European Journal of Cardiovascular Medicine

Sepsis is an innate immunological response of systemic inflammation to a confirmed or suspected microbial infection ^[1]. As per 2016 (SCCM and ESICM) definition, sepsis is a life-threatening organ dysfunction due to dysregulated host response to infection. Organ dysfunction is defined as an increase of ≥2 points in the SOFA score ^[2]. Sepsis and septic shock are among the major causes of mortality in critically ill patients. Historically, the mortality rates were 50 – 75%. After the antibiotics introduction, mortality rates associated with bloodstream infection were 30 to 50 % ^{[2, 3]}. Further advancement in treatment reduced mortality to around 18% but the number of patients dying from sepsis is increasing ^[2]. There is ambiguity in diagnosis of sepsis, delayed treatment, overuse, and misuse of antimicrobials that results in antimicrobial resistance (AMR) ^[4]. It is puzzling to identify sepsis from non-sepsis (non-infectious) in a Systemic Inflammatory Response Syndrome (SIRS) patient. So, there is a need for biomarkers to distinguish between sepsis & non-infectious SIRS and to curb antimicrobial resistance (AMR). Some of the serological markers like procalcitonin may even differentiate between bacterial, fungal, and viral etiology where its quantity is lesser in fungal and viral causes ^[5].

Blood culture is a gold standard test for the diagnosis of sepsis, but the turnaround time is longer (48hrs – 72 hrs). Blood culture as a diagnostic tool has less sensitivity, good specificity and doesn’t aid much in early diagnosis and treatment of sepsis. Molecular methods like PCR are available to detect bacteria and other microbes but it is little time consuming, and it can’t differentiate DNA sequencing from viable and non-viable bacteria ^[6]. The traditional biomarkers of systemic inflammation, like total leucocytes, ESR, C-reactive protein sometimes influenced by other parameters and are not that specific. They may be released slowly during an infection. The culture results may be positive due to contamination and negative results do not rule out sepsis. As these common lab measurements lack specificity other tests are needed to give an early marker of systemic inflammatory response of an infectious cause.

The aim is to study the role of biomarkers in the diagnosis of sepsis with objectives of detecting biomarkers like C-Reactive Protein (CRP), procalcitonin (PCT) and presepsin (PSP: N terminal fragment of soluble CD14-ST subtype) correlating with SOFA (Sequential Organ Failure Assessment) score and aerobic blood culture that helps in early accurate diagnosis of sepsis. Thus, it helps in decreasing the mortality and morbidity.

This prospective study is conducted in a tertiary care hospital. Institutional ethical committee permission was granted. Sample size was calculated based on the formula (Daniel,1999) n = Z²P(1-P)/d² where Z is statistic for a level of confidence (Z value is 1.96), P is expected prevalence and d is precision (in proportion of one; if 9%, d=0.09). A total of 112 patients were registered in this study after informed consent. This study required Chemiluminescence Immunoassay (CLIA) automated analyser system, Enzyme Linked Immunosorbent Assay (ELISA) apparatus, testing reagents, micropipettes, culture media. As it is a tertiary care hospital, many patients come in final stages of their illnesses. Blood samples from consecutive adult patients admitted in the medical & surgical ICU’s presenting with two or more features of sepsis like fever or hypothermia, tachycardia, tachypnoea, leucocytosis, or leukopenia was collected before antibiotic therapy for the detection of biomarkers of sepsis and aerobic culture. The adult patients on antibiotic therapy or immunocompromised were excluded. The samples used were (a) serum for testing serological markers and (b) blood for culture. 2 - 3 ml blood in vacutainer was left for 2hrs at room temperature in the lab. Then centrifuged at 2000-3000 rpm for 15 – 20 min to separate serum. A drop of serum was used for testing of CRP. Remaining serum was stored in aliquots after proper labelling to test for presepsin and procalcitonin (PCT) later in batches. Serum CRP levels and blood culture was done at the time of collection of samples. Serum Presepsin and Serum Procalcitonin was tested later within a month in batches.

Latex slide agglutination immunoassay was done using serum CRP latex agglutination kit (ARKRAY HEALTH CARE LTD/ASPEN) on slides. CLIA procedure was done using serum Procalcitonin CLIA kit (Siemens) in a CLIA automated analyser. ELISA procedure was done using serum Presepsin (P-SEP) ELISA kit (Sincere Biotech Co., Ltd) in ELISA apparatus. Blood culture was done where 8 - 10 ml of blood dispensed in a pair of medical flat bottles with Brain Heart Infusion (BHI) broth and microorganism was identified based on standard microbiological techniques. SOFA score was calculated based on respiratory (PaO2/FiO2), CNS (Glasgow Coma Scale), CVS (Mean arterial pressure or administration of vasopressors required), serum bilirubin, coagulation (platelet count), and renal system (creatinine) parameters.

Storage of serum aliquots was done at -20^oC. Presepsin ELISA kit, Procalcitonin CLIA kit, CRP latex agglutination kit and reagents were stored at 2 to 8^oC.

In the 112 patients, the adults were divided into young age adults 18 – 30 yrs., middle-aged adults 31 – 45 yrs. and old age adults 46 – 65 yrs. ^[7]

Table 1 Total number of positive biomarkers and blood culture

Out of them, 18 (16%) were culture positive, 94 (83.9%) were CRP positive (cut off value 12µg/ml), 78 (69.6%) were PCT positive (cut off value of 0.5ng/ml), 92 (82.1%) were presepsin positive (cut off value 820pg/ml).

Table 2 Age wise distribution of positive percentages of biomarkers & blood culture

The middle-aged adults followed by old age adults were more prone to sepsis as evident from table 2 showing higher positive percentages of all biomarkers and aerobic culture. The biomarkers were higher among male than female in this study.

The positive biomarkers and blood culture were relatively higher among surgical cases than medical cases. The surgical and medical cases were divided based on the ward, presenting complaints and provisional diagnosis. Surgical cases include patients with fever, along with either pain abdomen, lump abdomen, burst abdomen, leg cellulitis, infected ulcer foot, enterocutaneous fistula, diabetic foot, post operative infection, fracture with AKI. Medical cases include patients with fever or hypothermia, along with either shortness of breath, vomiting, altered sensorium, cough, hematemesis, oliguria, anuria, loose stools, seizures, diabetes.

Table 3 Distribution of blood culture isolates

Staphylococcus aureus was the most common isolate followed by Klebsiella pneumoniae, CONS.

Sepsis, defined as systemic inflammatory response syndrome (SIRS), commonly induced by severe bacterial infection. Delay in diagnosis and treatment usually results in rapid progression to shock, multiple organ failure, and finally death. Early identification significantly reduces mortality from sepsis-related multiple organ dysfunction. However, sepsis can sometimes be difficult to assess, and its distinction from non-infectious conditions in critically ill patients is often a challenge ^[11].

The mean age of sepsis in this study was 45.64 yrs., and the biomarkers relatively raised in male patients that correlates with Sudhir U et al study ^[5]. Procalcitonin positive percentage of present study correlated with Rowther et al and Shuhua et al studies as shown in table 4.

Table 4 Comparison of biomarkers positivity in other studies

The value of procalcitonin is less in fungal infection (mean PCT: 0.18ng/ml) compared to bacterial infection (mean PCT: 17.64ng/ml) like Shuhua et al. study ^[12]. Their mean PCT value is 0.6 ng/ml in fungal sepsis and 7.47 ng/ml in bacterial sepsis. This implies that PCT can be used to distinguish between bacterial & fungal sepsis. PCT mean values in Gram negative sepsis (20.78 ng/ml) was higher than Gram positive sepsis (14.68 ng/ml) like Shuhua et al. study ^[12].

Based on severity of sepsis the procalcitonin range was higher, and it matched with higher SOFA score. A greater number of patients with low SOFA score had low PCT values and a greater number of patients with high SOFA score had high PCT values as evident in the table 5.

Table 5 Procalcitonin range in relation to SOFA score

Table 6 Comparison of procalcitonin and presepsin statistical data in previous studies for different thresholds

In this study, the sensitivity of CRP is 85.71% and specificity is 54.76%; sensitivity of presepsin is 83% and specificity is 34%.  The sensitivity and specificity of biomarker presepsin in this study correlated with Brenner et al study. In this study the sensitivity & specificity of procalcitonin was 72% & 75% respectively for a cut off value of 0.5 ng/ml and best correlated with Brenner et al. ^[16] study, Takahashi et al. ^[17], Romualdo et al. and ^[18] and Liu et al. ^[19] study.

CRP is frequently used in clinical practice because of its greater availability though it is often reported as inferior compared with procalcitonin in terms of sepsis diagnosis. The study of CRP in time course helps to evaluate the response to therapy in septic patients.

Several studies support the important role of procalcitonin as biomarker for the diagnosis of sepsis. In this study a well-known biomarker CRP and a relatively recent presepsin biomarker has been tested along with procalcitonin and their role was studied in the early diagnosis of sepsis. The time required for automated CLIA of procalcitonin is around 20 min which is best compared to ELISA done for presepsin. Procalcitonin levels raised among most of the patients who had a greater SOFA score. Procalcitonin is a promising marker for diagnosis of sepsis as presepsin or CRP based on the results, but these results should be interpreted cautiously. Procalcitonin may be useful in combination with some sensitive biological markers. Continuing re-evaluation during sepsis is advisable. Presepsin has high sensitivity though specificity is low. Further evaluation is required to understand its usefulness in Indian settings.

The management of critically ill patients due to sepsis requires clinical & lab diagnosis based on biomarkers, SOFA score, and blood culture. Procalcitonin is more useful for ruling out than for confirming systemic bacterial infection. Higher procalcitonin levels (≥ 10 ng/ml) may imply serious bacterial infection and sepsis, that predicts worse outcome. Serial low procalcitonin values in the absence of positive cultures indicate non-bacterial sepsis and is enough reason to discontinue antimicrobial therapy. No single biomarker has sufficient sensitivity and specificity to stand on its own. However, some biomarkers like procalcitonin support antimicrobial stewardship determining duration of antimicrobial therapy, ruling out rather than confirming sepsis. Sepsis is not yet a laboratory-based diagnosis, but a laboratory supported diagnosis.

1. Chan T, Gu F. Early diagnosis of sepsis using serum biomarkers. Expert Rev Mol Diagn. 2011 Jun; 11(5): 487-96.
2. Mehta Y & Kochhar G. Sepsis and Septic Shock. J Card Crit Care TSS 2017; 1:1–3.
3. Tille P. Bailey & Scott’s Diagnostic Microbiology. Elsevier Publishers. 2017; 14th edition.
4. Reinhart K, Bauer M, Riedemann NC, Hartog CS. New Approaches to Sepsis: Molecular Diagnostics and Biomarkers. Clinical Microbiology Reviews, 2012; 25(4): 609-634.
5. Sudhir U, Venkatachalaiah R, Anil Kumar T, Yogesh Rao et al. Significance of serum procalcitonin in sepsis. Indian Journal of Critical Care Medicine 2011 January-March; 15(1): 1-5.
6. WuJ, HuL, ZhangG, WuF, et al. Accuracy of Presepsin in Sepsis Diagnosis: A Systematic Review and Meta-Analysis. PLoSONE 2015; 10(7): e0133057.
7. Dipali B, Patil V.K. Human Age Group Classification Using Facial Features. IJMTER June– 2016; 03 (6) ISSN (Online):2349–9745; ISSN (Print):2393-8161.
8. Rowther F B, Rodriguer CS, Deshmukh S, Kapadia F et al. Prospective Comparison of Eubacterial PCR, and measurement of Procalcitonin Levels with Blood Culture for Diagnosing Septicemia in Intensive Care Unit Patients. Journal of clinical microbiology. 2009; 47(9): 2964 – 2969.