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Multiplex PCR assay underreports true bloodstream infections with coagulase-negative staphylococci in hematological patients with febrile neutropenia
Diagnostic Microbiology and Infectious Disease xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Note
Multiplex PCR assay underreports true bloodstream infections with coagulase-negative staphylococci in hematological patients with febrile neutropenia Yvonne Reers a, Evgeny A. Idelevich a, Hanna Pätkau a, Maria Cristina Sauerland b, Sascha Tafelski c, Irit Nachtigall c, Wolfgang E. Berdel d, Georg Peters a, Gerda Silling d, Karsten Becker a,⁎, for the Molecular Diagnostics of Sepsis Study Group a
Institute of Medical Microbiology, University Hospital Münster, Domagkstr. 10, 48149 Münster, Germany Institute of Biostatistics and Clinical Research, University of Münster, Schmeddingstraße 56, 48149 Münster, Germany c Department of Anesthesiology and Intensive Care Medicine, Campus Charité Mitte and Campus Virchow-Klinikum, Charité - University Medicine Berlin, Charitéplatz 1, 10117 Berlin, Germany d Department of Medicine A, University Hospital Münster, Albert-Schweitzer-Campus 1/A1, 48149 Münster, Germany b
a r t i c l e
i n f o
Article history: Received 2 January 2016 Received in revised form 26 April 2016 Accepted 30 April 2016 Available online xxxx
a b s t r a c t SeptiFast multiplex PCR assay was evaluated for detecting true bloodstream infections (BSIs) with coagulasenegative staphylococci (CoNS) in neutropenic hematological patients. Sensitivity for samples representing true CoNS-BSIs was 23.3% with an integrated cutoff and increased to 83.3% if the cutoff was neglected. Hence, the cutoff may prohibit timely targeted antimicrobial therapy. © 2016 Elsevier Inc. All rights reserved.
Keywords: Bloodstream infections Coagulase-negative staphylococci Multiplex PCR Cutoff Febrile neutropenia
Bloodstream infections (BSIs) are among the leading clinical complications in cancer patients (Williams et al., 2004; Gaieski et al., 2013). They are supported by low leukocyte counts, insertion of central venous catheters (CVCs) as well as the administration of chemotherapeutics (Pagano et al., 1997; Bucaneve et al., 2005; Freifeld et al., 2011). Coagulase-negative staphylococci (CoNS), though common bacteria of the skin microbiota and hence frequent blood culture (BC) contaminants (Becker et al., 2014; Kaspar et al., 2015), are also the most common etiological agents of significant bacteremia (based on clinical signs of infection and multiple CoNS-positive BCs) in cancer patients (Cherif et al., 2003; Marra et al., 2011; Passerini et al., 2011). Early pathogen identification is crucial since time to administration of targeted antimicrobial therapy inversely correlates with survival (Kumar et al., 2006). The study´s objective was to assess the appropriateness of a semi-quantitative, analytical cutoff applied by a multiplex PCR assay (LightCycler® SeptiFast Test) for detecting true CoNS-BSIs among neutropenic hematological patients.
⁎ Corresponding author. Tel.: +49-251 83-55375; fax: +49-251-83-55350. E-mail address: [email protected] (K. Becker).
This analysis employed study data of adult hematological patients with febrile neutropenia (Idelevich et al., 2015). Prior to the initiation of empirical antimicrobial therapy, a BC set (aerobic, anaerobic and fungal BC bottles) as well as EDTA blood samples for PCR analysis were collected from peripheral veins and/or CVCs/ports wherever possible. PCR samples were analyzed according to the manufacturer’s instructions (Roche Diagnostics GmbH, 2011). If patients continued to be febrile beyond 72 hours, a second samples set was obtained. In total, this analysis is based on 253 paired BC and PCR samples from 150 patients. Further BCs and CVC tips were obtained as deemed clinically necessary. The integrated cutoff for CoNS by the SeptiFast Identification Software (SIS) automatically omitted CoNS results if they occurred after 20 of the overall 30 amplification cycles. For the purpose of this study, CoNS detection was considered a true BSI if one of the initial BCs and at least one additional microbiological culture within five days (BC or CVC tip/port) from a different location turned positive with the same species during the same infective episode (modified from the CDC recommendations) (Beekmann et al., 2005; Horan et al., 2008). Thirty-seven of 253 BCs were CoNS-positive (27/150 patients) (Table 1). Four samples had CoNS positive PCR results despite negative BCs (three patients). Twenty-five of 37 CoNS positive BCs were obtained
http://dx.doi.org/10.1016/j.diagmicrobio.2016.04.024 0732-8893/© 2016 Elsevier Inc. All rights reserved.
Please cite this article as: Reers Y, et al, Multiplex PCR assay underreports true bloodstream infections with coagulase-negative staphylococci in hematological patients with ..., Diagn Microbiol Infect Dis (2016), http://dx.doi.org/10.1016/j.diagmicrobio.2016.04.024
2
Y. Reers et al. / Diagnostic Microbiology and Infectious Disease xxx (2016) xxx–xxx
Table 1 Patient characteristics. Characteristics Central venous catheter Stem cell transplantation Average leucocyte count at fever onset, cells/μL
Table 3 Detection of all CoNS and true CoNS-BSIs by PCR with and without cutoff. CoNS positive BCs (n = 27)
CoNS negative BCs (n = 123)
p-value (two-sided)
23 (85.2%) 19 (70.4%) 97.4 ± 143.1
87 (70.7%) 71 (57.7%) 203.8 ± 242.1
.125 .223 .025
from CVCs or ports (15/27 patients). Twenty (80.0%) central cultures also had correspondent CoNS-positive peripheral BCs (Table 2; 10/15 patients, 66.7%). Patients with CoNS-positive BCs frequently had CVCs (85.2%), underwent stem cell transplantation (70.4%) and had significantly lower leucocyte counts as compared to patients with other or not microbiologically documented infections (p = 0.025; Table 1). CoNS were frequently resistant towards common antimicrobial agents (resistance rates to oxacillin, gentamicin and teicoplanin were 86.5%, 64.9% and 27.0%, respectively); all isolates were vancomycin-susceptible. Further microbiological evidence was found for 20/27 patients (74.1%), suggesting true CoNS-BSIs in the majority of cases (Table S1). With the integrated cutoff in place, only seven of these 30 samples with true CoNS-BSIs (5/20 patients, 25.0%) also had CoNS-positive PCR results. If the cutoff was neglected, true CoNS-BSIs were confirmed by PCR for 25 samples (83.3%; 17 patients, 85.0%; Table 3). Changes to an adequate antibiotic therapy due to Gram stain, identification and antibiogram took place in 7/20 patients with true CoNS-BSIs (35.0%). Corresponding PCR tests were only once positive and six times omitted by the cutoff. A detailed breakdown of all 37 CoNS-positive BC samples with their respective amplification results as well as their identification as true CoNS-BSI or contamination is demonstrated in Fig. 1. The median number of amplification cycles for samples accounting for true CoNS-BSIs was 21. Hence, the majority of PCR results for true BSIs occurred barely after the cutoff. Overall, the cutoff omitted 18 of 30 PCR results accounting for true CoNS-BSIs (60%). Sensitivity for the detection of samples with true CoNS-BSIs by PCR was 23.3% with the cutoff and 83.3% without the cutoff (Table 3). In contrast, supposed CoNS contamination in BCs was truly recognized by PCR in six of seven samples with the cutoff, yielding a specificity of 85.7%. If the cutoff was neglected, specificity decreased to 14.3%. Our data indicate that CoNS frequently account for true BSIs in this patient population. This is probably due to frequent CVC usage as well as extremely low leucocyte counts. Furthermore, CoNS isolates are frequently resistant to common antibiotic treatment regimens (de Naurois et al., 2010; Freifeld et al., 2011; Penack et al., 2011). Therefore, PCR detection of CoNS may be particularly important for early targeted treatment in these immunocompromised patients. Concerning CoNS identification by PCR, this study supports data from previous studies which showed poor sensitivity for Gram-
PCR detection rate
Patients
Samples
Of all CoNS detected by BC confirmed by PCR with cutoff confirmed by PCR without cutoff Of true CoNS-BSIs confirmed by PCR with cutoff confirmed by PCR without cutoff
n = 27 6 (22.2%) 17 (63.0%) n = 20 5 (25.0%) 17 (85.0%)
n = 37 8 (21.6%) 31 (83.8%) n = 30 7 (23.3%) 25 (83.3%)
positive organisms (0.39) in neutropenic patients (Paolucci et al., 2013), supposedly due to the established cutoff criteria (Varani et al., 2009; Lamoth et al., 2010; Maubon et al., 2010; Josefson et al., 2011). When the cutoff was neglected, sensitivity substantially rose while specificity considerably decreased, leading to the reporting of more contaminants. Our patient group had higher CoNS isolation rates (18.0% vs. 7.7%) and more true CoNS-BSIs (74.1% vs. 16.7%), compared to ICU patients from a study on postoperative pulmonary or abdominal sepsis (Tafelski et al., 2015). Hence, the introduced cutoff might be more appropriate for postoperative patients who appear to have fewer true CoNS-BSIs. In contrast, true CoNS-BSIs appear significantly more often in hematological patients with febrile neutropenia and regularly require a change in the initial empiric antimicrobial therapy. Therefore, decreasing specificity by eliminating the cutoff may just have to be the necessary compromise to detect clinically relevant infections and timely administer appropriate therapy in these high-risk patients. Nonetheless, in postoperative ICU patients, true CoNS-BSIs were also omitted due to the cutoff (16.7%, n = 1). Decreased test specificity might lead to an increased use of vancomycin. Therefore, physicians should perform a critical appraisal of patient’s data to decide on the relevance of the finding. In conclusion, our results suggest that the integrated cutoff omits numerous true CoNS-BSIs in hematological patients with neutropenia and possibly also in postoperative ICU patients. Hence, the applied cutoff value, which was based on data from infection-free individuals (Lehmann et al., 2008), is not appropriate for this high-risk patient population. Instead, all positive PCR results should be indicated and communicated to the clinician, optionally with a comment regarding potential contamination. Omitting the cutoff would allow this relevant information to become available for clinical decision making, next to other microbiological and laboratory data, clinical signs of infection, and local epidemiological patterns.
Conflict of interest YN, EAI, ST, IN, GS, WEB and KB received lecture fees from Pfizer Deutschland GmbH; ST, IN, WEB and KB received lecture fees from Roche Diagnostics. The randomized controlled study, which generated data used for this analysis, was partly supported by Roche Diagnostics GmbH, Mannheim, Germany (supplied the LightCycler® SeptiFast Kits), and Pfizer GmbH (provided restricted grant for study activities). No support has been provided for this study’s activities. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.diagmicrobio.2016.04.024.
Table 2 CoNS positive BCs according to the withdrawal site, patients and respective samples. CoNS positive BCs
Patients (n = 27)
Samples (n = 37)
Central and peripheral obtained Both positive Only peripheral positive Only central positive Only peripheral obtained, positive Only central obtained, positive
15 (55.5%) 10 (37.0%) 4 (14.8%) 1 (3.7%) 8 (29.6%) 4 (14.8%)
25 (67.6%) 20 (54.1%) 4 (10.8%) 1 (2.7%) 8 (21.6%) 4 (10.8%)
Acknowledgments This study is part of a joined project of the Molecular Diagnostics of Sepsis Study Group aimed to the improvement of sepsis management. The authors are grateful to all members of the study group including M. Deja (Berlin), C. Spies (Berlin), H. Bunzemeier (Münster), R. Grube (Munich), D. Voss (Münster), M. Wilke (Munich) and J. Schulze (Berlin).
Please cite this article as: Reers Y, et al, Multiplex PCR assay underreports true bloodstream infections with coagulase-negative staphylococci in hematological patients with ..., Diagn Microbiol Infect Dis (2016), http://dx.doi.org/10.1016/j.diagmicrobio.2016.04.024
Y. Reers et al. / Diagnostic Microbiology and Infectious Disease xxx (2016) xxx–xxx
3
Fig. 1. Corresponding PCR results of all samples with CoNS-positive blood cultures plotted by the number of amplification cycles until positivity and stratified for true bloodstream infections and contaminations (samples listed at 25 amplification cycles include all samples that were detected between 25 to 30 cycles; samples listed under n.d. = not detected, remained negative).
References Becker K, Heilmann C, Peters G. Coagulase-negative staphylococci. Clin Microbiol Rev 2014;27:870–926. Beekmann SE, Diekema DJ, Doern GV. Determining the clinical significance of coagulasenegative staphylococci isolated from blood cultures. Infect Control Hosp Epidemiol 2005;26:559–66. Bucaneve G, Micozzi A, Menichetti F, Martino P, Dionisi MS, Martinelli G, et al. Levofloxacin to prevent bacterial infection in patients with cancer and neutropenia. N Engl J Med 2005;353:977–87. Cherif H, Kronvall G, Bjorkholm M, Kalin M. Bacteraemia in hospitalised patients with malignant blood disorders: a retrospective study of causative agents and their resistance profiles during a 14-year period without antibacterial prophylaxis. Hematol J 2003;4: 420–6. de Naurois J, Novitzky-Basso I, Gill MJ, Marti FM, Cullen MH, Roila F. Management of febrile neutropenia: ESMO Clinical Practice Guidelines. Ann Oncol 2010;21(Suppl. 5): v252–6. Freifeld AG, Bow EJ, Sepkowitz KA, Boeckh MJ, Ito JI, Mullen CA, et al. Clinical practice guideline for the use of antimicrobial agents in neutropenic patients with cancer: 2010 update by the Infectious Diseases Society of America. Clin Infect Dis 2011;52:e56–93. Gaieski DF, Edwards JM, Kallan MJ, Carr BG. Benchmarking the incidence and mortality of severe sepsis in the United States. Crit Care Med 2013;41:1167–74. Horan TC, Andrus M, Dudeck MA. CDC/NHSN surveillance definition of health careassociated infection and criteria for specific types of infections in the acute care setting. Am J Infect Control 2008;36:309–32. Idelevich EA, Silling G, Niederbracht Y, Penner H, Sauerland MC, Tafelski S, et al. Impact of multiplex PCR on antimicrobial treatment in febrile neutropenia: a randomized controlled study. Med Microbiol Immunol 2015;204:585–92. Josefson P, Stralin K, Ohlin A, Ennefors T, Dragsten B, Andersson L, et al. Evaluation of a commercial multiplex PCR test (SeptiFast) in the etiological diagnosis of community-onset bloodstream infections. Eur J Clin Microbiol Infect Dis 2011;30:1127–34. Kaspar U, Kriegeskorte A, Schubert T, Peters G, Rudack C, Pieper DH, et al. The culturome of the human nose habitats reveals individual bacterial fingerprint patterns. Environ Microbiol 2015. http://dx.doi.org/10.1111/1462-2920.12891. Kumar A, Roberts D, Wood KE, Light B, Parrillo JE, Sharma S, et al. Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock. Crit Care Med 2006;34:1589–96.
Lamoth F, Jaton K, Prod'hom G, Senn L, Bille J, Calandra T, et al. Multiplex blood PCR in combination with blood cultures for improvement of microbiological documentation of infection in febrile neutropenia. J Clin Microbiol 2010;48:3510–6. Lehmann LE, Hunfeld KP, Emrich T, Haberhausen G, Wissing H, Hoeft A, Stuber F. A multiplex real-time PCR assay for rapid detection and differentiation of 25 bacterial and fungal pathogens from whole blood samples. Med Microbiol Immunol 2008;197: 313–24. Marra AR, Camargo LF, Pignatari AC, Sukiennik T, Behar PR, Medeiros EA, et al. Nosocomial bloodstream infections in Brazilian hospitals: analysis of 2,563 cases from a prospective nationwide surveillance study. J Clin Microbiol 2011;49:1866–71. Maubon D, Hamidfar-Roy R, Courby S, Vesin A, Maurin M, Pavese P, et al. Therapeutic impact and diagnostic performance of multiplex PCR in patients with malignancies and suspected sepsis. J Infect 2010;61:335–42. Pagano L, Tacconelli E, Tumbarello M, Laurenti L, Ortu-La BE, Antinori A, et al. Bacteremia in patients with hematological malignancies. Analysis of risk factors, etiological agents and prognostic indicators. Haematologica 1997;82:415–9. Paolucci M, Stanzani M, Melchionda F, Tolomelli G, Castellani G, Landini MP, et al. Routine use of a real-time polymerase chain reaction method for detection of bloodstream infections in neutropaenic patients. Diagn Microbiol Infect Dis 2013;75:130–4. Passerini R, Ghezzi T, Sandri M, Radice D, Biffi R. Ten-year surveillance of nosocomial bloodstream infections: trends of aetiology and antimicrobial resistance in a comprehensive cancer centre. Ecancermedicalscience 2011;5:191. Penack O, Buchheidt D, Christopeit M, von Lilienfeld-Toal M, Massenkeil G, Hentrich M, et al. Management of sepsis in neutropenic patients: guidelines from the infectious diseases working party of the German Society of Hematology and Oncology. Ann Oncol 2011;22:1019–29. Roche Diagnostics GmbH. LightCycler® SeptiFast Test MGrade. Package Insert 04623886001–06. Mannheim, Germany; 2011. Tafelski S, Nachtigall I, Adam T, Bereswill S, Faust J, Tamarkin A, et al. Randomized controlled clinical trial evaluating multiplex polymerase chain reaction for pathogen identification and therapy adaptation in critical care patients with pulmonary or abdominal sepsis. J Int Med Res 2015;43:364–77. Varani S, Stanzani M, Paolucci M, Melchionda F, Castellani G, Nardi L, et al. Diagnosis of bloodstream infections in immunocompromised patients by real-time PCR. J Infect 2009;58:346–51. Williams MD, Braun LA, Cooper LM, Johnston J, Weiss RV, Qualy RL, et al. Hospitalized cancer patients with severe sepsis: analysis of incidence, mortality, and associated costs of care. Crit Care 2004;8:R291–8.
Please cite this article as: Reers Y, et al, Multiplex PCR assay underreports true bloodstream infections with coagulase-negative staphylococci in hematological patients with ..., Diagn Microbiol Infect Dis (2016), http://dx.doi.org/10.1016/j.diagmicrobio.2016.04.024
Contents lists available at ScienceDirect
Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Note
Multiplex PCR assay underreports true bloodstream infections with coagulase-negative staphylococci in hematological patients with febrile neutropenia Yvonne Reers a, Evgeny A. Idelevich a, Hanna Pätkau a, Maria Cristina Sauerland b, Sascha Tafelski c, Irit Nachtigall c, Wolfgang E. Berdel d, Georg Peters a, Gerda Silling d, Karsten Becker a,⁎, for the Molecular Diagnostics of Sepsis Study Group a
Institute of Medical Microbiology, University Hospital Münster, Domagkstr. 10, 48149 Münster, Germany Institute of Biostatistics and Clinical Research, University of Münster, Schmeddingstraße 56, 48149 Münster, Germany c Department of Anesthesiology and Intensive Care Medicine, Campus Charité Mitte and Campus Virchow-Klinikum, Charité - University Medicine Berlin, Charitéplatz 1, 10117 Berlin, Germany d Department of Medicine A, University Hospital Münster, Albert-Schweitzer-Campus 1/A1, 48149 Münster, Germany b
a r t i c l e
i n f o
Article history: Received 2 January 2016 Received in revised form 26 April 2016 Accepted 30 April 2016 Available online xxxx
a b s t r a c t SeptiFast multiplex PCR assay was evaluated for detecting true bloodstream infections (BSIs) with coagulasenegative staphylococci (CoNS) in neutropenic hematological patients. Sensitivity for samples representing true CoNS-BSIs was 23.3% with an integrated cutoff and increased to 83.3% if the cutoff was neglected. Hence, the cutoff may prohibit timely targeted antimicrobial therapy. © 2016 Elsevier Inc. All rights reserved.
Keywords: Bloodstream infections Coagulase-negative staphylococci Multiplex PCR Cutoff Febrile neutropenia
Bloodstream infections (BSIs) are among the leading clinical complications in cancer patients (Williams et al., 2004; Gaieski et al., 2013). They are supported by low leukocyte counts, insertion of central venous catheters (CVCs) as well as the administration of chemotherapeutics (Pagano et al., 1997; Bucaneve et al., 2005; Freifeld et al., 2011). Coagulase-negative staphylococci (CoNS), though common bacteria of the skin microbiota and hence frequent blood culture (BC) contaminants (Becker et al., 2014; Kaspar et al., 2015), are also the most common etiological agents of significant bacteremia (based on clinical signs of infection and multiple CoNS-positive BCs) in cancer patients (Cherif et al., 2003; Marra et al., 2011; Passerini et al., 2011). Early pathogen identification is crucial since time to administration of targeted antimicrobial therapy inversely correlates with survival (Kumar et al., 2006). The study´s objective was to assess the appropriateness of a semi-quantitative, analytical cutoff applied by a multiplex PCR assay (LightCycler® SeptiFast Test) for detecting true CoNS-BSIs among neutropenic hematological patients.
⁎ Corresponding author. Tel.: +49-251 83-55375; fax: +49-251-83-55350. E-mail address: [email protected] (K. Becker).
This analysis employed study data of adult hematological patients with febrile neutropenia (Idelevich et al., 2015). Prior to the initiation of empirical antimicrobial therapy, a BC set (aerobic, anaerobic and fungal BC bottles) as well as EDTA blood samples for PCR analysis were collected from peripheral veins and/or CVCs/ports wherever possible. PCR samples were analyzed according to the manufacturer’s instructions (Roche Diagnostics GmbH, 2011). If patients continued to be febrile beyond 72 hours, a second samples set was obtained. In total, this analysis is based on 253 paired BC and PCR samples from 150 patients. Further BCs and CVC tips were obtained as deemed clinically necessary. The integrated cutoff for CoNS by the SeptiFast Identification Software (SIS) automatically omitted CoNS results if they occurred after 20 of the overall 30 amplification cycles. For the purpose of this study, CoNS detection was considered a true BSI if one of the initial BCs and at least one additional microbiological culture within five days (BC or CVC tip/port) from a different location turned positive with the same species during the same infective episode (modified from the CDC recommendations) (Beekmann et al., 2005; Horan et al., 2008). Thirty-seven of 253 BCs were CoNS-positive (27/150 patients) (Table 1). Four samples had CoNS positive PCR results despite negative BCs (three patients). Twenty-five of 37 CoNS positive BCs were obtained
http://dx.doi.org/10.1016/j.diagmicrobio.2016.04.024 0732-8893/© 2016 Elsevier Inc. All rights reserved.
Please cite this article as: Reers Y, et al, Multiplex PCR assay underreports true bloodstream infections with coagulase-negative staphylococci in hematological patients with ..., Diagn Microbiol Infect Dis (2016), http://dx.doi.org/10.1016/j.diagmicrobio.2016.04.024
2
Y. Reers et al. / Diagnostic Microbiology and Infectious Disease xxx (2016) xxx–xxx
Table 1 Patient characteristics. Characteristics Central venous catheter Stem cell transplantation Average leucocyte count at fever onset, cells/μL
Table 3 Detection of all CoNS and true CoNS-BSIs by PCR with and without cutoff. CoNS positive BCs (n = 27)
CoNS negative BCs (n = 123)
p-value (two-sided)
23 (85.2%) 19 (70.4%) 97.4 ± 143.1
87 (70.7%) 71 (57.7%) 203.8 ± 242.1
.125 .223 .025
from CVCs or ports (15/27 patients). Twenty (80.0%) central cultures also had correspondent CoNS-positive peripheral BCs (Table 2; 10/15 patients, 66.7%). Patients with CoNS-positive BCs frequently had CVCs (85.2%), underwent stem cell transplantation (70.4%) and had significantly lower leucocyte counts as compared to patients with other or not microbiologically documented infections (p = 0.025; Table 1). CoNS were frequently resistant towards common antimicrobial agents (resistance rates to oxacillin, gentamicin and teicoplanin were 86.5%, 64.9% and 27.0%, respectively); all isolates were vancomycin-susceptible. Further microbiological evidence was found for 20/27 patients (74.1%), suggesting true CoNS-BSIs in the majority of cases (Table S1). With the integrated cutoff in place, only seven of these 30 samples with true CoNS-BSIs (5/20 patients, 25.0%) also had CoNS-positive PCR results. If the cutoff was neglected, true CoNS-BSIs were confirmed by PCR for 25 samples (83.3%; 17 patients, 85.0%; Table 3). Changes to an adequate antibiotic therapy due to Gram stain, identification and antibiogram took place in 7/20 patients with true CoNS-BSIs (35.0%). Corresponding PCR tests were only once positive and six times omitted by the cutoff. A detailed breakdown of all 37 CoNS-positive BC samples with their respective amplification results as well as their identification as true CoNS-BSI or contamination is demonstrated in Fig. 1. The median number of amplification cycles for samples accounting for true CoNS-BSIs was 21. Hence, the majority of PCR results for true BSIs occurred barely after the cutoff. Overall, the cutoff omitted 18 of 30 PCR results accounting for true CoNS-BSIs (60%). Sensitivity for the detection of samples with true CoNS-BSIs by PCR was 23.3% with the cutoff and 83.3% without the cutoff (Table 3). In contrast, supposed CoNS contamination in BCs was truly recognized by PCR in six of seven samples with the cutoff, yielding a specificity of 85.7%. If the cutoff was neglected, specificity decreased to 14.3%. Our data indicate that CoNS frequently account for true BSIs in this patient population. This is probably due to frequent CVC usage as well as extremely low leucocyte counts. Furthermore, CoNS isolates are frequently resistant to common antibiotic treatment regimens (de Naurois et al., 2010; Freifeld et al., 2011; Penack et al., 2011). Therefore, PCR detection of CoNS may be particularly important for early targeted treatment in these immunocompromised patients. Concerning CoNS identification by PCR, this study supports data from previous studies which showed poor sensitivity for Gram-
PCR detection rate
Patients
Samples
Of all CoNS detected by BC confirmed by PCR with cutoff confirmed by PCR without cutoff Of true CoNS-BSIs confirmed by PCR with cutoff confirmed by PCR without cutoff
n = 27 6 (22.2%) 17 (63.0%) n = 20 5 (25.0%) 17 (85.0%)
n = 37 8 (21.6%) 31 (83.8%) n = 30 7 (23.3%) 25 (83.3%)
positive organisms (0.39) in neutropenic patients (Paolucci et al., 2013), supposedly due to the established cutoff criteria (Varani et al., 2009; Lamoth et al., 2010; Maubon et al., 2010; Josefson et al., 2011). When the cutoff was neglected, sensitivity substantially rose while specificity considerably decreased, leading to the reporting of more contaminants. Our patient group had higher CoNS isolation rates (18.0% vs. 7.7%) and more true CoNS-BSIs (74.1% vs. 16.7%), compared to ICU patients from a study on postoperative pulmonary or abdominal sepsis (Tafelski et al., 2015). Hence, the introduced cutoff might be more appropriate for postoperative patients who appear to have fewer true CoNS-BSIs. In contrast, true CoNS-BSIs appear significantly more often in hematological patients with febrile neutropenia and regularly require a change in the initial empiric antimicrobial therapy. Therefore, decreasing specificity by eliminating the cutoff may just have to be the necessary compromise to detect clinically relevant infections and timely administer appropriate therapy in these high-risk patients. Nonetheless, in postoperative ICU patients, true CoNS-BSIs were also omitted due to the cutoff (16.7%, n = 1). Decreased test specificity might lead to an increased use of vancomycin. Therefore, physicians should perform a critical appraisal of patient’s data to decide on the relevance of the finding. In conclusion, our results suggest that the integrated cutoff omits numerous true CoNS-BSIs in hematological patients with neutropenia and possibly also in postoperative ICU patients. Hence, the applied cutoff value, which was based on data from infection-free individuals (Lehmann et al., 2008), is not appropriate for this high-risk patient population. Instead, all positive PCR results should be indicated and communicated to the clinician, optionally with a comment regarding potential contamination. Omitting the cutoff would allow this relevant information to become available for clinical decision making, next to other microbiological and laboratory data, clinical signs of infection, and local epidemiological patterns.
Conflict of interest YN, EAI, ST, IN, GS, WEB and KB received lecture fees from Pfizer Deutschland GmbH; ST, IN, WEB and KB received lecture fees from Roche Diagnostics. The randomized controlled study, which generated data used for this analysis, was partly supported by Roche Diagnostics GmbH, Mannheim, Germany (supplied the LightCycler® SeptiFast Kits), and Pfizer GmbH (provided restricted grant for study activities). No support has been provided for this study’s activities. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.diagmicrobio.2016.04.024.
Table 2 CoNS positive BCs according to the withdrawal site, patients and respective samples. CoNS positive BCs
Patients (n = 27)
Samples (n = 37)
Central and peripheral obtained Both positive Only peripheral positive Only central positive Only peripheral obtained, positive Only central obtained, positive
15 (55.5%) 10 (37.0%) 4 (14.8%) 1 (3.7%) 8 (29.6%) 4 (14.8%)
25 (67.6%) 20 (54.1%) 4 (10.8%) 1 (2.7%) 8 (21.6%) 4 (10.8%)
Acknowledgments This study is part of a joined project of the Molecular Diagnostics of Sepsis Study Group aimed to the improvement of sepsis management. The authors are grateful to all members of the study group including M. Deja (Berlin), C. Spies (Berlin), H. Bunzemeier (Münster), R. Grube (Munich), D. Voss (Münster), M. Wilke (Munich) and J. Schulze (Berlin).
Please cite this article as: Reers Y, et al, Multiplex PCR assay underreports true bloodstream infections with coagulase-negative staphylococci in hematological patients with ..., Diagn Microbiol Infect Dis (2016), http://dx.doi.org/10.1016/j.diagmicrobio.2016.04.024
Y. Reers et al. / Diagnostic Microbiology and Infectious Disease xxx (2016) xxx–xxx
3
Fig. 1. Corresponding PCR results of all samples with CoNS-positive blood cultures plotted by the number of amplification cycles until positivity and stratified for true bloodstream infections and contaminations (samples listed at 25 amplification cycles include all samples that were detected between 25 to 30 cycles; samples listed under n.d. = not detected, remained negative).
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Please cite this article as: Reers Y, et al, Multiplex PCR assay underreports true bloodstream infections with coagulase-negative staphylococci in hematological patients with ..., Diagn Microbiol Infect Dis (2016), http://dx.doi.org/10.1016/j.diagmicrobio.2016.04.024