Sepsis has been derived from a Greek word which means “decomposition” or “decay” and is described as a medical emergency arising due to the body’s systemic immunological response to an infection leading to end-stage organ dysfunction or even death. Sepsis is “a life-threatening organ dysfunction caused by dysregulated host response to infection”. This is as per the third international consensus definitions for sepsis and septic shock 2016 ^[1]

Sepsis is observed very frequently among critically ill patients and is a common cause of death in hospitals’ intensive care units. The death toll has been reported to be comparable to that seen in patients with myocardial infarction and has been referred to as a public health disaster. ^[2] It is one of the top 10 leading causes of death in the world. ^[3] As per the data from the Surviving Sepsis Campaign 2012, mortality rates from sepsis were 41% approximately in Europe and 28.3% approximately in the United States. ^[1]

Usually, sepsis is due to an exuberant response of inflammation ^[5], but it may also be caused due to the development of global tissue hypo-perfusion and oxidative damage which results in morbidity and mortality in sepsis. Tissue hypoperfusion and hypoxia leads to mitochondrial dysfunction and widespread reactive oxygen species (ROS) production, causing extensive oxidative stress damage to cells and tissues. Ischemia and oxidative damage induced by sepsis credibly play a key role in the pathogenesis of organ dysfunction and are assumed to be the prelude to the development of multiple organ failure and death. Timely recognition of the above progress may be helpful for judicious clinical intervention. ^[7]

Early categorization and identification of patients who are at higher risk of death, pose serious clinical challenge for healthcare workers, as timely decisions on the best therapeutic approach and appropriate site of care are both crucial in healthcare settings. It is therefore imperative to understand and elucidate the pathophysiological mechanisms which occur in patients with sepsis. Concurrently, various biomarkers have been proposed among which the most extensively studied biomarker of sepsis or septic shock is procalcitonin (PCT). Although, PCT is coupled with the severity of sepsis and existence of organ dysfunction, certain outcomes can be predicted through dynamic changes of PCT levels in sepsis. ^[9]

PCT is a 116 amino acid residues containing polypeptide, produced as the precursor of calcitonin by C cells of thyroid gland. Almost all of the PCT is broken down to form the hormone calcitonin, within the thyroid gland itself. Therefore, the plasma levels of PCT in healthy subjects are generally low. The levels of PCT increase in patients with sepsis and correlates with the severity of the disease process. PCT is regularly being used as a gold standard marker for diagnosis of sepsis in patients admitted to the ICU having a sensitivity of 85 % and specificity of 91 %. ^[3]

A novel biomarker, which has been shown to be useful in predicting ischemic oxidative stress is Ischemia modified albumin (IMA). It is a modification of human serum albumin (HSA) caused by ischemia and has been recently proposed as a serum biomarker of myocardial ischemia. IMA is useful in early detection of myocardial ischemia. ^[4] IMA is a specific and sensitive marker for ischemic process resulting from oxidative damage of tissues. ^[12] IMA is a form of HSA where the N-terminal amino acids cannot bind to transition metals. Free radicals generation, development of acidosis and release of free iron and copper ions occurs in myocardial ischemia. HSA scavenges divalent metal ions. Therefore, it may be postulated that in ischemia, these processes do not let N-terminus to bind to transition metal ions and release of these ions probably initiates one potential pathway for IMA generation. ^[13]

This study seeks to highlight the importance of simultaneously quantifying and analyzing the following tests namely serum PCT and IMA in patients of sepsis. ^[2]

A positive correlation between PCT and IMA might give us an additional parameter which will be cost effective for routine monitoring of the disease process and severity. Moreover, IMA levels in sepsis may help in early recognition of patients at risk of irreversible ischemic damage to vital organs thereby preventing poor outcome.

This observational follow up study was carried out in a tertiary care hospital in collaboration with Department of Biochemistry from December 2019 to October 2021. Overall, 63 patients (35 males and 28 females) were selected for the study with age ranging from 46 years to 75 years. Purposive sampling method - based on physician’s provisional diagnosis at the time of admission, was used for selecting the study subjects.

Patients who were suspected to have bacterial infection and having SOFA (Sequential Organ Failure Assessment) score of 2 or more points from the baseline were diagnosed with sepsis by the physician. ^[9]

A written informed consent was obtained from all the participants after explaining the need and possible outcomes of the study. Ethical approval for the study was obtained from the institutional ethical committee.

All patients with sepsis between 46 to 75 years of age and patients who provided written informed consent were included in the study. Patients with pre-existing renal disease, heart disease, liver disease, HIV, cancer and COVID-19 infection were excluded from the study.

Sample collection

Blood samples were collected on two occasions: firstly, on admission of patient to I.C.U and secondly, when the patient was on recovery phase from sepsis. 5ml of blood was drawn from the peripheral vein of patients under all aseptic precautions and was collected in plain vacutainers. After allowing to stand for 30 minutes, serum was separated by centrifugation at 3000 rpm for 10 minutes. Serum obtained was used to measure PCT and IMA

Tests and equipments

1. Procalcitonin

PCT was measured by a rapid quantitative test based on fluorescence immunoassay technologyon Finecare FIA system using Finecare PCT kit. The method used in Finecare PCT Rapid Quantitative test is a sandwich immunodetection method. Values above 0.5 ng/ml were considered as significant ^[10]

The Signal intensity of fluorescence of detector antibodies reflect the amount of captured PCT.

Reference range:   0 - 0.5 ng/ml ^[49]

2. Serum Ischemia Modified Albumin

Method: IMA was measured by albumin cobalt binding (ACB) assay.  ^[4]

Human serum albumin has an intrinsic property of combining with metals at its N-terminal region, like cobalt & nickel etc. When ischemia occurs, changes occur in the N-terminal portion of albumin because of acetylation or depletion of one or more amino acids resulting in loss of its ability to bind cobalt and other metals. This modified form of albumin is known as Ischemia Modified Albumin (IMA). Cobalt acts as an indicator in this assay. The principle of this test is based on adding a known amount of cobalt to serum sample and measuring unbound cobalt by the intensity of coloured complex formed, after reacting with dithiothreitol (DTT) by spectrophotometer. ^[4]

Statistical analysis

The data collected was coded and entered in excel spread sheet and further analysed with SPSS 20.0 version statistical software. Students paired‘t’ test and Pearson product moment correlation coefficient was used for data analysis. Statistically significant values were the P value of ≤ 0.05.

The most common cause of sepsis in these patients was infection of the lungs followed by infection of abdomen or pelvis and urinary tract. Mortality of the patients was mostly related to septic shock or severe form of sepsis where multi-organ dysfunction was more pronounced. After exclusions, 63 patients (35 males and 28 females) were enrolled in the study with age ranging between 46 – 75 years.

There was no significant difference in laboratory values of male and female patients. Neither, the difference in laboratory values of participants of different age groups was significant. The laboratory findings notably differed on comparing pre-treatment values with post-treatment values in the study participants.

Table 1 shows a highly significant difference (p = 0.000) between the mean ± SD values of serum PCT in patients of sepsis before treatment (40.37 ± 11.86 ng/ml) and after treatment (1.99 ± 1.07 ng/ml). For IMA also, in patients of sepsis the before treatment mean value was 1.44 ± 0.27 ABSU and after treatment mean value was 0.05 ± 0.03 ABSU. The difference was significantly high (p = 0.000) on comparing the two.

Table 1: Laboratory findings of S.PCT and S.IMA in patients of sepsis before and after treatment and their comparison applying paired ‘t’ test

S.PCT, S.IMA – before treatment values of patients of sepsis

S.PCT(A), S.IMA(A) – after treatment values of patients of sepsis

Figure 1: Bar diagram showing mean and SD of PCT and IMA before and after treatment

Also, a moderate positive correlation was observed between S. PCT and S. IMA (r=0.485, p=0.000) as depicted in Table 2

Table 2: Pearson’s coefficient of correlation of PCT with IMA

***Moderate correlation

Figure 2: Scatter diagram showing a positive moderate correlation between S.PCT and S.IMA

The purpose of this study was to measure and compare the levels of inflammation, ischemia and tissue hypoperfusion in patients of sepsis before and after treatment.

The mean value of PCT in patients of sepsis before treatment, in the present study was reported to be 40.37 ± 11.86 ng/ml which was considerably higher than the normal range (0 - 0.5 ng/ml). The mean PCT value after treatment was 1.9 ± 1.07 ng/ml. The difference in the values of PCT before treatment and after treatment, was highly significant (p=0.000).

High levels of PCT indicate flared up inflammatory processes in the patients of sepsis in response to infection, which start subsiding on appropriate treatment and the values reduce as patients start recovering. In sepsis, systemic infection and severe inflammation cause the serum PCT levels to upsurge distinctly, reaching values of tens, to hundreds, to thousands-fold that of normal levels. ^[12]

Similar mean value of PCT before treatment (44.05 ± 31.6 ng/ml) was seen in a study done by F. Hu¨meyra Yerlikaya et al ^[13], but it was done in premature babies of 7–28 days of age. Such similarity in values of PCT in adults and premature babies might be because of the fact that the PCT values obtained were from the premature babies who were in late stages of sepsis where the disease process might have been more advanced and profound, whereas in the present study the adult patients were enrolled and before treatment PCT values were obtained at the time of admission when the disease process might have just set in or was in the early stage. The above study enrolled 21 healthy controls for comparing the results and the difference was found to be highly significant (p<0.001), whereas in our study the comparison was made between the before treatment and after treatment values (1.705±1.99 ng/ml) for which the difference was also significantly high (p=0.000).

In a study done by Dong Wook Jekarl et al ^[9], the mean value of PCT in the patients of sepsis was 10.2 ± 24.5 ng/mL and in those who survived sepsis, the mean and standard deviation for PCT was 8.1 ± 19.5 ng/ml. The values observed were lower than that in the present study. The reason for this might also be the age of the subjects. In this study, patients of age >18 years have been enrolled which involves a significant young age group from 19 to 45 years whereas, in our study the patients enrolled were between 45 and 76 years of age. In the above study, comparison was made between the sepsis and non-sepsis group and also between the survivors and non-survivors of sepsis. The difference in the mean values of PCT between sepsis and non-sepsis (3.2 ± 11.7 ng/ml) group was highly significant (p<0.001) and between sepsis survivors and non survivors (23.2 ± 43.2 ng/ml), it was significant (p=0.005).

In a study by Naher BS et al ^[14] and Nilgün Köksal et al ^[15], the values of PCT in patients with highly probable sepsis were 5.85 ± 1.39 ng/ml and 9.3 ± 9.9 ng/ml, respectively. In the probable sepsis group, the mean values were 5.3636 ± 1.35 ng/ml and 1.8 ± 1.7 ng/ml, respectively. Both the studies show lower mean PCT values in comparison to present study values. The probable reason for this might be the study population selected for these studies is newborn infants whereas, in our study adult population was enrolled. In the study by Naher BS et al ^[14], the PCT values of both the groups were compared with that of patients not having sepsis and it was observed that the difference was significantly high (p=0.001. In a study by Nilgün Köksal et al ^[15] on comparing the PCT value of group 1 (9.3±9.9 ng/ml) with that of group 4 (0.4±0.3 ng/ml), the difference was found to be significantly high (p=0.000).

Regarding IMA, the mean value in the present study before treatment was observed to be 1.44 ± 0.28 ABSU which was significantly high (p = 0.000) when compared with after treatment values (0.05 ± 0.03 ABSU). IMA has been seen to be a useful in identifying ischemic oxidative stress ^[2]. Its elevation in sepsis indicates existence of hypoxia and oxidative stress in patients ^[4]. Levels of IMA indicate the level of inflammation as during inflammation the capacity of albumin to bind cobalt and other metals reduces. ^[12].

Similar mean values of IMA (1.47 ± 0.25 ABSU) in sepsis patients before treatment, was also seen in a study done by F. Hu¨meyra Yerlikaya et al ^[12] which was significantly higher (p<0.01) when compared with the control group (1.23 ± 0.36 ABSU). But this study was done in premature babies of 7–28 days of age. Such similarity in values of IMA in adults and premature babies might be because of the fact that the premature babies were in late stages of sepsis and the harm caused to the body might have been more profound.

In the study done by A.K. Prashanth et al ^[3], as compared to our study lower value of IMA was reported in the patients with sepsis (1.087 ± 0.786 ABSU) but when compared to those without sepsis (0.085 ABSU ± 0.234), the difference was found to be highly significant with a p value < 0.0001.

As compared to our study, lower values of IMA were also reported in studies by B. Hekimoğlu et al ^[16] (0.71 ± 0.08 ABSU) and D.U Işık et al ^[17] (0.89 ± 0.62 ABSU). The difference was statistically high (p < 0.05, p=0.04 respectively) when compared to IMA values of controls (0.60 ± 0.05 ABSU and 0.54 ± 0.37 ABSU respectively). The reason behind lower values reported in these studies might be the age of the study population which was less than 28 days (neonates).

In the present study, apart from comparison in before and after treatment values of PCT and IMA, correlation between PCT and IMA was also observed. A moderate positive correlation was seen between PCT and IMA (r=0.485, p=0.000) indicating that IMA is associated with inflammation which reduces the capacity of albumin to bind cobalt ^[12].

Positive correlation between PCT and IMA (r = 0.404, p<0.05) was also observed in a study by F. Hu¨meyra Yerlikaya et al ^[12] in the sepsis group before treatment.

From the results of our study it can be concluded that in sepsis, inflammation and ischemic oxidative stress go hand in hand causing global tissue hypo-perfusion indicating that inflammation, alone does not contribute to the pathophysiology of the disease. PCT is an established sensitive marker of inflammation and its upsurge in sepsis has been witnessed in several studies, therefore elevation and decline in the levels of IMA along with PCT, indicate that these changes are purely sepsis induced. A positive correlation of PCT with IMA show that the ischemic processes, tissue hypoxia and oxidative stress occur simultaneously along with inflammation as a response to infection implying that inflammation is not the only culprit causing damage in sepsis. Since, the values of IMA were observed to rise and decline with values of PCT, IMA can be considered to be used in place of PCT in sepsis for monitoring purpose as a cost effective routine laboratory investigation, although an extensive research work will be needed for its implementation. Future research can be done to determine the diagnostic and prognostic utility of IMA in sepsis and once established as a sensitive and specific marker of sepsis it may replace other costly investigations. Studies also show that IMA is an early marker of ischemia as it starts rising as soon as ischemic processes starts in the body and its levels are detectable much before actual damage is done. Therefore, the levels of IMA may be helpful in monitoring and stratifying the patients according to the severity followed by timely measures, thereby preventing serious complications. Very few studies have been done in this aspect particularly in India and therefore this study would add to our knowledge about the same.

Affiliation where the work was primarily carried out (institution to which the work should be credited): Krishna Vishwa Vidyapeeth Deemed to be University, Karad, Maharashtra Formerly known as Krishna Institute of Medical sciences deemed to be university, Karad, Maharashtra

Source(s) of Support and Funding: Krishna Vishwa Vidyapeeth deemed to be University Formerly known as Krishna Institute of Medical sciences deemed to be university, Karad, Maharashtra

Acknowledgements: I thank all the members of the Department of Biochemistry of KVV , KARAD for the support and guidance they provided for the study. I thank research committee and finance of department KVV, Karad to support me with the funds to carry out the study. Also, I acknowledge the support of central store of KVV, Karad for timely arranging for the kits, reagents and required material for the project.

Conflict of Interest statement: None

Declaration Statement: The manuscript has been read and approved by all the authors, that the requirements for authorship as stated earlier in this document have been met, and that each author believes that the manuscript represents honest work

Ethical Approval and/or Institutional Review Board (IRB): Approval received from Institutional and Ethical committee of Krishna Vishwa Vidyapeeth Deemed to be University, KARAD previously known as Krishna institute of medical sciences deemed to be university, KARAD

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