Correlation of Interleukin-6 Levels With Disease Severity in Rhino-Orbito-Cerebral Mucormycosis: A Prospective Study

Abstract

Background and Objectives

Rhino-orbito-cerebral mucormycosis (ROCM) shows elevated levels of the pro-inflammatory biochemical marker interleukin-6 (IL-6) compared with healthy subjects, and this increase is significantly correlated with morbidity and disease outcome. Surgical debridement has been shown to provide significant protection against mortality in patients with ROCM. This study aimed to identify serum IL-6 levels in patients with sino-nasal mucormycosis with or without orbital/cerebral involvement, both before and after operative interventions, and to explore their possible role in disease severity.

Methods

A single-centre prospective cohort study was conducted from June 2021 to December 2022. A total of 40 patients met the inclusion and exclusion criteria. Surgical debridement combined with antifungal therapy was performed as part of the management protocol. Pre-operative and post-operative IL-6 levels were assessed, with post-operative measurement performed on day 7. Patients were further subcategorised into three groups according to radiological extent, and separate analyses were conducted for these subgroups.

Results

Among the 40 patients, 21 (52.5%) had sinus involvement, 12 (30%) had sinus with orbital involvement, and 7 (17.5%) had sinus with orbital and cerebral involvement. Pre-operative IL-6 levels were significantly correlated with disease extent (p<0.001). In addition, there was a significant reduction between pre-operative and post-operative IL-6 levels (p<0.001).

Conclusion

The study indicates that IL-6 levels are correlated with disease severity. This marker may therefore serve as a valuable tool for pre-operative counselling, prognosis prediction, and guiding future treatment outcomes.

INTRODUCTION

Mucormycosis is a highly aggressive and rapidly progressive disease that requires urgent management due to its life-threatening nature. The sino-nasal subtype is particularly dangerous, as it can readily spread to the cerebral compartment, raising the mortality rate from 34% in the localised sinus subtype to 67% in the rhino-orbito-cerebral subtype [1]. The high mortality rate in rhino-orbito-cerebral mucormycosis (ROCM) has also been confirmed in a study from India by Chakrabarti et al. [2]. Mucormycosis is caused by fungi belonging to the class Zygomycetes and represents the third most serious invasive fungal infection after candidiasis and aspergillosis [3]. Patients with comorbidities such as diabetes mellitus, diabetic ketoacidosis, iron overload, cardiovascular disease, haemochromatosis, organ transplantation, or malignancy are predisposed to mucormycosis. Additional risk is associated with the concurrent use of systemic corticosteroids, immunosuppressive agents, or desferrioxamine therapy [4,5].

Elevated IL-6 stimulates ferritin synthesis, which leads to increased intracellular iron content. Accumulated intracellular iron promotes the production of reactive oxygen species, resulting in tissue destruction and the release of free iron into the bloodstream. Elevated serum iron, along with hyperglycaemia and ketoacidosis, enhances fungal growth and induces the expression of endothelial glucose-regulated protein 78 (GRP78) and spore-coating protein CotH. The interaction between GRP78 and CotH promotes angioinvasion and thrombosis, ultimately leading to tissue necrosis and death [6,7].

The interplay between pro-inflammatory and anti-inflammatory markers determines disease progression and outcome [8]. However, research on this aspect has been limited. Previous studies have demonstrated elevated IL-6 levels in patients with COVID-19-associated mucormycosis (CAM) and pulmonary mould infections [9-11]. Reddy et al. [6] reported that the median IL-6 level was significantly higher in patients with CAM compared with the COVID-19 patients. Nevertheless, the precise role of this pro-inflammatory marker in disease pathogenesis remains unclear. The present study was conducted following the epidemic of ROCM in India during late 2021 and 2022, at a tertiary healthcare centre in Western Rajasthan. At that time, limited literature was available and the evolving course of the disease remained unpredictable. This study aimed to measure immune biomarkers in non-COVID-19 patients with sino-nasal mucormycosis, with or without orbital/cerebral involvement, before and after operative intervention, and to explore their possible role in disease pathogenesis.

METHODS

Study design and patient selection

This was a prospective cohort study conducted at a tertiary care centre in North-Western India. Patients who developed ROCM between June 2021 and December 2022 and met the inclusion and exclusion criteria were enrolled. The study was approved by the Institutional Ethics Committee under certificate reference number AIIMS/IEC/2021/3645. Patients aged >18 years undergoing surgery for sino-nasal mucormycosis with or without orbital or cerebral involvement were eligible for inclusion. Patients who tested positive for COVID-19 by reverse transcription polymerase chain reaction (RT-PCR) within the previous 15 days, or those with active tuberculosis or autoimmune disorders, were excluded to minimise the possibility of confounding IL-6 levels.

Procedures

Contrast-enhanced computed tomography of the paranasal sinuses (CECT-PNS) was performed pre-operatively for all patients, while contrast-enhanced magnetic resonance imaging (CE-MRI) was performed in cases with suspected intra-orbital or intracranial extension.

Patients were categorised into three groups according to radiological extent, based on the classification used by Singh et al. [12]:

• Set 1: nose and paranasal sinus (PNS) involvement

• Set 2: nose and PNS involvement with orbital extension

• Set 3: nose, PNS, and orbital involvement with intracranial extension

Surgical debridement, determined by disease extent and tissue viability, was performed alongside antifungal therapy with liposomal amphotericin B. The drug was administered at a dose of 5–10 mg/kg/day (10 mg/kg/day for intracranial involvement). The maximum cumulative dose was 5 g, except in patients with intracranial involvement, where the maximum dose was up to 10 g.

All included patients had histologically confirmed mucor infection according to the 2020 Revision and Update of the Consensus Definitions of Invasive Fungal Disease by the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium (EORTC/MSGERC) [13]. Pre-operative blood samples were collected prior to surgery, and post-operative samples were collected on day 7. The choice of post-operative day 7 was based on the study by Cruickshank et al. [14], which demonstrated that IL-6 levels require 100–150 hours after surgery to return to baseline. This time point was therefore selected to minimise the confounding influence of surgery on serum IL-6 measurement.

Data collection

Biochemical testing for IL-6 estimation was performed in the Research Laboratory, Department of Biochemistry, using serum samples. Blood was obtained via venepuncture under universal precautions. Samples were left at room temperature for 20 minutes to clot, followed by centrifugation for 8 minutes at 4,000 revolutions per minute (RPM). The resulting serum supernatant was collected in aliquot vials and preserved at -80°C until analysis. All samples were analysed in batch following completion of collection. Prior to analysis, aliquots were brought to room temperature and homogenised using a Remi CM-101 Plus Cyclo Mixer, a laboratory vortex mixer (Fig. 1). Estimation of IL-6 was performed pre-operatively and on post-operative day 7 using the ADVIA Centaur^® Interleukin-6 chemiluminescence assay (Siemens Healthineers).

Fig. 1.

Process of homogenisation using a vortex mixture.

Statistical analysis

Data were entered into Microsoft Excel 2019 and analysed using SPSS version 23 for Windows (IBM Corp.). Categorical variables were compared using the chi-square test or the Fisher exact test, as appropriate. Analysis of variance was used to compare mean values between groups. Since pre-operative and post-operative IL-6 levels did not follow a normal distribution, non-parametric tests were applied. The Wilcoxon signed-rank test was used to compare pre- and post-operative IL-6 levels, while the Kruskal–Wallis test was employed to assess the association between pre-operative IL-6 levels and radiological extent of disease. Multivariate regression analysis was conducted to identify predictors of pre-operative IL-6. A p-value of <0.05 was considered statistically significant.

RESULTS

A total of 40 patients with sino-nasal mucormycosis with or without orbital or cerebral involvement were included in the study, comprising 29 males and 11 females. The mean age at presentation was 51.1±11.8 years, with a range of 24–73 years. At presentation, 42.5% (17/40) of patients had diabetes, while 27.5% (11/40) had diabetes and an additional history of steroid intake. All 28 of these patients had uncontrolled blood sugar levels at the time of admission. Among the 12 patients without diabetes, 6 had a history of steroid intake, while the remaining 6 had no such history (Table 1).

Table 1.

Baseline characteristics and distribution of patients according to radiological involvement of disease and findings of radiological subsets

History	Overall (n=40)	Radiological extent†			p-value
		Set 1 (n=21)	Set 2 (n=12)	Set 3 (n=7)
Sex					0.410
Male	29 (72.5)	17 (81.0)	8 (66.7)	4 (57.1)
Female	11 (27.5)	4 (19.0)	4 (33.3)	3 (42.9)
Age (yr)	51.1±11.8	51.0±12.0	54.4±11.3	45.9±11.4	0.318
HbA1c (%)	6.9±1.0	6.4±0.7	7.3±0.9	7.9±1.1	<0.001*
History of COVID-19 RT-PCR test positive	19 (47.5)	13 (61.9)	4 (33.3)	2 (28.6)	0.156
History of steroid intake	6 (15.0)	4 (19.0)	2 (16.7)	0	0.465
History of DM	17 (42.5)	5 (23.8)	8 (66.7)	4 (57.1)	0.039*
History of both steroid intake and DM	11 (27.5)	9 (42.9)	1 (8.3)	1 (14.3)	0.070
No history of steroid intake or DM	6 (15.0)	3 (14.3)	1 (8.3)	2 (28.6)	0.487

Values are presented as n (%) or mean±standard deviation.

^*p<0.05;

^†Set 1: nose and paranasal sinus (PNS) involvement; Set 2: nose and PNS involvement with orbital extension; Set 3: nose, PNS, and orbital involvement with intracranial extension.

HbA1c, haemoglobin A1c; RT-PCR, reverse-transcription polymerase chain reaction; DM, diabetes mellitus.

Nineteen patients had a history of COVID-19 RT-PCR positivity, while 21 patients had no such history. Of the 19 patients with a positive COVID-19 history, 18 were classified as having late CAM, as defined by Muthu et al. [15] in a 2021 systematic review. As defined by those authors, late CAM refers to mucormycosis detected between 7 days and 3 months after COVID-19 confirmation. In keeping with the study’s exclusion criteria, none of these 18 patients had tested COVID-19 RT-PCR positive within the preceding 15 days. The remaining patient with prior COVID-19 positivity developed mucormycosis more than 3 months after infection.

The frequency distribution of presenting complaints is summarised in Table 2. On diagnostic nasal endoscopy (DNE), 20 of 40 patients showed discoloured mucosa or discharge, while the remaining 20 had no gross abnormalities. Vision testing revealed reduced vision in 9 of 40 patients, while the rest had normal vision. Oral cavity examination revealed that 9 patients had loose teeth, 8 had palatal discolouration, and 6 had both loose teeth and palatal discolouration. Two patients presented with a palatal fistula, while the remaining 15 had normal findings (Table 2).

Table 2.

Distribution of patients according to their presenting complaints and examination finding

Complaints	Value (n=40)
Nasal complaints	9 (22.5)
Ophthalmic complaints	17 (42.5)
Headache	15 (37.5)
Facial pain	38 (95.0)
Dental complaints	24 (60.0)
Fever	2 (5.0)
Diagnostic nasal endoscopic findings
Normal	20 (50.0)
Discoloration/Discharge	20 (50.0)
Vision examination
Normal	31 (77.5)
Reduced vision	9 (22.5)
Oral cavity examination
Normal	15 (37.5)
Loose tooth	9 (22.5)
Palate discoloration	8 (20.0)
Both loose tooth and palate discoloration	6 (15.0)
Palatal fistula	2 (5.0)

Values are presented as n (%).

Radiological evaluation demonstrated that all 40 patients (100%) had involvement of two or more paranasal sinuses. Classification of disease extent was based on the criteria outlined by Singh et al. [12] in non-COVID patients, dividing cases into three subsets according to radiological involvement (Table 3).

Table 3.

Comparison between pre-operative and post-operative levels of IL-6, and associations of pre-operative and post-operative IL-6 levels with the radiological extent of disease

	Overall (n=40)	Radiological extent‡			p-value*
		Set 1 (n=21)	Set 2 (n=12)	Set 3 (n=7)
Pre-operative IL-6 (pg/mL)	48.0 (29.9–122.4)	32.7 (24.5–48.0)	96.8 (40.8–164.3)	181.9 (122.4–273.2)	<0.001
Post-operative IL-6 (pg/mL)	12.0 (6.5–59.3)	6.5 (5.8–11.1)	34.8 (12.1–68.2)	73.8 (50.9–107.5)	<0.001
p-value†	<0.001	<0.001	0.002	0.018

Values are presented as median (interquartile range).

^*Kruskal-Wallis test;

^†Wilcoxon signed-rank test;

^‡Set 1: nose and paranasal sinus (PNS) involvement; Set 2: nose and PNS involvement with orbital extension; Set 3: nose, PNS, and orbital involvement with intracranial extension.

IL-6, interleukin-6.

The majority of patients fell into set 1, followed by sets 2 and 3, respectively (Table 1). Further analysis revealed that most patients in each subset had a history of either steroid intake or diabetes mellitus. Disease extent showed a strong correlation with diabetes mellitus and glycated haemoglobin (HbA1c) levels (Table 1).

Comparison of pre-operative and post-operative levels of the pro-inflammatory cytokine IL-6 demonstrated a statistically significant difference (p<0.05) in both overall and subset analyses (Table 3 and Fig. 2). Median IL-6 levels also increased with disease progression from stage 1 to stage 3, a trend found to be statistically significant (Table 3). On multivariate regression analysis, sex and mucormycosis were identified as significant predictors of pre-operative IL-6 levels (Table 4).

Fig. 2.

Box-whisker plot showing the comparison between pre-operative and post-operative levels of interleukin-6 (IL-6) overall and in a sub-group analysis. Radiological extent. Set 1: nose and paranasal sinus (PNS) involvement; Set 2: nose and PNS involvement with orbital extension; Set 3: nose, PNS, and orbital involvement with intracranial extension.

Table 4.

Multivariate regression analysis for predictors of pre-operative IL-6 level

Independent variable	Unstandardized coefficients		Standardized coefficients	p-value
	B	Std. error	Beta
Age	-0.58	0.64	-0.26	0.365
Sex	-42.85	18.21	-0.50	0.025
History of RT-PCR positivity	43.12	37.78	0.60	0.262
History of steroid use	-80.99	39.89	-1.15	0.050
HbA1c	14.54	7.29	0.87	0.054
Mucormycosis involvement	80.18	13.08	1.26	<0.001

RT-PCR, reverse-transcription polymerase chain reaction; HbA1c, haemoglobin A1c.

DISCUSSION

Sino-nasal mucormycosis is a highly fatal disease owing to its angio-invasive nature, and it requires urgent intervention. Mortality increases significantly when the disease extends to the brain [1]. A reliable biochemical marker that can be easily isolated from a patient’s blood would be valuable for assessing disease status, monitoring progression or regression, and evaluating treatment effectiveness. In line with previous studies [16], our findings confirmed a male preponderance of sino-nasal mucormycosis. Diabetes mellitus emerged as the most common risk factor, consistent with earlier reports [1,15,17]. In our study, 70% of patients had diabetes mellitus as an underlying condition. Most patients in our cohort fell into set 1, followed by sets 2 and 3, a distribution that contrasts with the study by Singh et al. [12], in which the majority of patients presented in set 3, followed by sets 2 and 1. Importantly, our study demonstrated a statistically significant association between serum IL-6 levels and the radiological extent of disease [9,18]. A meta-analysis by Said et al. [19] reported a pooled estimate of IL-6 in healthy individuals as 5.186 pg/mL (95% confidence interval: 4.631–5.740). In contrast, in our study, the median pre-operative IL-6 level was 48.0 pg/mL (interquartile range [IQR], 29.9–122.4), which decreased to 12.0 pg/mL (IQR, 6.5–59.3) on post-operative day 7. Both values were markedly higher than those reported in healthy individuals.

Reddy et al. [6] demonstrated that IL-6 levels were significantly elevated in patients with CAM compared with nonmucor COVID-19 patients. They reported a median (IQR) IL-6 level of 56.37 pg/mL (30.6–203.89) in CAM patients, compared with 29.4 pg/mL (15.2–54.7) in non-mucor COVID-19 patients. Similarly, in our study, the median pre-operative IL-6 level was 48.000 pg/mL (29.875–122.375), which decreased to 12.000 pg/mL (6.500–59.275) on post-operative day 7. In all three subsets, pre-operative IL-6 levels were consistently higher than those on postoperative day 7. To date, there has been limited literature comparing pre-operative and post-operative IL-6 levels in patients with sino-nasal mucormycosis with or without orbital/cerebral involvement. Our findings demonstrated a statistically significant reduction in IL-6 levels from pre-operative to post-operative day 7 in both overall and subset analyses.

Limitation

This study has certain limitations. First, the absence of a healthy control group precluded direct comparison of serum IL-6 levels between patients and healthy individuals. Second, the follow-up period was relatively short, limiting the assessment of long-term outcomes. As this was a single-centre prospective study with a small sample size, the findings require further validation through multi-centre studies with larger cohorts and longer follow-up. Such studies would help confirm these results, identify trends, and establish IL-6 as a predictor of survival, mortality, and complications. The primary objective of this study was to compare pre-operative IL-6 levels with post-operative day 7 levels. No long-term follow-up was performed to evaluate IL-6 as a prognostic marker.

Conclusion

The study concluded that median serum IL-6 levels in patients with ROCM were markedly higher than pooled estimates reported in healthy individuals. A statistically significant reduction was observed between pre-operative IL-6 levels and post-operative day 7 levels across the cohort. To our knowledge, this is the first study to compare pre-operative and post-operative IL-6 levels in patients with sino-nasal mucormycosis with or without orbital or cerebral involvement. The findings indicate that IL-6 may serve as a useful biomarker for monitoring disease severity and treatment response.