Abstract
Annually, around half a million people with severe dengue require hospitalization, all over the globe, with around 12,500 (2.5%) succumbing to the illness. In this prospective observational study, we recruited 74 patients with dengue infection, 28 had severe dengue and 46 had dengue with warning signs. sIL-2R levels were significantly raised in the severe dengue group, compared to the warning signs group. Using an ROC curve (receive operator characteristic), at a cutoff of 5.379 ng/ml, it predicted the severe dengue classification with a p-value of < 0.001. As a marker for predicting hemophagocytic lymphohistiocytosis (HLH), the ROC curve revealed a cutoff of ≥ 5.379 ng/ml for sIL-2R levels with the AUROC being 0.83, suggesting a strong diagnostic performance(p-value < 0.001). sIL-2R levels can be used for predicting severe dengue classification with moderate sensitivity and specificity. Secondary HLH is an under-reported entity in dengue infection and early surveillance with the help of sIL-2R may be helpful in-patient care management.
Introduction
Interleukin 2, (IL-2), being a significant cytokine of the immune system1, is involved in the modulation of protective immunity and sustaining immune tolerance by CD4 + regulatory T lymphocytes (Tregs)2,3,4. A dissoluble form of IL-2 receptor (sIL-2R) is secreted upon the activation of T- cells. Increased levels of this receptor in the blood are found in various immunological diseases5.
Soluble IL2 receptors
During the process of immune activation, lymphocytes undergo a myriad of changes viz., molecular, biochemical and, cellular, evolving from a resting, uncommitted state to a state of maturity and functional integrity3. In addition to the morphologic changes, including changes in cell size and chromatin configuration, various lymphotropic factors that interact with new cell surface determinants are secreted, often either before or at the time of DNA synthesis and cellular proliferation6.
The origin of sIL-2R is quite likely due to the enzymatic cleavage of IL-2R from the cell membrane. sIL-2R is currently regarded as a marker of T cell activation7,8,9. The IL-2R is composed of a minimum of three distinct polypeptide subunits -IL-2R-α, IL-2R-β, and IL-2R-ϒ. This cytokine is predominantly synthesized by activated T-cells, serving as a self-stimulating growth factor for T-cells and, concurrently, a neighbouring growth factor for NK-cells. Its significance lies in fostering immune tolerance through generation and maintenance processes10. Soluble IL-2-R-α chains, also called, sIL-2R are plentiful in the bloodstream of individuals in good health, but their levels are elevated even more in individuals dealing with infections, inflammation, and autoimmune conditions.
Soluble IL-2R levels in dengue
In a study conducted at a Bangkok Children hospital during 1987 and 1988 it was found that, levels of sIL-2R, sCD4, sCD8, IL-2 and IFN-ϒ were significantly higher in the acute sera of patients with dengue hemorrhagic fever than in the sera of healthy children (P < 0.001 for all markers)7. The acute sera of patients with Dengue fever (DF) were containing higher levels of sIL-2R, sCD4, IL-2 and IFNϒ than the sera of healthy children, but didn’t have elevated levels of sCD8.The levels of sIL-2R, sCD4 and sCD8 were higher in Dengue Hemorrhagic fever (DHF) than in DF on days 3–4 after the onset of fever. No significant differences in the levels of sIL-2R, sCD4, sCD8, IL-2 and IFNϒ among grades 1, 2 and 3 of DHF were noted. These observations led to the following conclusions; T-lymphocytes are activated and produce IL-2 and IFN-ϒ in vivo during DHF and DF. CD4 + T lymphocytes are activated both in DHF and DF, but to a lesser extent in DF. Activation of CD8 + T lymphocytes is evident in DHF but not in DF7.
Role of sIL-2R in the diagnosis of secondary hemophagocytic lymphohistiocytosis
Serum sIL-2R, a marker of excessively activated T cells is strongly elevated in primary and secondary forms of HLH. The updated HLH -2004 criteria have incorporated sIL-2R levels of more than 2400 U/ml as one of the criteria for diagnosis of HLH. Sensitivity is around 93% and specificity is 100% for pediatric HLH.
In addition to its application in diagnosing HLH, serum sIL-2R level may also act as an instrument to monitor disease activity in HLH as serum sIL-2R levels decline with clinical improvement vis a vis increasing sIL-2R levels are associated with clinical deterioration. Moreover, higher baseline levels of sIL-2R are reported to be associated with reduced survival compared to those having low levels11,12.
Secondary hemophagocytic lymphohistiocytosis (HLH) and dengue
Despite the fact that dengue is usually a self-limiting disease, many complications may occur among which Hemophagocytic Lymphohistiocytosis (HLH) has been increasingly reported13. Diagnosis can become grueling as it can easily be mistaken for other clinical entities like sepsis/other systemic inflammatory response syndrome (SIRS)14. There are numerous similarities between dengue and HLH, which include pronounced increase in cytokine levels, thrombocytopenia, increased levels of ferritin and evidence of hemophagocytosis may be present in dengue even in the absence of HLH itself. While most HLH cases associated with dengue were observed in dengue with warning signs/severe disease it may also present in classic dengue without warning signs15. There were few studies in pediatric age group where role of Soluble Interleukin-2 Receptor Level as marker of dengue severity were studied16.
This study aimed at using levels of a biomarker-soluble IL-2 receptor (sIL-2R) as a marker of severity in dengue infection in adult age group. The progression to severe dengue can also be attributed to the development of secondary hemophagocytic lymphohistiocytosis (HLH), which if detected early can be treated with HLH-directed therapy. The objectives of our study were to measure sIL-2R levels in all patients with dengue with warning signs and check the role of sIL-2R levels in predicting dengue severity.
Method
This is a prospective observational study conducted at a tertiary healthcare facility in north India from July 2022 to October 2023. Patients with age of ≥ 12 years having an acute febrile illness with thrombocytopenia and duration of illness less than and equal to seven days with a positive laboratory test for dengue infection (NS1 antigen or Dengue IgM antibody with clinical feature or Dengue virus RT PCR test) were recruited in this study. Patient were classified based upon dengue severity as per WHO 2009 dengue severity classification17. Patient without warning sign of dengue infection, established coinfection with other tropical infection or on immunosuppressive medication or malignancy were excluded. Patient without warning sign of dengue have mild illness with no major increase in the inflammatory marker and they recover spontaneously. We want to predict the Role of sIL-2R in sick patient who were admitted in the ward, especially for cost effective analysis of this test. Unnecessary testing patient with mild illness will not serve the purpose. Hence patient without warning signs were excluded. This study was approved by Postgraduate Institute of Medical Education and Research Chandigarh (PGIMER) Institutional Ethics Committee with No: INT/IEC/2022/SPL-1744 dated 15/11/22. All research was performed in accordance with relevant guidelines/regulations. Informed consent was obtained from all participants. Primary objective of this research was to see the role of sIL-2R in predicting severity of dengue infection. Role of sIL-2R in Predicting secondary HLH is secondary objective.
We also calculated H-Score in our patients to look for the incidence of HLH in dengue infection as the secondary outcome of our study18. H-Score was calculated in the patients using the web-based H-Score calculator as described by Fardet et al. for reactive hemophagocytic lymphohistiocytosis (HLH). Out of the nine variables, eight variables were used for calculating the H-Score, as bone marrow evaluation (for evaluation of Hemophagocytosis features) was not done19. An H-Score of 169 was initially validated for predicting HLH by Fardet, having a sensitivity of 93%, specificity of 86%, and accurately classifying 90% of patients. But it corresponds to a probability of 40–54% for hemophagocytic syndrome20. We used an H-Score of 191 (corresponding to the probability of 80–88% for hemophagocytic syndrome) to diagnose reactive or secondary HLH in our patients21.
Ninety-one patients were screened, out of which 17 were excluded from the study—four patients came out to be positive for scrub typhus, three patients although dengue positive were on immunosuppressants, for two patients none of the dengue tests as mentioned in methodology came out to be positive and eight patients weren’t included as their samples for sIL-2r couldn’t be collected because of logistic issues (Fig. 1).
Fig. 1
figure 1
Flowchart of patient recruitment.
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Cytokine assay
Samples were taken on the day of admission and were stored at − 70 °C.Human soluble IL-2 receptor levels were analyzed in serum samples using commercially available quantikine solid phase sandwich ELISA (Cat-BMS212-2 and BMS212-2TEN, ThermoFisher SCIENTIFIC). All reagents, standards, samples were prepared as per manufacturer’s recommendations. The detection rate of the kit was 0.312 ng/ml − 20 ng/ml with a minimum detectable limit of 0.21 ng/ml. Intra-assay and Inter-assay precision were 7.7% and 10% respectively. We used an ELISA kit for detecting sIL-2R levels with a range of 0.632–3.226 ng/ml among healthy controls22.
Statistical analysis
To analyse the data, SPSS (statistical package for social sciences) version 23 (IBM Corp.) was utilised. Descriptive statistics were utilized to determine the means/standard deviations and medians/IQRs for continuous variables”, as well as” frequencies and percentages for categorical variables”. Independent sample 't' tests were used to compare two groups for continuous data.” In case the data were non-normally distributed, Mann–Whitney U test was used. “For categorical data, non-parametrical tests like Chi-squared test were used”. Results having p-value < 0.05 were deemed significant. ROC analysis was performed for predicting an optimal cutoff for a continuous predictor predicting a binary outcome.
Results
Out of the 74 patients, 50 (67.6%) were males and 24(32.4%) were females. Total 26 (35%) patients were in the 21–30 years of age group. Forty five (60.8%) patients were below 30 years of age. Symptoms like headache, shortness of breath and altered mental status were significantly more common in the severe dengue group (P-value < 0.05). The presence of hepatomegaly was also associated with severe dengue (P-value < 0.05). (Table 1) Overall, in the SD group liver involvement was significant as compared to that in the DWS group with an increase in transaminases and bilirubin associated with severe disease. A higher Total leukocyte count (TLC) was also associated with severe disease. (Table 2) The median values of various inflammatory markers in the SD group were significantly higher as compared to those in the DWS group (Table 3). Using an H-Score of 191 as a cutoff for HLH, 13(17.6%) of our patients had HLH. Around 40% of our patients with severe dengue had HLH. The mortality rate among patients with HLH in our study was 46.2%. Values of all the inflammatory markers were stastically significant and higher in the SD group (Table 3).
Table 1 Patient symptom and sign at the time of admission in PGIMER Chandigarh.
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Table 2 Baseline laboratory parameter at the time of admission in PGIMER Chandigarh.
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Table 3 Comparison of various inflammatory markers between the two groups at time of admission in PGIMER Chandigarh.
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ROC curves were constructed for the assessing the performance of “sIL-2R levels in predicting the severity of disease in dengue patients. As a marker for predicting severe disease, the ROC curve revealed a cutoff of ≥ 5.379 ng/ml for sIl-2 levels with the area under the ROC curve (AUROC) being 0.84, thus demonstrating strong diagnostic performance (P-value < 0.001). The “sensitivity and “specificity” of this cutoff came out to be 64% and 87% respectively. (Fig. 2).
Fig. 2
figure 2
An ROC curve analysis showing the diagnostic performance of soluble IL-2R (ng/mL) in predicting dengue severity.
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We used an H- Score of 191 (corresponding to a probability of 80–88% for hemophagocytic syndrome) to diagnose reactive or secondary HLH in our patients.
With a cutoff of ≥ 5.379 ng/ml for sIL-2r levels, HLH can be predicted with a sensitivity of 85% and a specificity of 79%. The specificity for sIL-2R reaches almost 100% at levels of around 8.75 ng/ml (Fig. 3).
Fig. 3
figure 3
An ROC curve analysis showing diagnostic performance of soluble IL-2R (ng/mL) in predicting secondary HLH.
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Discussion
In this prospective observational study, soluble IL-2R level found to have moderate sensitivity and specificity in predicting severity of dengue infection. Levels of soluble IL-2R were found to be significantly higher in patient with severe dengue compared to dengue with warning sign group. In a study carried out in 2007, using the earlier classification for dengue, levels of soluble IL-2R were correlated with severity. Increased levels were related to the Dengue haemorrhagic fever (DHF). (P-value < 0.05)23. Another study conducted in Thailand on childern upto age 14 yrs where increased levels of sIL-2R were found to be directly linked to the severity of dengue7 (Table 4).
Table 4 Summary of some earlier studies on sIL-2r and dengue severity.
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As a marker for predicting HLH the ROC curve revealed a cutoff of ≥ 5.379 ng/ml for sIL-2R levels with the AUROC being 0.83, suggesting a strong diagnostic performance. A study done by Mahabala et al. in South India26, using sIL-2R level as a marker for dengue-associated HLH took an H-Score > 185 as a cutoff for HLH, it found the coefficient of correlation between sIL-2R and H-Score to be 0.9 and a cutoff value of 10,345 pg/ml(10.345 ng/ml) for predicting HLH with a sensitivity and specificity of 100%. The contradictory results between our and the aforementioned study can be clarified by the fact that the average duration of fever in our patients was five days with the maximum being seven days, however, in the aforementioned study, the cutoff for recruitment was dengue with more than 5 days of fever. As the duration of illness increases the probability of infections by other organisms especially opportunistic pathogens increases which may lead to raised levels of cytokine-like sIL-2R.In such a scenario the raised incidence of HLH may be explained by other infections27. Being a premier institute of national importance there is potential for referral bias and patient of dengue without warning sign were not included this may be limitation of this study. In conclusion raised levels of sIL-2R are associated with development of severe disease in the first week of dengue infection. Secondary HLH is an under-reported entity in dengue infection and early surveillance with the help of sIL-2R may be helpful in-patient care management.
Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.
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Authors and Affiliations
Department of Internal Medicine, PGIMER Chandigarh, Room No.11, 4th Floor F Block Nehru Hospital, Chandigarh, India
Arkit Deep Singh, Atul Saroch, Ashok Kumar Pannu, Saurabh C. Sharda & Mandeep Bhatia
Department of Experimental Medicine and Biotechnology, PGIMER Chandigarh, Chandigarh, India
Ashok Kumar Yadav & Kajal Kamboj
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Arkit Deep Singh
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2. Atul Saroch
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3. Ashok Kumar Pannu
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4. Ashok Kumar Yadav
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5. Saurabh C. Sharda
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Contributions
ADS: Conceptualization, Methodology, AS: Data curation, Writing—Original draft preparation and review. AKP: Formal analysis, Supervision: AKY: Investigation, Software, Validation.: SCS: Methodology, Writing—Reviewing and Editing, MB: Validation Conceptualization, KK: Investigation, Data curation. All authors reviewed the manuscript.
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Correspondence to Atul Saroch.
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Singh, A.D., Saroch, A., Pannu, A.K. et al. Role of soluble interleukin-2 receptor (sIL-2R) as a predictor for severe dengue infection. Sci Rep 15, 10834 (2025). https://doi.org/10.1038/s41598-025-91274-8
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Received:03 September 2024
Accepted:19 February 2025
Published:29 March 2025
DOI:https://doi.org/10.1038/s41598-025-91274-8
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