Clinical Utility of the Time-to-Positivity/ Procalcitonin Ratio to Predict Bloodstream Infection Due to Coagulase-Negative Staphylococci

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1 Clinical Utility of the Time-to-Positivity/ Procalcitonin Ratio to Predict Bloodstream Infection Due to Coagulase-Negative Staphylococci Binghuai Lu, MS, 1* Lina Shi, BS, 1 Fengxia Zhu, BS, 1 Hua Zhao, BS 1 ABSTRACT Objective: To develop a novel clinical diagnostic method to distinguish bacteremia from blood contamination due to coagulase-negative staphylococci (CNS) by using the ratio of the time to positivity (TTP) of a blood culture with positive results to its serum procalcitonin (PCT) level. Methods: We retrospectively reviewed the clinical and microbiological records of 102 patients who had 1 or more blood cultures that tested positive for CNS from August 2007 through February Results: Receiver operating characteristic (ROC) curve analysis indicated that, at a cutoff of 1.24 ng per ml, 21.5 hours, and 22.5 (hours ml)/ng,the TTP to PCT ratio, PCT level, and TTP yielded the most favorable discrimination for bacteremia caused by CNS, with sensitivities of 85.7%, 78.6%, and 61.9%,respectively, and specificities of 80.0%, 78.3%, and 61.7%,respectively. The TTP to PCT ratio was the most accurate of the studied variables in predicting CNS-triggered bacteremia levels. Conclusion: The TTP to PCT ratio is a useful predictor to differentiate the culture samples that test positive, revealing the presence of CNS bacteremia, from those that are merely contaminated. Keywords: procalcitonin, time to positivity, bacteremia, TTP/PCT ratio, coagulase-negative staphylococci, blood culture contamination Bacteremia is one of the most common causes of mortality in hospitalized patients. Blood culture is currently the recommended method for diagnosing bacteremia. However, a positive result via blood culture does not always mean that bacteremia is present. For example, common skin flora, particularly coagulase-negative staphylococci (CNS), may contaminate blood cultures. It is important to determine DOI: /LM6Y0IALOGL4KAEC Abbreviations BSI, bloodstream infection; CNS, coagulase-negative staphylococci; TTP, time to positivity; PCT, procalcitonin; IQR, interquartile range; FA, fluorescent aerobic; FN, fluorescent anaerobic; WBCs, white blood cells; CRP, C-reactive protein; HAI, hospital-acquired infection; CDC, Centers for Disease Control and Prevention; SIRS, systemic inflammatory response syndrome; ROC, receiver operating characteristic; AUC, area under the curve; OR, odds ratio; CI, confidence interval; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus 1 Department of Laboratory Medicine, Civil Aviation General Hospital, Beijing, China *To whom correspondence should be addressed. zs25041@126.com whether a positive blood culture reflects a severe threat of sepsis, or occurs due to contaminant of the specimen by normal skin flora. 1 Incorrect interpretation of CNS-positive blood cultures results in unnecessary administration of antibiotics, increases healthcare costs, prolongs hospitalization, and promotes antibiotic resistance in microorganisms. 2 Certain factors help health care professionals determine whether CNS detected via blood culture represents true bacteremia or merely reveals contamination in the specimen. 1 For example, the time to positivity (TTP), which is mainly influenced by the species of bacteria and the original bacterial load, has been used in the interpretation of blood cultures that test positive for CNS. 3 Further, an elevated level of serum procalcitonin (PCT) has been associated with true CNS bacteremia. 4,5,6 Patients with CNS bacteremia have an elevated PCT level and a shortened TTP. We hypothesized that the ratio of TTP to PCT might be a more accurate predictor of CNS bacteremia, compared to PCT, or TTP alone. We retrospectively reviewed blood cultures from 102 individuals with positive results due to CNS to assess the clinical utility of the TTP to PCT ratio in predicting true bacteremia. Fall 2013 Volume 44, Number 4 Lab Medicine Science_Fall.indd 313

2 Materials and Methods Subjects We reviewed the clinical characteristics and laboratory data from patients with CNS-positive blood cultures at Civil Aviation General Hospital, a 550-bed tertiary teaching hospital in Beijing, China, from August 2007 through February A total of 102 hospitalized patients with suspected bacteremia (average age, 72 years, interquartile range [IQR], years) were enrolled in the study; all had 2 or more blood cultures drawn during a 48 hour period, at least 1 of which was positive for CNS. The exclusion criteria included inhalational injury, burn injury, pancreatitis, mechanical trauma, extensive surgery, C-cell carcinoma of the thyroid, and heatstroke, all of which cause elevations in serum PCT. 4 Laboratory Procedures We used the BacT/ALERT 3D blood culture microbial detection system (biomérieux SA, Marcy l Étoile, France) with activated charcoal medium, fluorescent aerobic (FA), and fluorescent anaerobic (FN). A set of blood cultures consisted of a total of 20 ml of blood; the blood was inoculated equally into culture media in aerobic and anaerobic tubes, 10 ml per tube. We then analyzed the following data: Demographic characteristics (age and sex), medical history, symptoms (fever, chill, and/or altered mental status), and test results (TTP and presence of microorganisms). We also examined the white blood cell (WBC) count and concentrations of C-reactive protein (CRP) and PCT; PCT was measured by an enzyme-linked fluorescence immunoassay on the mini VIDAS B.R.A.H.M.S (biomérieux SA) with a detection limit of 0.05 ng per ml. If PCT was undetectable, a value of 0.05 ng per ml was assigned to the specimen. Definitions Previous studies 6,7 have suggested that bacteremia involving common skin contaminants can be identified using certain criteria: (1) the patient has at least 1 of the following clinical signs or symptoms: temperature higher than 38 C, chills, or hypotension at the time of blood-culture sample collection; (2) clinical signs, symptoms, and positive laboratory results that are not related to an infection at another anatomical site; (3) positive cultures (we were only concerned with CNS in the current study) in 2 or more blood specimens drawn on separate occasions during a 48-hour period; and/or (4) diagnosis of CNS bacteremia by an infectious-disease specialist who is unaware of the PCT value or TTP of the sample in question. Herein, hospitalacquired infection (HAI) is defined according to the criteria of the Centers for Disease Control and Prevention (CDC). 7 Systemic inflammatory response syndrome (SIRS) is defined according to the criteria of the American College of Chest Physicians and the Society of Critical Care Medicine consensus. 8 TTP is defined as the time elapsed from the start of incubation to the detection of microbial growth. After blood collection, the tubes were immediately transferred to the microbiological laboratory. If multiple cultures tested positive, the shortest TTP among the cultures was used for data analysis. Statistical Analysis We evaluated differences among groups via the Mann- Whitney U test for continuous variables (expressed as the median [IQR]) and χ 2 or Fisher exact tests for categorical variables, as appropriate. Statistical analyses and data sorting were conducted using SPSS software, version 10.0 (SPSS Inc, Chicago, IL). To compare the diagnostic values of individual laboratory markers for predicting bacteremia, we performed receiver operating characteristic (ROC) curve analysis. We used the area under the curve (AUC) to assess the accuracy of laboratory parameters in differentiating bacteremia from contaminated specimens. We calculated the ROC using Medcalc for Windows (version ; Medcalc Software bvba, Ostend, Belgium). A P value of less than.05 was considered statistically significant. Results Of the 102 subjects whose blood cultures tested positive for CNS, 54 (52.9%) were men and 48 (47.1%) were women. The overall prevalence of true bacteremia caused by CNS was 41.2%. The 3 CNS subtypes most commonly detected were Staphylococcus epidermidis (79 [77.5%]), S. saprophyticus(12 [11.8%]) and S. warneri (5[4.9%]). Of the 42 cases of CNS, Staphylococcus subtypes S. epidermidis, S. saprophyticus, and S. Warneri accounted for 29 (69.0%), 7 (16.7%) and 2 (4.8%) cases of bacteremia, respectively. The comorbidities of the subjects in this study included respiratory tract infection (n = 49), diabetes mellitus (n = 50), cancer (n = 6), autoimmune diseases (n = 4), and renal failure (n = 33); none of these were statistically related to 314 Lab Medicine Fall 2013 Volume 44, Number Science_Fall.indd 314

3 Table 1. Baseline Characteristics of 102 Subjects With Blood Culture Positivity for CNS a Demographic Variable Total (n = 102) Bacteremia (n = 42) Sample Contamination (n = 60) P Value Median age, y (IQR) 72 (61-77) 72 (47-79) 72 (65-77) NS Sex (male/female) 54/48 25/17 29/31 NS HAI, no. (%) 57 (55.9) 23 (54.8) 34 (56.7) NS Clinical Characteristics Diabetes mellitus, no. (%) 50 (49.0) 20 (47.6) 30 (50.0) NS Cancer, no. (%) 6 (5.9) 2 (4.8) 4 (6.7) NS Respiratory tract infection, no. (%) 49(48.0) 18 (42.9) 31 (51.7) NS Renal failure, no. (%) 33 (32.4) 13 (31.0) 20 (33.3) NS Fever (>38 C), no. (%) 71 (69.6) 29 (69.0) 42 (70.0) NS Altered mental state,no. (%) 23 (22.5) 8 (19.0) 15 (25.0) NS Chills, no. (%) 17 (16.7) 11 (26.2) 6 (10.0).03 Death, no. (%) 14 (13.7) 9 (21.4) 5 (8.3) NS Hypotension, no. (%) 13 (12.7) 7 (16.7) 6 (10.0) NS Autoimmune diseases, no. (%) b 4 (3.9) 2 (4.8) 2 (3.3) NS Indwelling catheter, no. (%) 20 (19.6) 18 (42.9) 2 (3.3) <.001 SIRS, no. (%) 67 (65.7) 40 (95.2) 27 (45.0) <.001 Laboratory Parameters PCT (ng/ml) 0.86 ( ) 3.84 ( ) 0.43 ( ) <.001 TTP (h) ( ) ( ) 22.2 ( ) <.001 TTP/PCT ratio (h ml)/ng ( ) 4.59 ( ) 80.4 ( ) <.001 CRP (nmol/l) ( ) ( ) ( ) <.001 WBC count ( 10 9 /L) 10.2 ( ) 10.3 ( ) 10.3 ( ) NS Leucopenia (WBC < ),no. (%) 10 (9.8) 7 (16.7) 3 (5.0) NS Leucocytosis (WBC> ),no. (%) 44 (43.1) 27 (64.3) 17 (28.3) NS a Continuous data are expressed as the median (interquartile range). P >.05 was considered nonsignificant. b Rheumatoid arthritis (RA), systemic lupus erythematosus (SLE). CNS, coagulase-negative staphylococci; IQR, interquartile range; NS, nonsignificant; HAI, hospital-acquired infection; PCT, procalcitonin; TTP, time-to-positivity; CRP, C-reactive protein; WBC, white blood cell. the presence of bacteremia (all P >.05). The clinical symptoms of the study subjects, including fever, hypotension, and altered mental status, were also statistically unrelated to CNS positivity (P >.05). However, patients with an indwelling catheter or experiencing chills had a greater risk of bacteremia (indwelling catheters: odds ratio [OR], [95% confidence interval (CI), ]; chills: [ ]). Among the 102 subjects, there were 67 episodes of SIRS, of which 40 were confirmed to be bacteremia (95.2%). In samples from 33 patients with renal failure, the PCT values were significantly higher in the bacteremia group than in the contamination group (P <.01; Mann-Whitney U test, 3.38 [ ]vs0.54 [ ] ng/ml). A full listing of the baseline characteristics of the study subjects is shown in Table 1. Among 102 patients whose blood cultures tested positive due to CNS, ROC analysis demonstrated that the TTP to PCT ratio, PCT level, and TTP were all significant (all P <.05) for the prediction of bacteremia. The distributions of the TTP to PCT ratio in the subjects are presented in Figure 1. Table 2 includes accuracy data for the prediction of bacteremia via TTP, PCT level, and TTP to PCT ratio. The TTP to PCT ratio had a stronger predictive value than TTP and PCT by themselves (AUC: TTP/PCT ratio, 0.850[95%CI, ] vs TTP, 0.713[ ] and PCT, 0.812[ ], respectively; P <.05 for each). Pairwise comparison of the AUC of PCT level and TTP with the TTP to PCT ratio indicated significant differences (P =.02 and P =.01, respectively). Patients with CNS-caused bacteremia had shorter TTP, higher PCT levels, and a lower TTP to PCT ratio than patients without this condition. With a rising bacteremia rate, TTP decreased and PCT values increased, resulting in significant differences in the TTP to PCT ratio among the 2 groups. For the TTP to PCT ratio, the optimal threshold for sensitivity and specificity was 22.5 hours ml ng -1 for the prediction of true bacteremia. With this value, as many as 85.7% (36 of 42) and 80.0% (48 of 60) patients could be correctly classified as having confirmed bacteremia or blood-culture contamination, respectively. We present the accuracy indices for the prediction of bacteremia via TTP, PCT levels, and TTP to PCT ratios in Table 2 and Figure 2. Fall 2013 Volume 44, Number 4 Lab Medicine Science_Fall.indd 315

4 Figure 1 Box-plot diagram showing TTP/PCT ratios in true bacteremia and * in blood contamination due to CNS. Differences between the 2 groups are considered significant (P <.05) in comparison with the results of a nonparametric Mann-Whitney U test. TTP/PCT Ratio * * 0 Blood Contamination (n = 60) 1 Bacteremia (n = 42) Table 2. Accuracy of TTP, PCT Level, and TTP/PCT Ratio in 102 Patients With Suspected Bacteremia Due to CNS Variable TTP PCT TTP/PCT Ratio Most favorable cut-off value 21.5 h 1.24ng/mL 22.5 (h ml)/ng Sensitivity (95%CI) 61.9 ( ) 78.6 ( ) 85.7 ( ) Specificity, (95%CI) 61.7 ( ) 78.3 ( ) 80.0( ) Positive likelihood ratio Negative likelihood ratio NPV,% PPV,% Exclusion cases, no. (%) 37 (36.3%) 47 (46.1%) 48 (47.1%) Diagnostic cases, no. (%) 26 (25.5%) 33 (32.4%) 36 (35.3%) AUC (95%CI) 0.713( ) ( ) ( ) TTP, time-to-positivity; PCT, procalcitonin; NPV, negative predictive value; PPV, positive predictive value; AUC, area under [receiver operating characteristic] curve. Figure 2 Receiver operating characteristic (ROC) curve for PCT level, TTP, and TTP/PCT ratio, as used to differentiate bloodstream infections from incidents of sample contamination. The area under the curve was (95%CI, ) for TTP, ( ) for PCT level, and ( ) for TTP/PCT ratio. Sensitivity PCT TTP TTP/PCT Specificity 316 Lab Medicine Fall 2013 Volume 44, Number Science_Fall.indd 316

5 Discussion Blood culture is the most common method used to diagnose and treat patients with suspected bacteremia. Use of prosthetic devices and indwelling vascular catheters creates a risk for bacteremia caused by common skincolonizing bacteria, especially CNS. 9,10 Improvements in commercially available blood culture systems now make it possible, during blood sample collection, to detect small amounts of skin bacteria that contaminate blood specimens during collection. 11 Strategies to distinguish between positive blood culture results due to bacteremia and positive cultures resulting from contamination remain a subject of active research. Several factors should be considered: The species of organisms identified in the specimens, the number of blood cultures that test positive, and other clinical and laboratory clues. 11,12 TTP is a convenient way to evaluate initial bacterial load. Kassis et al 3 demonstrated, using the BACTEC 9240 blood culture system (Becton, Dickinson, and Company; Franklin Lakes, NJ) that TTP could be used to differentiate CNS bacteremia (TTP 16 hours) from contaminated specimens using a TTP threshold of 20 hours). 3 Our results indicated that a TTP of 21.5 hours or longer yields the most favorable sensitivity (61.9%) and specificity (61.7%) for predicting true bacteremia. The culture differences in media and supplements in various blood culture systems may explain the differences in TTP thresholds. For example, Viganò et al 13 found that the TTP of CNS as detected via the BACTEC 9240 was significantly shorter than when the BacT/ALERT (Becton, Dickinson, and Company) system was used; (15 strains of S. epidermidis; BACTEC, 17.1 hours vs BacT/ ALERT, 23.3 hours). Mirrett et al 14 demonstrated that the TTP of CNS-positive cultures, using the BACTEC 9240 system, was shorter than cultures using the BacT/ALERT but the differences were not significant (for 66 cases of CNS, BACTEC, 19 [3-68] hours vs BacT/ALERT, 21 [4-55 ]hours). However, PCT is also a marker of inflammation or infection 4 and elevated PCT levels are highly suggestive of bacteremia. Schuetz et al 6 demonstrated that PCT values can be used to discriminate between blood-sample contamination and CNS bacteremia. We also confirmed the predictive value of PCT in our study: If the PCT level was elevated to 1.24 ng per ml in a patient with a CNS-positive blood culture, the PCT level had a specificity of 78.3% and a sensitivity of 78.6% to predict true bacteremia. Because shortened TTP and an elevated PCT level are both associated with a higher possibility of true bacteremia caused by CNS, the TTP to PCT ratio might be a more favorable predictor for CNS bacteremia. Our study demonstrates that a CNS-positive blood culture with a TTP to PCT ratio of less than or equal to 22.5 (h ml)/ng is highly associated with true bacteremia (specificity of 80.0% and sensitivity of 85.7%). ROC analysis indicate that the TTP to PCT ratio has significantly higher predictive value than TTP or PCT by itself. Our study is limited by several factors. First, the sample size is small. Although a small sample size is common in this type of study, 6 the conclusions should be interpreted with caution. In our hospital, blood cultures are often not performed according to strict guidelines. Most patients suspected of harboring bacteremia have only 1 blood culture collected; therefore, these individuals were not included in our study, per our exclusion criteria. Second, no widely accepted criteria are available to distinguish true pathogens from contaminants. 3,6 In our study, we define the bacteremia according to literature reports including clinical signs or symptoms and a confirmed diagnosis from an experienced physician. 6,7 Third, conditions that elevate the PCT level, such as inhalational injury, burn injury, pancreatitis, mechanical trauma, and extensive surgery; were excluded from thisstudy. 4 Steinbach et al 15 demonstrated that in patients with acute and chronic renal insufficiency, PCT levels were not significantly affected by renal disease but were markedly elevated in the presence of infections. We obtained similar results in our study. Regarding autoimmune diseases, we observed 3 cases of rheumatoid arthritis (RA) and 1 of systemic lupus erythematosus (SLE), distributed in the contamination and bacteremia groups. The patient with SLE was a 35-year-old woman with a high PCT level (1.52ng/mL); her blood sample was categorized in the contaminated-sample group because we discovered S. epidermidisin only 1 aerobic culture. Further, 2 of 3 patients with rheumatoid arthritis (RA) in the bacteremia group had elevated PCT levels (3.21 and 2.98 ng/ ml); the patient with RA in the contaminated-sample group had a normal PCT level (<0.05ng/mL). Joo et al 16 found that patients with SLE, RA, and other autoimmune diseases had higher PCT levels in the in the presence of bacteremia than those in a disease-flare group, for example, those with a worsening of SLW, RA, and other autoimmune diseases that, if persistent, would in most cases lead to initiation or change of therapy. Our analysis yielded comparable results. Conclusion Our study has potential application to the clinical interpretation of blood culture results and the detection of CNS Fall 2013 Volume 44, Number 4 Lab Medicine Science_Fall.indd 317

6 bacteremia. Recently, direct identification of pathogens in positive blood cultures without previous isolation of the organism has been reported. 17 A positive blood culture result can quickly be interpreted as stemming from the presence of CNS. However, when a positive CNS culture result is detected in a single blood culture, as is often the case with pediatric patients, or in cases where the earliest culture with a positive result reveals CNS when 2 or more blood cultures have been collected, healthcare professionals are still at a loss to determine is clinical significance. 1,11 We found that TTP to PCT ratio is a useful measurement to predict true bacteremia and to rule out contamination. LM Acknowledgments This study was supported by Civil Aviation General Hospital Research Funds (grant no ). We thank Yongzhong Ning, MD, for his helpful suggestions on clinical aspects of the study. References To read this article online, scan the QR code, ascpjournals.org/content/44/4/313. full.pdf+html 1. Hall KK, Lyman JA. Updated review of blood culture contamination. Clin Microbiol Rev. 2006;19: Souvenir D, Anderson DE Jr, Palpant S, et al. Blood cultures positive for coagulase-negative staphylococci: antisepsis, pseudobacteremia, and therapy of patients. J Clin Microbiol. 1998; 36: Kassis C, Rangaraj G, Jiang Y, Hachem RY, Raad I. Differentiating culture samples representing coagulase-negative staphylococcal bacteremia from those representing contamination by use of timeto-positivity and quantitative blood culture methods. J Clin Microbiol. 2009;47: Becker KL, Snider R, Nylen ES. Procalcitonin assay in systemic inflammation, infection, and sepsis: clinical utility and limitations. Crit Care Med. 2008;36: Charles PE, Ladoire S, Aho S, et al. Serum procalcitonin elevation in critically ill patients at the onset of bacteremia caused by either Gram negative or Gram positive bacteria. BMC Infect Dis. 2008;8: Schuetz P, Mueller B, Trampuz A. Serum procalcitonin for discrimination of blood contamination from bloodstream infection due to coagulase-negative staphylococci. Infection. 2007;35: Horan TC, Andrus M, Dudeck MA. CDC/NHSN surveillance definition of health care associated infection and criteria for specific types of infections in the acute care setting. Am J Infect Control. 2008;36: Levy MM, Fink MP, Marshall JC, et al SCCM/ESICM/ACCP/ ATS/SIS International Sepsis Definitions Conference. Intensive Care Med. 2003;29: Schifman RB, Strand CL, Meier FA, Howanitz PJ. Blood culture contamination: a College of American Pathologists Q-Probes study involving 640 institutions and specimens from adult patients. Arch Pathol Lab Med. 1998;122: Wisplinghoff H, Seifert H, Wenzel RP, Edmond MB. Current trends in the epidemiology of nosocomial bloodstream infections in patients with hematological malignancies and solid neoplasms in hospitals in the United States. Clin Infect Dis. 2003;36: Weinstein MP. Blood culture contamination: persisting problems and partial progress. J Clin Microbiol. 2003;41: Mirrett S, Weinstein MP, Reimer LG, Wilson ML, Reller LB. Relevance of the number of positive bottles in determining clinical significance of coagulase-negative staphylococci in blood cultures. J Clin Microbiol. 2001;39: Viganò EF, Vasconi E, Agrappi C, Clerici P. Use of simulated blood cultures for time to detection comparison between BacT/ALERT and BACTEC9240 blood culture systems. Diagn Microbiol Infect Dis. 2002;44: Mirrett S, Reller LB, Petti CA, et al. Controlled clinical comparison of BacT/ALERT standard aerobic medium with BACTEC standard aerobic medium for culturing blood. J Clin Microbiol. 2003;41: Steinbach G, Bölke E, Grünert A, Störck M, Orth K. Procalcitonin in patients with acute and chronic renal insufficiency [in German]. Wien Klin Wochenschr. 2004;116: Joo K, Park W, Lim MJ, Kwon SR, Yoon J. Serum procalcitonin for differentiating bacterial infection from disease flares in patients with autoimmune diseases. J Korean Med Sci. 2011;26: Christner M, Rohde H, Wolters M, Sobottka I, Wegscheider K, Aepfelbacher M. Rapid identification of bacteria from positive blood culture bottles by use of matrix-assisted laser desorption-ionization time of flight mass spectrometry fingerprinting. J Clin Microbiol. 2010;48: Lab Medicine Fall 2013 Volume 44, Number Science_Fall.indd 318