Kwa, F. J. Vos, C. J. Bartels, and T. Schulin Performance of two tube coagulase methods for rapid identification of Staphylococcus aureus from blood cultures and their impact on antimicrobial management. Clin Microbiol Infect 14: van der Heijden, Y. F., G. Miller, P. W. Wright, B. E. Shepherd, T. L. Daniels, and T. R. Talbot Clinical impact of blood cultures contaminated with coagulase-negative staphylococci at an academic medical center. Infect Control Hosp Epidemiol 32:

18 van Hal, S. J., S. O. Jensen, V. L. Vaska, B. A. Espedido, D. L. Paterson, and I. B. Gosbell Predictors of mortality in Staphylococcus aureus Bacteremia. Clin Microbiol Rev 25: Wolk, D. M., M. J. Struelens, P. Pancholi, T. Davis, P. Della-Latta, D. Fuller, E. Picton, R. Dickenson, O. Denis, D. Johnson, and K. Chapin Rapid detection of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) in wound specimens and blood cultures: multicenter preclinical evaluation of the Cepheid Xpert MRSA/SA skin and soft tissue and blood culture assays. J Clin Microbiol 47: Wuppenhorst, N., C. Consoir, D. Lorch, and C. Schneider Direct identification of bacteria from charcoal-containing blood culture bottles using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Eur J Clin Microbiol Infect Dis 31:

19 Figure 1 QFT. Upper: green fluorescence of Staphylococcus aureus; bottom: red fluorescence of a Staphylococcus epidermidis strain 19

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21 TABLES AND FIGURES Identification Staphylococcus QuickFISH DTCT + GF RF Negative 4h 24h S. aureus 35 * S. epidermidis 0 86 *,# S. hominis 0 27 # S. capitis S. haemolyticus S. warneri S. lugdunensis S. simulans Micrococcus luteus Corynebacterium striatum Not identified Table 1 Study synopsis. DTCT = direct tube coagulase test; GF = green fluorescence; RF = red fluorescence; * = one sample yielded S. aureus and S. epidermidis; # = five samples yielded S. epidermidis and S. hominis; = one sample yielding both S. aureus and S. epidermidis was positive to DTCT at 4h and 24h.

22 QFT Standard ID (culture) GF RF NF Sensitivity SA = = 100 CNS 141 = ,56 Others 3 = = Total Table 2 Comparison between the final ID with conventional techniques and QFT results: GF = green fluorescence, RF = red fluorescence; NF = absence of fluorescence. Other strains are 2 Micrococcus luteus and 1 Corynebacterium striatum.