Journal of Critical Care xxx (2014) xxx–xxx
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Procalcitonin as a rapid diagnostic biomarker to differentiate between culture-negative bacterial sepsis and systemic inflammatory response syndrome: A prospective, observational, cohort study☆ Dimple Anand, MSc a, Sabari Das, PhD a, Seema Bhargava, MD, PhD a, Lalit Mohan Srivastava, PhD a, Ashish Garg, MD b, Niraj Tyagi, MD b, Saurabh Taneja, MD b, Sumit Ray, MD b,⁎ a b
Department of Biochemistry, Sir Ganga Ram Hospital, New Delhi, 110060, India Department of Critical Care and Emergency Medicine, Sir Ganga Ram Hospital, New Delhi, 110060, India
a r t i c l e
i n f o
Keywords: Culture negative Sepsis Procalcitonin (PCT) Interleukin 6 (IL-6) Systemic inflammatory response syndrome (SIRS) Negative predictive value (NPV)
a b s t r a c t Purpose: Differentiation between culture-negative sepsis and noninfectious systemic inflammatory response syndrome (SIRS) remains a diagnostic challenge for clinicians, both conditions having similar clinical presentations. Therefore, a swift accurate diagnostic tool, which helps differentiate these 2 conditions would immensely aid appropriate therapeutic continuum. This prospective study was conducted to evaluate the potential diagnostic role of biomarkers, procalcitonin (PCT) and interleukin 6 (IL-6), in culture-negative sepsis patients. Methods: Enrolled patients (208) included 46 noninfectious SIRS, 90 culture-negative sepsis, and 72 culturepositive sepsis. Culture, PCT, and IL-6 estimations were performed on day 1 of intensive care unit admission. Results: Procalcitonin and IL-6 levels were significantly higher (P b .001) in both culture-negative and culturepositive groups as compared with SIRS group. Procalcitonin was a better predictor of sepsis in both culturenegative (area under curves 0.892 vs 0.636) and culture-positive (area under curves 0.959 vs 0.784) groups as compared with IL-6. In culture-negative group, the best cutoff point for PCT was at 1.43 ng/mL (92% sensitivity; 83% negative predictive value), best cutoff point for IL-6 was at 219.85 pg/mL (47% sensitivity and 42% negative predictive value). Conclusions: Procalcitonin can accurately differentiate culture-negative sepsis from noninfectious SIRS and thereby contribute to early diagnosis and effective management of these conditions. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Across the world, various epidemiological studies have revealed an increase in the incidence of sepsis during the last decade [1-3]. In the United States, the hospitalization rate due to sepsis as principal diagnosis has increased by more than 2-fold, extending from 11.6 to 24.0 per 10 000 population between the years 2001 and 2008 [4]. In recent years, improved sepsis management has decreased the case fatality rate in sepsis patients, but owing to the increase in number of cases, the overall mortality is on the rise [5]. Conceptually, sepsis is defined as a systemic inflammatory host response to infection and characterized by alterations in physiologic parameters such as temperature, heart rate, respiratory rate, etc [6]. In clinical practice, such changes in physiologic parameters are nonspecific, and may manifest in the other noninfectious systemic inflammatory
☆ We are grateful to the Indian Council of Medical Research, New Delhi, India, for financial support through project no. 52/10/08. ⁎ Corresponding author at: Department of Critical Care and Emergency Medicine, Sir Ganga Ram Hospital, New Delhi, 110060, India. Tel.: +91 98 10614433; fax: +91 11 45088437. E-mail address: [email protected] (S. Ray).
response (SIRS) conditions such as trauma, burns, pancreatitis, etc [7,8]. According to the currently used definition of sepsis, the presence of documented or suspected infection is the key demarcating characteristic between sepsis and other noninfectious SIRS conditions. The major challenge, however, is to diagnose infection in sepsis patients. Although, microbiological culture is considered as the criterion standard for the diagnosis of infection, the major concern is lack of sensitivity, specificity, and delay in reporting of culture results. The Sepsis Occurrence in Acutely Ill Patients study revealed that approximately 40% of the sepsis patients remain culture negative [9]. The Extended Prevalence of Infection in the intensive care unit (ICU) study has also reported a prevalence of culture-negative infection at 30% of overall infections [10]. Most of these clinically suspected sepsis patients who remain culture-negative pose a challenge in the decision making of antibiotic administration. Hence, it is of utmost importance to differentiate these patients from those with noninfectious SIRS, as both disease conditions require different therapeutic regimens. According to the Surviving Sepsis Campaign recommendation, antibiotics should be administered within 1 hour of septic shock onset [11]. Every hour of delay in antibiotic administration demonstrated an increase in mortality of 7.6% in septic shock [12]. Therefore, early empirical antibiotic administration is crucial for improving outcome in sepsis. This may get delayed due to misdiagnosis of culture-negative sepsis
http://dx.doi.org/10.1016/j.jcrc.2014.08.017 0883-9441/© 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Anand D, et al, Procalcitonin as a rapid diagnostic biomarker to differentiate between culture-negative bacterial sepsis and systemic inflammatory..., J Crit Care (2014), http://dx.doi.org/10.1016/j.jcrc.2014.08.017
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patients as SIRS. On the other hand, noninfectious SIRS patients misdiagnosed as sepsis might get inappropriately treated with broad spectrum antibiotics, which may contribute to the emergence of antibiotic resistance—a growing health problem [13]. Thus, a specific biomarker with high sensitivity and negative predictive value (NPV), which can diagnose or exclude suspected sepsis early (within 1 hour of sampling) in culture-negative patients would be of vital importance for timely institution of antibiotic treatment or avoidance thereof. Inflammatory mediators in the serum, which are altered in septic conditions, have been studied for their potential in diagnosis or prognosis of sepsis. Among these, procalcitonin (PCT) and interleukin 6 (IL-6) have been investigated widely in relation to severity and organ failure in sepsis patients [14-16]. Various studies, including several metaanalyses have assessed the usefulness of PCT as a diagnostic tool in differentiating sepsis from noninfectious SIRS [17-22]. Although PCT has been regarded as a sensitive marker for the prediction of sepsis in culture-positive patients, its role in culture-negative sepsis is scarcely known. To the best of our knowledge, no study in the past has focused on the diagnostic accuracy of PCT and IL-6 in culture-negative sepsis, and therefore, no optimal cutoff value has been proposed for differentiating culture-negative sepsis from noninfectious SIRS. The current prospective observational study sought to investigate the diagnostic accuracy of serum PCT and IL-6 to differentiate between culturenegative sepsis and noninfectious SIRS. We also studied their diagnostic efficacy in culture-positive sepsis. 2. Materials and methods The study protocol was approved by institutional ethics committee; and the prospective, observational, single-center study was conducted at a 675-bedded superspeciality hospital in New Delhi (India) over a period of 1 year and 5 months (January 2013 to May 2014). Informed consent was taken from each patient or their next of kin if patient was unconscious or not in a state to give consent. Our inclusion criteria were as follows: adult patients (aged N 18 years) admitted from the community to the ICU were screened; and patients diagnosed with noninfectious SIRS, sepsis, severe sepsis, or septic shock (according to the established consensus sepsis definition) were enrolled in this study [23]. Exclusion criteria were patients who had received prior antibiotics (last 3 months), transferred from other ICUs; having conditions, which were considered lethal in the next 24 hours; postoperative; immunocompromised; and with malignancy were not included in the study. Patients with bilateral pneumonia (suspected viral infection) and diagnosed tropical diseases such as malaria, dengue, Leptospira, and rickettesiae were also excluded. Patient's demographics, principal diagnosis, and all clinical parameters were recorded at the time of enrollment. Initial severity of illness was determined using the Acute Physiology and Chronic Health evaluation score (APACHE II) at 24 hours in all patients. Patients were followed up for 28 days to observe survival or mortality. 2.1. Sample collection and processing Blood samples were assayed for microbiological culture and biomarkers on the day of ICU admission. Two sets of blood cultures, urine culture, sputum culture (in nonintubated patients), endotracheal culture (in intubated patients), and high vaginal swab culture (where puerperal sepsis was suspected) were sent before the commencement of antibiotic therapy. For blood cultures, samples were obtained in both aerobic and anaerobic BacT/Alert bottles and performed by BacT/Alert method (BioMerieux, Marcy l' Etiole, France). Urine and sputum specimen were procured in sterile containers. For endotracheal and vaginal swab cultures, specimens were collected in mucus trap and swab container, respectively. Positive cultures were further processed for the identification of organisms using standard laboratory methods.
Blood samples for PCT and IL-6 estimation were obtained in serumevacuated separator tubes and centrifuged for the separation of serum and processed on the same day. Procalcitonin estimation was done with timeresolved amplified cryptate emission technology by measuring the signal that is emitted from an immunocomplex with time delay (Kryptor PCT; BRAHMS, Henningsdorf, Germany). The assay time of this estimation takes less than half an hour. Interleukin 6 estimation was done by solid-phase Chemiluminescent Access Immunoassay System (Beckman Coulter, Inc, Brea, California, USA). 2.2. Classification of patient groups Enrolled patients were classified into SIRS and suspected sepsis at the time of enrollment based on clinical presentation by 4 clinicians (also coauthors). Culture-negative and culture-positive groups were defined, once the microbiological results were available. (i) Group I (noninfectious systemic inflammatory response group): Included patients with 2 or more signs of SIRS with recent onset pancreatitis and trauma (within 24 hours) without any evidence of infection. (ii) Group II (culture-negative sepsis group): Patients with 2 or more signs of SIRS and clinical suspicion of infection with negative culture results. The diagnosis of bacterial infection in these patients was done based on findings of a clinical focus of infection. Intraabdominal infection was diagnosed in case of exudative ascitic tap with increased polymorphonuclear cell count. Bacterial pneumonia was confirmed by x-ray showing lobar infiltrate. Urosepsis was suspected with signs of urinary tract infection and with a raised leukocyte count in the urine (N10 pus cells/high-power field) and signs of pyelonephritis by ultrasonography. Cellulitis was diagnosed by the skin signs, that is, lesions. Puerperal sepsis was suspected in peripartum patients with signs of pelvic pain and abnormal or foul smelling vaginal discharge (presence of pus). (iii) Group III (culture-positive sepsis group): This group consisted of the patients who had microbiologically documented source of infection with 2 or more of the SIRS criteria. A blood culture was considered positive if any significant pathogenic bacterial organism was grown from twin cultures taken from different sites. Respiratory secretions were considered positive for infection if many polymorpohonuclear cells were present along with colony count more than 10 5. Urine culture was considered positive if there were more than 10 pus cells/highpower field, along with single organism cultured with more than 10 5 colony-forming units/mL. 2.3. Statistical analysis Statistical analyses were performed using SPSS, version 17.0 (SPSS, Chicago, IL). Continuous variables are presented as mean (range) or median (interquartile range) as appropriate. Categorical variables are presented as absolute numbers and percentage. For multiple group comparisons, one-way analysis of variance with post hoc comparison or nonparametric Kruskal-Wallis with Mann-Whitney U test was used. Categorical variables were analyzed using the χ2 statistic. The diagnostic performance of biomarkers was demonstrated with receiver operating characteristics (ROC) analysis. The Youden index with highest sum of sensitivity and specificity was used to determine the optimal cutoff values [24]. Positive predictive value, NPV, positive likelihood ratio, and negative likelihood ratios were calculated. For all statistical tests, a P value b .05 was considered significant. 3. Results The study was conducted in a total of 208 patients prospectively. As defined in methodology, enrolled patients were classified into following
Please cite this article as: Anand D, et al, Procalcitonin as a rapid diagnostic biomarker to differentiate between culture-negative bacterial sepsis and systemic inflammatory..., J Crit Care (2014), http://dx.doi.org/10.1016/j.jcrc.2014.08.017
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Table 1 Demographics and outcome of the study patient groups Characteristics
Group I (SIRS)
Group II (culture negative)
Group III (culture positive)
P
No. of patients Sex, M/F Age (y), Mean (range) Mortality, n (%) APACHE II, Mean (range)
46 33/13 42 (19-80) 8 (17.3%) 15.9 (4-46)
90 49/41 53.7 (19-84) 17 (18.8%) 22.9 (8-42)
72 40/32 54 (17-85) 21 (29.1%) 25.8 (13-56)
– NSa ,b .001⁎ NSa ,b b.001⁎
Abbreviations: M, male; F, female. Data are presented as mean (range), n (percentage) as appropriate. Significance testing was performed by: a χ2 test. b One-way analysis of variance with Bonferroni correction. ⁎ P b .005 was considered highly significant.
3 groups. Group I, noninfectious SIRS (n = 46); group II, culturenegative sepsis (n = 90); and group III, culture-positive sepsis (n = 72). Of 162 clinically suspected bacterial sepsis patients, 55.5% (90/ 162) were culture negative and 44.5% (72/162) were culture positive. The demographic profile and outcome of different patient groups are shown in Table 1.
3.1. Characteristics of the enrolled subjects No statistically significant sex difference was observed between the study groups. Mean age was significantly lower in SIRS as compared with culture-negative and culture-positive groups (P = .004 and P = .003, respectively). Median APACHE II score was significantly different in all 3 groups, being highest (25) in group III, which also had the highest mortality (29.1%). Although overall mortality was 22.1%, there was no statistically significant difference observed between the groups (Table 1).
3.2. Source of infection The foci of clinically suspected bacterial infection in culture-negative sepsis patients were predominantly abdominal (43%) and respiratory (37%) (Fig.1A). In culture-positive patients, the most common site of infection found was blood stream (43%), followed by respiratory tract (22%) (Fig.1B). Of 72 patients enrolled in culture-positive group, 84.7% (61/72) had Gram-negative and 15.3% (11/72) had Gram-positive infections. Fiftysix patients in Gram-negative group had bacilli (Escherichia coli in 40 cases, Klebsiella pneumoniae in 11 cases, and Pseudomonas aeruginosa in 5 cases). Among Gram-positive, 6 had Staphylococcus aureus, 2 had Streptococcus pneumoniae, and 3 had Enterococcus faecalis. Although levels of PCT were higher in Gram-positive group and levels of IL-6 were higher in Gram-negative group, this difference between 2 groups was not statistically significant (P = .748 and P = .937, respectively). Median PCT levels in Gram-positive and Gram-negative groups were 26 ng/mL (Interquartile range [IQR], 6.94-54) vs 21.6 ng/mL (IQR, 5.0-94), whereas IL-6 levels were 649 pg/mL (IQR, 216-1594) vs 959 pg/mL (IQR, 120.9-1582).
3.3. Serum levels of PCT and IL-6 in study groups Procalcitonin and IL-6 levels were measured and analyzed in all enrolled patients on the day of admission. In culture-negative patients, the median PCT levels were significantly high (P b .001) approximately 8fold than noninfectious SIRS patients, whereas IL-6 levels showed only 1.7-fold increase, which was also statistically significant (P = .01) in the culture-negative patients as compared with SIRS. However, levels of both the parameters were highest in culture-positive patients (Fig. 2 A and B). In trauma patients (n = 15), the median PCT and IL-6 levels were 0.76 ng/mL and 65.6 ng/mL, respectively similar to those found in remaining patients of SIRS group (P = .453 and .223 for PCT and IL-6, respectively). 3.4. Discriminative performance of PCT and IL-6 Culture negative vs SIRS: ROC curve analysis for PCT and IL-6 between culture-negative and SIRS groups for the prediction of systemic infection were performed (Fig. 3A). The area under curve (AUCs) for PCT and IL-6 were 0.892 (confidence interval [CI], 0.836-0.947; P = .028) and 0.636 (CI, 0.540-0.731; P = .049), respectively. For PCT, the optimal cutoff point was 1.43 ng/mL with a sensitivity of 92.2% and NPV of 82.5%. The best cutoff point for IL-6 was 219.85 pg/mL with a sensitivity of 47% and NPV of 41.5%, which was much lower than PCT (Table 2). Culture positive vs SIRS: Similarly, ROC analysis to distinguish between patients with noninfectious SIRS and culture-positive sepsis, the PCT had an AUC of 0.959 (CI, 0.920-0.988; P = .017), and IL-6 had an AUC of 0.784 (CI, 0.701-0.867; P = .042) (Fig. 3B). At the optimum cutoff level of 2.49 ng/mL, PCT demonstrated a sensitivity of 94.4% and NPV of 91.5%, respectively. Interleukin 6 demonstrated a sensitivity of 63.9% and NPV of 63.7% at the optimum cutoff of 423.5 pg/mL (Table 2). 4. Discussion To overcome the inherent inadequacies of culture as a diagnostic tool of differentiation between SIRS and sepsis, PCT and IL-6 have been studied and found useful for early diagnosis of sepsis [25,26]. Yet, one subset of patients—the culture-negative sepsis patient—still
Fig. 1. A, Suspected source of infection in culture-negative sepsis patients. B, Site of infection in culture-positive sepsis patients.
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Fig. 2. A, Graphical representation of median (IQR) levels of PCT in 3 study groups. B, Graphical representation of median (IQR) levels of IL-6 in 3 study groups.
remains difficult to diagnose. Presently, they are diagnosed primarily by clinical acumen—a very subjective method. The current study evaluates the ability of serum PCT and IL-6 in differentiating suspected culturenegative sepsis from noninfectious SIRS. Procalcitonin is a peptide precursor of hormone calcitonin, and it consists of 116 amino acids (molecular weight, 13 kd) [27]. Normally, PCT is synthesized by the C cells of thyroid gland, and levels are undetectable in healthy individuals. Abnormally raised PCT levels were first reported by Assicot et al [28] in children with bacterial infections. High PCT levels are detectable in plasma 2 to 3 hours after endotoxin injection in healthy volunteers; half-life of PCT in serum is 25 to 30 hours [29]. Cytokine IL-6 is a mediator of acute phase response, induced by tumor necrosis factor α and produced by various cell types such as monocytes, macrophages, and endothelial cells [15]. Literature has acknowledged IL-6 as a marker to predict sepsis severity and clinical outcome; however, it is also known to be relatively nonspecific and is elevated under a variety of conditions [30]. Review of literature revealed only 1 epidemiological study pertaining to differentiation of culture-positive from culture-negative sepsis with no conclusion regarding the differentiation of culturenegative sepsis from SIRS [31]. However, the latter differentiation is of utmost clinical importance, as a sepsis patient's course depends on timely institution of antibiotics and in SIRS patients' inappropriate antibiotic therapy may lead to emergence of antibiotic resistance. In the ICU setting, APACHE II score is an indicator of severity and mortality risk in critically ill patients [32]. In the current study, we observed that
APACHE scores were significantly higher in culture positive as compared with culture negative and in culture negative as compared with SIRS. The mortality rates (least in SIRS and maximum in culture positive) were not significantly different in the 3 groups. This was similar to the results of Phua et al [31], who reported lower mortality in culture-negative patients as compared with culture-positive patients. Furthermore, our study showed that culture-positive patients had predominantly (84.7%) Gram-negative infections corroborating previous reports [10]. However, the predominant sources of infection in culture-negative patients in our study were abdominal (43%) followed by respiratory (37%), which were in sharp contrast to the study by Phua et al [31] in which respiratory infections accounted for 75% of culture-negative patients. An interesting observation of our study was that PCT and IL-6 gave a graded differentiation between the 3 groups of patients, being highest in culture positive and lowest in SIRS. The use of these 2 markers in diagnosis of culture-negative sepsis would depend on the sensitivity and NPV of these biomarkers. The ROC analysis between culture-negative and SIRS patients demonstrated a superior AUC for PCT in comparison with IL-6. The sensitivity of PCT at the optimum cutoff value of 1.43 ng/mL was 92.2% with an NPV of 82.5%. The same for IL-6 were also much lower, 47% and 41.5%, respectively at the optimal cutoff value of 219.85 pg/mL. Higher sensitivity and NPV of PCT were also observed in culture-positive patients as seen in previous studies [33,34]. These biomarker characteristics clearly indicate the superiority of PCT over IL-6 in its ability to differentiate culture-negative sepsis from SIRS.
Fig. 3. A, Receiver operating characteristic curve for PCT (dashed line) and IL-6 (dotted line) between culture-negative and SIRS groups. B, Receiver operating characteristic curve for PCT (dashed line) and IL-6 (dotted line) between culture-positive and SIRS groups.
Please cite this article as: Anand D, et al, Procalcitonin as a rapid diagnostic biomarker to differentiate between culture-negative bacterial sepsis and systemic inflammatory..., J Crit Care (2014), http://dx.doi.org/10.1016/j.jcrc.2014.08.017
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Table 2 Diagnostic performance of PCT and IL-6 in culture-negative and culture-positive patients in comparison with SIRS Cutoffa
Sensitivity, %
Specificity, %
Positive predictive value (%)
NPV (%)
Positive likelihood ratio
Negative likelihood ratio
Culture negative in comparison with SIRS PCT 1.43 ng/mL 0.892 (0.836-0.947) IL-6 219.85 pg/mL 0.636 (0.540-0.731)
92.2 47
72 73
86.5 78
82.5 41.5
3.53 1.79
0.11 0.72
Culture positive in comparison with SIRS PCT 2.49 ng/mL 0.959 (0.926-0.991) IL-6 423.5 pg/mL 0.784 (0.701-0.867)
94.4 63.9
87 89
90.5 88.4
91.5 63.7
6.74 5.4
0.07 0.4
Variable
AUC (95% CI)
a Sensitivity, specificity, predictive values, and likelihood ratios were calculated for the PCT and IL-6 at the cutoff, which demonstrated the best discrimination of the study groups as derived from the ROC curves.
Key findings of the present study were that, in culture-negative sepsis patients, PCT and IL-6 levels were significantly higher than in noninfectious SIRS. In ROC curve analyses of PCT and IL-6 for the prediction of systemic infection, optimum cutoff point of PCT was associated with higher sensitivity, specificity, predictive values, and likelihood ratio values than IL-6. Determination of appropriate and timely treatment in sepsis patients depends on a swift diagnostic method. Procalcitonin analysis is simple to perform, done at most modern health care establishments, and results are rapidly available. The quantitative estimation of PCT using kryptate emission technology can be performed in less than half an hour. The superior sensitivity and NPV as compared with IL-6 and the ease of performance contribute to the early, accurate, and rapid differentiation of sepsis, both culture negative and positive, much before the availability of culture results. The strength of our study is that we have included only patients who were admitted from the community with no exposure to a health care facility in the last 3 months, which could have been a confounding factor. Patients with nonbacterial sepsis like viral infections (especially culture-negative patients with bilateral pneumonia) and tropical diseases were also excluded from the study. All the patients enrolled in culture-negative group had a clear source of suspected infection. Moreover, we obtained the samples before the initiation of antibiotic therapy and processed on the same day. We included patients with trauma only in first 24 hours and recent onset pancreatitis in SIRS group to avoid the possibility of infection. As pointed out by Mimoz et al [35] and Reinhart et al [36], postsurgical and trauma patients could also confound our results. But in the present study, we have not included any surgical patients to avoid confounding and had only 15 trauma patients in the SIRS group. These latter subjects exhibited PCT and IL-6 levels similar to those observed in remaining patients in the SIRS group and hence did not confound our results. The main limitation of this study is that it is a single-center study. A multicenter study may be required to extrapolate our findings to other clinical settings. Although we attempted to exclude all viral infections, this was based primarily on clinical acumen. Therefore, PCT should be interpreted in context with information from careful medical history and physical examination; continuing reevaluation during the course of disease is advisable. Thus, our study establishes the possible role of surrogate markers to simplify the challenge of identification of culture-negative sepsis patients. These findings could probably be extrapolated to diagnose secondary infection in-hospital settings.
5. Conclusions Our study indicates that PCT has the potential to aid in rapid differentiation of culture-negative sepsis from SIRS at a cutoff of 1.43 ng/mL (sensitivity, 92%; NPV, 83%), thus helping initiate timely antibiotic therapy, whereas reducing inappropriate use of antibiotics in critical care settings. However, because the number of patients in our study was 208, further multicentric studies would be required to substantiate our findings.
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Procalcitonin as a rapid diagnostic biomarker to differentiate between culture-negative bacterial sepsis and systemic inflammatory response syndrome: A prospective, observational, cohort study☆ Dimple Anand, MSc a, Sabari Das, PhD a, Seema Bhargava, MD, PhD a, Lalit Mohan Srivastava, PhD a, Ashish Garg, MD b, Niraj Tyagi, MD b, Saurabh Taneja, MD b, Sumit Ray, MD b,⁎ a b
Department of Biochemistry, Sir Ganga Ram Hospital, New Delhi, 110060, India Department of Critical Care and Emergency Medicine, Sir Ganga Ram Hospital, New Delhi, 110060, India
a r t i c l e
i n f o
Keywords: Culture negative Sepsis Procalcitonin (PCT) Interleukin 6 (IL-6) Systemic inflammatory response syndrome (SIRS) Negative predictive value (NPV)
a b s t r a c t Purpose: Differentiation between culture-negative sepsis and noninfectious systemic inflammatory response syndrome (SIRS) remains a diagnostic challenge for clinicians, both conditions having similar clinical presentations. Therefore, a swift accurate diagnostic tool, which helps differentiate these 2 conditions would immensely aid appropriate therapeutic continuum. This prospective study was conducted to evaluate the potential diagnostic role of biomarkers, procalcitonin (PCT) and interleukin 6 (IL-6), in culture-negative sepsis patients. Methods: Enrolled patients (208) included 46 noninfectious SIRS, 90 culture-negative sepsis, and 72 culturepositive sepsis. Culture, PCT, and IL-6 estimations were performed on day 1 of intensive care unit admission. Results: Procalcitonin and IL-6 levels were significantly higher (P b .001) in both culture-negative and culturepositive groups as compared with SIRS group. Procalcitonin was a better predictor of sepsis in both culturenegative (area under curves 0.892 vs 0.636) and culture-positive (area under curves 0.959 vs 0.784) groups as compared with IL-6. In culture-negative group, the best cutoff point for PCT was at 1.43 ng/mL (92% sensitivity; 83% negative predictive value), best cutoff point for IL-6 was at 219.85 pg/mL (47% sensitivity and 42% negative predictive value). Conclusions: Procalcitonin can accurately differentiate culture-negative sepsis from noninfectious SIRS and thereby contribute to early diagnosis and effective management of these conditions. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Across the world, various epidemiological studies have revealed an increase in the incidence of sepsis during the last decade [1-3]. In the United States, the hospitalization rate due to sepsis as principal diagnosis has increased by more than 2-fold, extending from 11.6 to 24.0 per 10 000 population between the years 2001 and 2008 [4]. In recent years, improved sepsis management has decreased the case fatality rate in sepsis patients, but owing to the increase in number of cases, the overall mortality is on the rise [5]. Conceptually, sepsis is defined as a systemic inflammatory host response to infection and characterized by alterations in physiologic parameters such as temperature, heart rate, respiratory rate, etc [6]. In clinical practice, such changes in physiologic parameters are nonspecific, and may manifest in the other noninfectious systemic inflammatory
☆ We are grateful to the Indian Council of Medical Research, New Delhi, India, for financial support through project no. 52/10/08. ⁎ Corresponding author at: Department of Critical Care and Emergency Medicine, Sir Ganga Ram Hospital, New Delhi, 110060, India. Tel.: +91 98 10614433; fax: +91 11 45088437. E-mail address: [email protected] (S. Ray).
response (SIRS) conditions such as trauma, burns, pancreatitis, etc [7,8]. According to the currently used definition of sepsis, the presence of documented or suspected infection is the key demarcating characteristic between sepsis and other noninfectious SIRS conditions. The major challenge, however, is to diagnose infection in sepsis patients. Although, microbiological culture is considered as the criterion standard for the diagnosis of infection, the major concern is lack of sensitivity, specificity, and delay in reporting of culture results. The Sepsis Occurrence in Acutely Ill Patients study revealed that approximately 40% of the sepsis patients remain culture negative [9]. The Extended Prevalence of Infection in the intensive care unit (ICU) study has also reported a prevalence of culture-negative infection at 30% of overall infections [10]. Most of these clinically suspected sepsis patients who remain culture-negative pose a challenge in the decision making of antibiotic administration. Hence, it is of utmost importance to differentiate these patients from those with noninfectious SIRS, as both disease conditions require different therapeutic regimens. According to the Surviving Sepsis Campaign recommendation, antibiotics should be administered within 1 hour of septic shock onset [11]. Every hour of delay in antibiotic administration demonstrated an increase in mortality of 7.6% in septic shock [12]. Therefore, early empirical antibiotic administration is crucial for improving outcome in sepsis. This may get delayed due to misdiagnosis of culture-negative sepsis
http://dx.doi.org/10.1016/j.jcrc.2014.08.017 0883-9441/© 2014 Elsevier Inc. All rights reserved.
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patients as SIRS. On the other hand, noninfectious SIRS patients misdiagnosed as sepsis might get inappropriately treated with broad spectrum antibiotics, which may contribute to the emergence of antibiotic resistance—a growing health problem [13]. Thus, a specific biomarker with high sensitivity and negative predictive value (NPV), which can diagnose or exclude suspected sepsis early (within 1 hour of sampling) in culture-negative patients would be of vital importance for timely institution of antibiotic treatment or avoidance thereof. Inflammatory mediators in the serum, which are altered in septic conditions, have been studied for their potential in diagnosis or prognosis of sepsis. Among these, procalcitonin (PCT) and interleukin 6 (IL-6) have been investigated widely in relation to severity and organ failure in sepsis patients [14-16]. Various studies, including several metaanalyses have assessed the usefulness of PCT as a diagnostic tool in differentiating sepsis from noninfectious SIRS [17-22]. Although PCT has been regarded as a sensitive marker for the prediction of sepsis in culture-positive patients, its role in culture-negative sepsis is scarcely known. To the best of our knowledge, no study in the past has focused on the diagnostic accuracy of PCT and IL-6 in culture-negative sepsis, and therefore, no optimal cutoff value has been proposed for differentiating culture-negative sepsis from noninfectious SIRS. The current prospective observational study sought to investigate the diagnostic accuracy of serum PCT and IL-6 to differentiate between culturenegative sepsis and noninfectious SIRS. We also studied their diagnostic efficacy in culture-positive sepsis. 2. Materials and methods The study protocol was approved by institutional ethics committee; and the prospective, observational, single-center study was conducted at a 675-bedded superspeciality hospital in New Delhi (India) over a period of 1 year and 5 months (January 2013 to May 2014). Informed consent was taken from each patient or their next of kin if patient was unconscious or not in a state to give consent. Our inclusion criteria were as follows: adult patients (aged N 18 years) admitted from the community to the ICU were screened; and patients diagnosed with noninfectious SIRS, sepsis, severe sepsis, or septic shock (according to the established consensus sepsis definition) were enrolled in this study [23]. Exclusion criteria were patients who had received prior antibiotics (last 3 months), transferred from other ICUs; having conditions, which were considered lethal in the next 24 hours; postoperative; immunocompromised; and with malignancy were not included in the study. Patients with bilateral pneumonia (suspected viral infection) and diagnosed tropical diseases such as malaria, dengue, Leptospira, and rickettesiae were also excluded. Patient's demographics, principal diagnosis, and all clinical parameters were recorded at the time of enrollment. Initial severity of illness was determined using the Acute Physiology and Chronic Health evaluation score (APACHE II) at 24 hours in all patients. Patients were followed up for 28 days to observe survival or mortality. 2.1. Sample collection and processing Blood samples were assayed for microbiological culture and biomarkers on the day of ICU admission. Two sets of blood cultures, urine culture, sputum culture (in nonintubated patients), endotracheal culture (in intubated patients), and high vaginal swab culture (where puerperal sepsis was suspected) were sent before the commencement of antibiotic therapy. For blood cultures, samples were obtained in both aerobic and anaerobic BacT/Alert bottles and performed by BacT/Alert method (BioMerieux, Marcy l' Etiole, France). Urine and sputum specimen were procured in sterile containers. For endotracheal and vaginal swab cultures, specimens were collected in mucus trap and swab container, respectively. Positive cultures were further processed for the identification of organisms using standard laboratory methods.
Blood samples for PCT and IL-6 estimation were obtained in serumevacuated separator tubes and centrifuged for the separation of serum and processed on the same day. Procalcitonin estimation was done with timeresolved amplified cryptate emission technology by measuring the signal that is emitted from an immunocomplex with time delay (Kryptor PCT; BRAHMS, Henningsdorf, Germany). The assay time of this estimation takes less than half an hour. Interleukin 6 estimation was done by solid-phase Chemiluminescent Access Immunoassay System (Beckman Coulter, Inc, Brea, California, USA). 2.2. Classification of patient groups Enrolled patients were classified into SIRS and suspected sepsis at the time of enrollment based on clinical presentation by 4 clinicians (also coauthors). Culture-negative and culture-positive groups were defined, once the microbiological results were available. (i) Group I (noninfectious systemic inflammatory response group): Included patients with 2 or more signs of SIRS with recent onset pancreatitis and trauma (within 24 hours) without any evidence of infection. (ii) Group II (culture-negative sepsis group): Patients with 2 or more signs of SIRS and clinical suspicion of infection with negative culture results. The diagnosis of bacterial infection in these patients was done based on findings of a clinical focus of infection. Intraabdominal infection was diagnosed in case of exudative ascitic tap with increased polymorphonuclear cell count. Bacterial pneumonia was confirmed by x-ray showing lobar infiltrate. Urosepsis was suspected with signs of urinary tract infection and with a raised leukocyte count in the urine (N10 pus cells/high-power field) and signs of pyelonephritis by ultrasonography. Cellulitis was diagnosed by the skin signs, that is, lesions. Puerperal sepsis was suspected in peripartum patients with signs of pelvic pain and abnormal or foul smelling vaginal discharge (presence of pus). (iii) Group III (culture-positive sepsis group): This group consisted of the patients who had microbiologically documented source of infection with 2 or more of the SIRS criteria. A blood culture was considered positive if any significant pathogenic bacterial organism was grown from twin cultures taken from different sites. Respiratory secretions were considered positive for infection if many polymorpohonuclear cells were present along with colony count more than 10 5. Urine culture was considered positive if there were more than 10 pus cells/highpower field, along with single organism cultured with more than 10 5 colony-forming units/mL. 2.3. Statistical analysis Statistical analyses were performed using SPSS, version 17.0 (SPSS, Chicago, IL). Continuous variables are presented as mean (range) or median (interquartile range) as appropriate. Categorical variables are presented as absolute numbers and percentage. For multiple group comparisons, one-way analysis of variance with post hoc comparison or nonparametric Kruskal-Wallis with Mann-Whitney U test was used. Categorical variables were analyzed using the χ2 statistic. The diagnostic performance of biomarkers was demonstrated with receiver operating characteristics (ROC) analysis. The Youden index with highest sum of sensitivity and specificity was used to determine the optimal cutoff values [24]. Positive predictive value, NPV, positive likelihood ratio, and negative likelihood ratios were calculated. For all statistical tests, a P value b .05 was considered significant. 3. Results The study was conducted in a total of 208 patients prospectively. As defined in methodology, enrolled patients were classified into following
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Table 1 Demographics and outcome of the study patient groups Characteristics
Group I (SIRS)
Group II (culture negative)
Group III (culture positive)
P
No. of patients Sex, M/F Age (y), Mean (range) Mortality, n (%) APACHE II, Mean (range)
46 33/13 42 (19-80) 8 (17.3%) 15.9 (4-46)
90 49/41 53.7 (19-84) 17 (18.8%) 22.9 (8-42)
72 40/32 54 (17-85) 21 (29.1%) 25.8 (13-56)
– NSa ,b .001⁎ NSa ,b b.001⁎
Abbreviations: M, male; F, female. Data are presented as mean (range), n (percentage) as appropriate. Significance testing was performed by: a χ2 test. b One-way analysis of variance with Bonferroni correction. ⁎ P b .005 was considered highly significant.
3 groups. Group I, noninfectious SIRS (n = 46); group II, culturenegative sepsis (n = 90); and group III, culture-positive sepsis (n = 72). Of 162 clinically suspected bacterial sepsis patients, 55.5% (90/ 162) were culture negative and 44.5% (72/162) were culture positive. The demographic profile and outcome of different patient groups are shown in Table 1.
3.1. Characteristics of the enrolled subjects No statistically significant sex difference was observed between the study groups. Mean age was significantly lower in SIRS as compared with culture-negative and culture-positive groups (P = .004 and P = .003, respectively). Median APACHE II score was significantly different in all 3 groups, being highest (25) in group III, which also had the highest mortality (29.1%). Although overall mortality was 22.1%, there was no statistically significant difference observed between the groups (Table 1).
3.2. Source of infection The foci of clinically suspected bacterial infection in culture-negative sepsis patients were predominantly abdominal (43%) and respiratory (37%) (Fig.1A). In culture-positive patients, the most common site of infection found was blood stream (43%), followed by respiratory tract (22%) (Fig.1B). Of 72 patients enrolled in culture-positive group, 84.7% (61/72) had Gram-negative and 15.3% (11/72) had Gram-positive infections. Fiftysix patients in Gram-negative group had bacilli (Escherichia coli in 40 cases, Klebsiella pneumoniae in 11 cases, and Pseudomonas aeruginosa in 5 cases). Among Gram-positive, 6 had Staphylococcus aureus, 2 had Streptococcus pneumoniae, and 3 had Enterococcus faecalis. Although levels of PCT were higher in Gram-positive group and levels of IL-6 were higher in Gram-negative group, this difference between 2 groups was not statistically significant (P = .748 and P = .937, respectively). Median PCT levels in Gram-positive and Gram-negative groups were 26 ng/mL (Interquartile range [IQR], 6.94-54) vs 21.6 ng/mL (IQR, 5.0-94), whereas IL-6 levels were 649 pg/mL (IQR, 216-1594) vs 959 pg/mL (IQR, 120.9-1582).
3.3. Serum levels of PCT and IL-6 in study groups Procalcitonin and IL-6 levels were measured and analyzed in all enrolled patients on the day of admission. In culture-negative patients, the median PCT levels were significantly high (P b .001) approximately 8fold than noninfectious SIRS patients, whereas IL-6 levels showed only 1.7-fold increase, which was also statistically significant (P = .01) in the culture-negative patients as compared with SIRS. However, levels of both the parameters were highest in culture-positive patients (Fig. 2 A and B). In trauma patients (n = 15), the median PCT and IL-6 levels were 0.76 ng/mL and 65.6 ng/mL, respectively similar to those found in remaining patients of SIRS group (P = .453 and .223 for PCT and IL-6, respectively). 3.4. Discriminative performance of PCT and IL-6 Culture negative vs SIRS: ROC curve analysis for PCT and IL-6 between culture-negative and SIRS groups for the prediction of systemic infection were performed (Fig. 3A). The area under curve (AUCs) for PCT and IL-6 were 0.892 (confidence interval [CI], 0.836-0.947; P = .028) and 0.636 (CI, 0.540-0.731; P = .049), respectively. For PCT, the optimal cutoff point was 1.43 ng/mL with a sensitivity of 92.2% and NPV of 82.5%. The best cutoff point for IL-6 was 219.85 pg/mL with a sensitivity of 47% and NPV of 41.5%, which was much lower than PCT (Table 2). Culture positive vs SIRS: Similarly, ROC analysis to distinguish between patients with noninfectious SIRS and culture-positive sepsis, the PCT had an AUC of 0.959 (CI, 0.920-0.988; P = .017), and IL-6 had an AUC of 0.784 (CI, 0.701-0.867; P = .042) (Fig. 3B). At the optimum cutoff level of 2.49 ng/mL, PCT demonstrated a sensitivity of 94.4% and NPV of 91.5%, respectively. Interleukin 6 demonstrated a sensitivity of 63.9% and NPV of 63.7% at the optimum cutoff of 423.5 pg/mL (Table 2). 4. Discussion To overcome the inherent inadequacies of culture as a diagnostic tool of differentiation between SIRS and sepsis, PCT and IL-6 have been studied and found useful for early diagnosis of sepsis [25,26]. Yet, one subset of patients—the culture-negative sepsis patient—still
Fig. 1. A, Suspected source of infection in culture-negative sepsis patients. B, Site of infection in culture-positive sepsis patients.
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Fig. 2. A, Graphical representation of median (IQR) levels of PCT in 3 study groups. B, Graphical representation of median (IQR) levels of IL-6 in 3 study groups.
remains difficult to diagnose. Presently, they are diagnosed primarily by clinical acumen—a very subjective method. The current study evaluates the ability of serum PCT and IL-6 in differentiating suspected culturenegative sepsis from noninfectious SIRS. Procalcitonin is a peptide precursor of hormone calcitonin, and it consists of 116 amino acids (molecular weight, 13 kd) [27]. Normally, PCT is synthesized by the C cells of thyroid gland, and levels are undetectable in healthy individuals. Abnormally raised PCT levels were first reported by Assicot et al [28] in children with bacterial infections. High PCT levels are detectable in plasma 2 to 3 hours after endotoxin injection in healthy volunteers; half-life of PCT in serum is 25 to 30 hours [29]. Cytokine IL-6 is a mediator of acute phase response, induced by tumor necrosis factor α and produced by various cell types such as monocytes, macrophages, and endothelial cells [15]. Literature has acknowledged IL-6 as a marker to predict sepsis severity and clinical outcome; however, it is also known to be relatively nonspecific and is elevated under a variety of conditions [30]. Review of literature revealed only 1 epidemiological study pertaining to differentiation of culture-positive from culture-negative sepsis with no conclusion regarding the differentiation of culturenegative sepsis from SIRS [31]. However, the latter differentiation is of utmost clinical importance, as a sepsis patient's course depends on timely institution of antibiotics and in SIRS patients' inappropriate antibiotic therapy may lead to emergence of antibiotic resistance. In the ICU setting, APACHE II score is an indicator of severity and mortality risk in critically ill patients [32]. In the current study, we observed that
APACHE scores were significantly higher in culture positive as compared with culture negative and in culture negative as compared with SIRS. The mortality rates (least in SIRS and maximum in culture positive) were not significantly different in the 3 groups. This was similar to the results of Phua et al [31], who reported lower mortality in culture-negative patients as compared with culture-positive patients. Furthermore, our study showed that culture-positive patients had predominantly (84.7%) Gram-negative infections corroborating previous reports [10]. However, the predominant sources of infection in culture-negative patients in our study were abdominal (43%) followed by respiratory (37%), which were in sharp contrast to the study by Phua et al [31] in which respiratory infections accounted for 75% of culture-negative patients. An interesting observation of our study was that PCT and IL-6 gave a graded differentiation between the 3 groups of patients, being highest in culture positive and lowest in SIRS. The use of these 2 markers in diagnosis of culture-negative sepsis would depend on the sensitivity and NPV of these biomarkers. The ROC analysis between culture-negative and SIRS patients demonstrated a superior AUC for PCT in comparison with IL-6. The sensitivity of PCT at the optimum cutoff value of 1.43 ng/mL was 92.2% with an NPV of 82.5%. The same for IL-6 were also much lower, 47% and 41.5%, respectively at the optimal cutoff value of 219.85 pg/mL. Higher sensitivity and NPV of PCT were also observed in culture-positive patients as seen in previous studies [33,34]. These biomarker characteristics clearly indicate the superiority of PCT over IL-6 in its ability to differentiate culture-negative sepsis from SIRS.
Fig. 3. A, Receiver operating characteristic curve for PCT (dashed line) and IL-6 (dotted line) between culture-negative and SIRS groups. B, Receiver operating characteristic curve for PCT (dashed line) and IL-6 (dotted line) between culture-positive and SIRS groups.
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Table 2 Diagnostic performance of PCT and IL-6 in culture-negative and culture-positive patients in comparison with SIRS Cutoffa
Sensitivity, %
Specificity, %
Positive predictive value (%)
NPV (%)
Positive likelihood ratio
Negative likelihood ratio
Culture negative in comparison with SIRS PCT 1.43 ng/mL 0.892 (0.836-0.947) IL-6 219.85 pg/mL 0.636 (0.540-0.731)
92.2 47
72 73
86.5 78
82.5 41.5
3.53 1.79
0.11 0.72
Culture positive in comparison with SIRS PCT 2.49 ng/mL 0.959 (0.926-0.991) IL-6 423.5 pg/mL 0.784 (0.701-0.867)
94.4 63.9
87 89
90.5 88.4
91.5 63.7
6.74 5.4
0.07 0.4
Variable
AUC (95% CI)
a Sensitivity, specificity, predictive values, and likelihood ratios were calculated for the PCT and IL-6 at the cutoff, which demonstrated the best discrimination of the study groups as derived from the ROC curves.
Key findings of the present study were that, in culture-negative sepsis patients, PCT and IL-6 levels were significantly higher than in noninfectious SIRS. In ROC curve analyses of PCT and IL-6 for the prediction of systemic infection, optimum cutoff point of PCT was associated with higher sensitivity, specificity, predictive values, and likelihood ratio values than IL-6. Determination of appropriate and timely treatment in sepsis patients depends on a swift diagnostic method. Procalcitonin analysis is simple to perform, done at most modern health care establishments, and results are rapidly available. The quantitative estimation of PCT using kryptate emission technology can be performed in less than half an hour. The superior sensitivity and NPV as compared with IL-6 and the ease of performance contribute to the early, accurate, and rapid differentiation of sepsis, both culture negative and positive, much before the availability of culture results. The strength of our study is that we have included only patients who were admitted from the community with no exposure to a health care facility in the last 3 months, which could have been a confounding factor. Patients with nonbacterial sepsis like viral infections (especially culture-negative patients with bilateral pneumonia) and tropical diseases were also excluded from the study. All the patients enrolled in culture-negative group had a clear source of suspected infection. Moreover, we obtained the samples before the initiation of antibiotic therapy and processed on the same day. We included patients with trauma only in first 24 hours and recent onset pancreatitis in SIRS group to avoid the possibility of infection. As pointed out by Mimoz et al [35] and Reinhart et al [36], postsurgical and trauma patients could also confound our results. But in the present study, we have not included any surgical patients to avoid confounding and had only 15 trauma patients in the SIRS group. These latter subjects exhibited PCT and IL-6 levels similar to those observed in remaining patients in the SIRS group and hence did not confound our results. The main limitation of this study is that it is a single-center study. A multicenter study may be required to extrapolate our findings to other clinical settings. Although we attempted to exclude all viral infections, this was based primarily on clinical acumen. Therefore, PCT should be interpreted in context with information from careful medical history and physical examination; continuing reevaluation during the course of disease is advisable. Thus, our study establishes the possible role of surrogate markers to simplify the challenge of identification of culture-negative sepsis patients. These findings could probably be extrapolated to diagnose secondary infection in-hospital settings.
5. Conclusions Our study indicates that PCT has the potential to aid in rapid differentiation of culture-negative sepsis from SIRS at a cutoff of 1.43 ng/mL (sensitivity, 92%; NPV, 83%), thus helping initiate timely antibiotic therapy, whereas reducing inappropriate use of antibiotics in critical care settings. However, because the number of patients in our study was 208, further multicentric studies would be required to substantiate our findings.
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