Sepsis is a life-threatening organ dysfunction resulting from a dysregulated host response to infection and one of the most common causes of death in ICUs globally [1]. Despite the increasing efforts to reduce the mortality of sepsis with early fluid resuscitation, timely and adequate antibiotic treatment and lung protective mechanical ventilation (LPV), mortality is still high over 30% and approaching 60% in septic shock [2,3]. Such high mortality rates are a reflection of the complicated and poorly understood pathophysiology of sepsis and emphasize the continued need for effective adjunctive therapy [4].

In sepsis, inflammatory response and immunosuppression are two simultaneous phenomena, which interact complexly in the pathophysiology of the disease [5, 6]. Following the development of sepsis there is a significant increase in the serum levels of tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-8 (IL-8), as well as many other cytokines and chemokines, so therapy might target the pathways that mediate these mediator's release or clearance [7,8]. The response of dysregulated inflammatory chain has thus become a targeted area of adjunctive sepsis therapy research [9].

Ulinastatin is a naturally occurring inhibitor of the serine proteases present in human blood and urine. Its anti-inflammatory activity has been proven in animal models through the inhibition of the TLR4/NF-kappaB pathway, as well as decreased levels of inflammatory mediators and has shown promise in the management of multiple organ dysfunction syndrome with sepsis [10,11]. The findings from clinical trials are inconsistent; some retrospective clinical studies have demonstrated a decrease in mortality in patients with sepsis who receive ulinastatin therapy [12] but other trials of sepsis in elderly patients with multiple organ failure have not shown any mortality benefit. In view of this variability in the available evidence and lack of randomised controlled trial data on the use of ulinastatin in post-surgical sepsis patients in the surgical intensive care unit (SICU) of tertiary care centres in Pakistan, this study was designed to assess how ulinastatin affects the clinical outcomes of these patients.

Post-surgical settings present a unique challenge. First, there is a high prevalence of septic attacks in post-surgical settings, and second, the capacity of critical care is limited in resource-limited, tertiary care facilities, where the chances of septic attacks are especially pronounced. Various scoring systems are established to objectively assess illness severity and to predict mortality, which was used as base-line risk assessment for this trial, including the Sequential Organ Failure Assessment (SOFA) and the Acute Physiology and Chronic Health Evaluation (APACHE) [6,7]. This study aimed to evaluate ulinastatin as an adjunctive drug for local sepsis management through evaluating the both in-hospital death and resource utilization outcomes such as the length of ICU stay and hospital stay.

Study Design and Setting The research was a randomized controlled trial that was performed in the Department of Anaesthesia of the Jinnah Post graduate Medical Center (JPMC), Karachi from 30th December 2022 to 30th June 2023. After providing written informed consent and thus meeting the inclusion criteria, post-surgical patients with sepsis were included in the study upon their admission to surgical intensive care unit. ETHICAL APPROVAL The study was executed after receiving approval from the Research Evaluation Unit (Ref: CPSP/REU/ANS-2021-186-2617) from the College of Physicians and Surgeons Pakistan (CPSP) & institutional approval of Jinnah Postgraduate Medical Centre, Karachi. All the participants or their caregivers consented to participate in the study through written consent prior to enrolment. Sample Size and Sampling Technique The sample size of 132 (66 in each group) was determined with WHO sample size calculator as follows: Frequency of mortality at ulinastatin (55%) vs control (31%) with power of 80% and confidence level of 95%. A strategy of non-probability consecutive sampling was used. Inclusion Criteria ● Age 18-70 years of either gender. ● Septic patients who were post surgery ICU admitted as per operational definition. ● ASA physical status I, II and III. Exclusion Criteria ● Immunocompromised patients. ● Known history of a chronic kidney disease, chronic liver disease or ischaemic heart disease. ● Pregnant women. ● Contraindications to ulinastatin (e.g., known allergy). ● Terminally ill patients who have a do not resuscitate order. ● Sepsis treated in other hospitals for >3 days before admission. Data Collection Procedure On a predesigned proforma, baseline information regarding age, gender, and residential status and time of onset of symptoms was obtained after informed consent. The patients were randomly divided into 2 groups using opaque sealed envelope technique among those who were enrolled. Group A Ulinastatin ($2 \times 10^5 \mathrm{IU})$ was administered intravenously 12 hourly for 5 days along with the conventional therapy, while Group B only received conventional therapy. Scores of APACHE (calculated by the online MDCalc) and SOFA score were acquired at admission. All patients were followed up for a period of 10 days for assessing in hospital mortality, length of ICU stay, length of hospital stay, duration of ventilation, duration of inotropic support all on proforma. Statistical Analysis SPSS version 26 was used to enter and analyse data. For continuous data normality was checked by Shapiro Wilk test. Mean ± SD or median with IQR, as appropriate, was calculated for age, SOFA score, APACHE score, duration of ventilation, duration of inotropic support, length of ICU stay and length of hospital stay. For gender, residential status, type of ventilation, use of inotropic support and mortality, frequencies and percentages were calculated. The chi square / Fisher's exact test was used to compare mortality and length of stay in the ICU and hospital was compared using t test for independent samples. Stratification was used to control for confounding variables and a p value of <0.05 was deemed significant. throughout.

A total of 132 patients of post-surgical sepsis were screened, 66 of them per group. The mean age was 53.12 ± 13.61 years in Group A (ulinastatin) and 49.53 ± 14.79 years in Group B (control). In Group A there were 28 males (42.4%) and 38 females (57.6%), while Group B comprised 26 males (39.4%) and 40 females (60.6%). The mean SOFA score was 8.64 ± 2.40 in Group A and 7.83 ± 1.85 in Group B, and the mean APACHE score was 20.83 ± 3.36 and 19.88 ± 4.66 respectively. The baseline characteristics are shown in Table 1.

Table 1: Baseline Demographic and Clinical characteristics of both groups (n=132).

The length of the ICU stay (mean ± SD) was significantly shorter for the ulinastatin group than for control group (3.29 ± 1.77 versus 4.00 ± 1.40 days; p=0.012). In a like manner, the hospitalization duration was substantially decreased for the ulinastatin group (4.79 ± 1.66 vs 9.65 ± 2.16 days; p < 0.001). Duration of ventilation did not differ significantly between the groups (2.14 ± 1.70 vs 2.26 ± 1.61 hours). These are shown in Figure 1.

Figure 1. Comparison of Clinical Outcomes between Ulinastatin and Control Groups.

Eleven more patients who received in-hospital ulinastatin died, showing a statistically significant difference (p=0.008), compared to those who did not (35 patients in the ulinastatin group vs 20 patients in the control group), as shown in Figure 2 and Table 2.

Figure 2. In-Hospital Mortality between Ulinastatin and Control Groups.

Table 2: Between Groups Primary Outcomes (n=132).

The study of 132 post-surgical sepsis patients is a randomized controlled trial with interesting and somewhat paradoxical findings: ulinastatin has been associated with reduced length of stay in the intensive care unit (3.29 vs 4.00 days; p value 0.012) and in hospital (4.79 vs 9.65 days; p value less than 0.001), while at the same time being associated with increased in-hospital mortality (53.0% vs 30.3%; p value 0.008) when compared to conventional therapy alone. These opposing results should be interpreted with caution. The decrease in length of stay is in line with the anti-inflammatory and immunomodulatory activity of ulinastatin seen in preclinical models, where blockade of the TLR4/NF-κB pathway has been shown to have a protective effect on inflammatory injury to organs and may help promote faster recovery post-injury [10,11]. The increase in deaths in the ulinastatin group does not fit very nicely with some studies that have been published. In a retro cohort study of ulinastatin in sepsis, Zhu et al., reported that the use of ulinastatin could be correlated with enhanced survival [12] and Xu et al., indicated that ulinastatin could decrease the mortality of septic patients who are critically ill [14]. The current result however is similar to that of Uchida et al., who had not observed a mortality reduction effect of ulinastatin in elderly multiple organ failure patients [13]. Such imbalance may have affected the results of still higher mortality independently of the intervention in the ulinastatin group in this study, which is reflected in the higher scores on the SOFA (8.64 vs 7.83) and APACHE (20.83 vs 19.88) scores and higher use of inotropic support (75.8% vs 63.6%). In part it may be attributable to the complex and poorly understood pathophysiology of sepsis which is characterized by hyper- and immunosuppression occurring simultaneously [5,7]. Theoretically, inhibiting the inflammatory cascade with known immunomodulatory drugs may be beneficial for hyperinflammatory patients but harmful for those patients who have arrived at the immunosuppressed phase of the disease that is susceptible to secondary infection. There may, therefore, be a future need to consider giving ulinastatin in relation to the phenotype of the sepsis for future trials [8,9] to be a key factor in study outcome. Nevertheless, the significantly reduced hospital stay among survivors treated with ulinastatin could be important for clinical practice and economic considerations in a resource-limited tertiary care environment with high pressure on the use of intensive care rooms and hospital beds, and with current guidelines for care following Surviving Sepsis Campaign [15]. But without the mortality signal the benefit of this must not be overlooked. These results reinforce the importance of using surrogate measures like length of stay always in conjunction with the hard endpoints like mortality, and of assuming that decreases in the surrogate cannot necessarily be inferred as benefits overall [3,4]. Mechanistically, the net effect of using a broad-spectrum protease inhibitor like ulinastatin might depend on the dominant immunological phase when it is used [8,9]. To prevent excessive protease and cytokine activation in the early hyperinflammatory period of illness, this could help to minimize organ damage and shorten length of stay in the survivors. On the other hand, in those patients that have reached an immunocompromised state, additional immunomodulation may be detrimental, and lead to a secondary nosocomial infection and death [7]. This, a later-on/before model, provides a possible explanation for the divergent outcomes found in this group of patients and highlights the need for patient selection based upon phenotype in future trials of ulinastatin in sepsis. Limitations of Study This is a single centre trial with a number of limiting factors. Within a group that were randomised there was a higher severity of illness (shown by a higher SOFA and APACHE scores) in the ulinastatin group at baseline which could have trigged the mortality comparison. These imbalances in the sample size restrain power to make adjustments in the multivariable analysis. The 10-day follow-up will not provide information about longer-term survival. Mechanistic insight was limited without measurement of inflammatory biomarkers, e.g., IL-6 and TNF-α. Further, larger multicentre, severity-stratified, randomised controlled trials with longer follow-up times and measurement of biomarkers are warranted before definite conclusions can be made about ulinastatin.

A randomized controlled trial showed that ulinastatin was correlated with shorter length of stay in the Intensive Care Unit and hospital, but with a surprising increase in in-hospital mortality in post-surgical sepsis. These findings should be viewed with some caution due to a possible baseline severity imbalance and dissimilarities among certain literature. Based on this study, ulinastatin cannot be routinely recommended as adjunctive therapy for sepsis, and larger, adequately powered, strata multinational trials are required to unravel the actual effect of ulinastatin on clinical outcomes.

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