Blog

Genetic sequencing of peptides in rebound virus in individuals with HIV who had analytic treatment interruptions (ATIs) confirmed the peptides’ expression in HIV-1 infection, according to data presented at the International AIDS Society Conference on HIV Science. 

Previous research has shown that HIV-specific CD8 T-cell responses directed against five genetically conserved HIV-1 protein regions (Gag, Pol, Vif, Vpr, and Env) are associated with viral control, wrote Josefina Marín-Rojas, PhD, Faculty of Medicine and Health, University of Sydney, Sydney, Australia, and colleagues in their abstract.

However, data on whether these peptides are expressed in rebound virus among individuals with HIV who experienced ATI are limited, they wrote.

The researchers applied an immunoinformatics analysis pipeline (IMAP) to select 182 peptides (IMAP-peptides) from structurally important and mutation-intolerant regions of HIV-1 proteins, said senior author Sarah Palmer, PhD, co-director of the Centre for Virus Research at the Westmead Institute for Medical Research and professor in the Faculty of Medicine and Health at the University of Sydney, in an interview.

“Our studies indicate if the immune system targets these structurally important and mutation-intolerant regions of HIV-1 proteins, this can contribute to virological control in the absence of HIV-1 therapy,” she explained.

The researchers reviewed data from the PULSE clinical trial, which included 68 men who have sex with men living with HIV in Australia. The study participants underwent three consecutive ATIs. A total of seven participants’ transiently controlled HIV rebound during the third ATI. The researchers examined whether the IMAP peptides were present in the HIV-1 RNA sequences of the rebound virus in four noncontrollers (patients who had viral rebound in all three ATIs) and five of the seven transient controllers who showed viral control during the third ATI.

The technique of near full-length HIV-1 RNA sequencing of rebound virus from three noncontrollers and two transient controllers identified the Gag, Pol, Vif, Vpr, and Env IMAP-peptides in 52%-100% of the viral sequences obtained from these participants across three ATI timepoints.

“We assumed that cells from people living with HIV that experience virological control after treatment interruption would have the immune response to our IMAP-peptides that we observed; however, we are amazed and encouraged by the level and extent of this immune response,” Palmer told Medscape Medical News.

The researchers also compared CD8 T-cell response between the IMAP peptides and a control peptide pool without the IMAP peptides.

The CD8 T-cells from three transient controllers had a 15- to 53-fold higher effector response to the IMAP-peptides than the CD8 T-cells from two noncontrollers, the researchers wrote in their abstract. The relative response to the IMAP-peptides in noncontrollers was 20 times lower than that to the control peptides, but the IMAP-peptide response in transient controllers group was similar to that in the control group, the authors noted.

The results highlight the potential of IMAP in developing treatment strategies. Although the results are too preliminary to impact clinical practice at this time, the findings from the current study could lead to the development of an mRNA vaccine to clear HIV-infected cells from people living with HIV, Palmer told Medscape Medical News.

“Our next steps include developing and testing mRNA vaccine constructs that contain our IMAP-peptides to assess the immune response of cells from people living with HIV to these vaccines,” Palmer told Medscape Medical News. “From there we will conduct studies of the most promising mRNA vaccine constructs in a humanized mouse model,” she said.

Data Enhance Understanding of Immunity

The current study may provide information that can significantly impact understanding of the immune responses to HIV, said David J. Cennimo, MD, associate professor of medicine & pediatrics in the Division of Infectious Disease at Rutgers New Jersey Medical School, Newark, New Jersey, in an interview.

“The investigators looked at highly conserved regions of multiple HIV proteins,” said Cennimo, who was not involved in the study. “Conserved regions and antibody responses to them may play a role in controlling HIV viral replication and rebound,” Cennimo told Medscape Medical News. “The investigators showed these regions were present in rebounding viremia, and individuals that exhibited greater immune recognition of these regions suppressed rebound viremia longer, and perhaps targeting these regions could impact HIV prevention or cure strategies,” he said.

Secondarily, the study showed the success of the novel technique (IMAP) to identify conserved peptides, said Cennimo. The technique could potentially be applied to other viruses that mutate to escape host response, he said.

The study was funded by the US National Institutes of Health, the Foundation for AIDS Research, the Australian National Health and Medical Research Council, and Sandra and David Ansley. 

The researchers disclosed no financial conflicts of interest. 

Cennimo had no financial conflicts to disclose.

Introduction

Vancomycin (VAN) is a narrow-spectrum glycopeptide antibiotic primarily used as first-line treatment for Gram-positive infections including pneumonia, endocarditis, osteomyelitis, and diarrhea.¹ VAN is essential in sepsis management protocols in ICU settings when Gram-positive infections are suspected or confirmed. In particular, critically ill patients show altered volume of distribution and impaired renal clearance which necessitates monitoring of VAN systemic levels.²

Neutrophils are the primary cells that respond to bacterial infections. A component of the innate immune response, neutrophils utilize several key mechanisms to combat pathogens including phagocytosis, degranulation, apoptosis, and NETosis. Bacteria counteract these mechanisms by inhibiting chemotaxis, resisting engulfment, neutralizing oxygen radicals, preventing phagolysosome fusion, cytotoxicity, anti-apoptotic activity, and degradation of extracellular traps. As such, capsules, biofilms, toxins, and DNases, constitute the major virulence factors that contribute to microbial success and establishment of persistent infections.^3–5 Although the reference interval for peripheral neutrophils is 3,000 to 6,000 cells per cubic milliliter, the maintenance of at least 500 cells per cubic milliliter has been demonstrated to be sufficient to mount a successful immune response.⁶ Additionally, lymphocytes participate in eliminating intracellular bacteria,⁷ although sepsis by Gram-positive organisms has been shown to suppress distinct lymphocyte subsets more strongly than that by Gram-negative organisms.⁸ Variations in systemic numbers of neutrophils and lymphocytes are thus a crucial aspect in the immune response against bacteria.

Neutrophil-lymphocyte ratio (NLR) is one of the most extensively studied systemic inflammatory markers, with recent evidence increasingly supporting its clinical application in the management of infectious disease. NLR predicts postoperative infection^9–11 and correlates with bacterial colonization in COPD,¹² endocarditis,¹³ severity of pneumonia,^14,15 and procalcitonin in sepsis.¹⁶ Moreover, it differentiates patients with Gram-positive infections from those with fungal infections¹⁷ and COVID-19 patients from their influenza counterparts.¹⁸ In critically ill COVID-19 patients, NLR is significantly elevated in subjects with low serum 25(OH)D compared to those with normal levels.¹⁹ Notably, NLR also predicts mortality after HBV infection,²⁰ COPD,²¹ COVID-19,^22,23 and bacteremia.²⁴ However, it fails to identify childhood brucellosis,²⁵ urinary tract infection,²⁶ or biofilm-forming pathogens in cystic fibrosis patients.²⁷

Compelling evidence underscores the need for rigorous validation of NLR as a biomarker of infection severity. This study was therefore designed to analyze the association between NLR and ICU status in adults undergoing VAN treatment which may help refine risk stratification in critical settings.

Materials and Methods

Patients and Data Collection

Ethical clearance for this study was issued by the Ethics Committee of King Saud University (E-25-9553). Consent was waived by the committee due to the retrospective nature of the study and patient data were anonymized to ensure confidentiality. The study was conducted according to the principles of the Declaration of Helsinki. The inclusion criteria were being an adult of at least 18 years of age and on VAN therapy. Patient charts for 300 subjects who were given VAN from January 2024 to February 2025 were reviewed and extracted. Five subjects were excluded due to missing NLR results, and data for 295 patients were analyzed. Based on the distribution of NLR and ICU status, the study had over 90% power (α = 0.05) to detect the observed association between NLR and critical illness. Laboratory data were collected along with VAN trough level determination which is immediately before the fourth dose. Isolated organisms and antimicrobial profiles of patients are shown in supplementary Table S1 and S2, respectively. Neutrophils and lymphocytes were counted using XN-2000 hematology analyzer (Sysmex Corporation, Kobe, Hyogo, Japan) based on fluorescence flow cytometry, and NLR was calculated as follows:

The cutoff for elevated NLR was determined at >5.58 based on ROC curve analysis (Youden’s index = 0.35) which is consistent with previous studies.^28,29

Statistical Analysis

All analyses were performed by Prism 9.0 (GraphPad Software, Inc., San Diego, CA, USA). Nonparametric tests were used since the data were skewed as demonstrated by the Shapiro–Wilk test. Medians were compared by the Mann–Whitney U-test and prevalence rates were assessed using the Chi-square test. Spearman correlation analysis and regression models were employed to assess the relationship between NLR and other factors and covariates. Prevalence ratio (PR) and odds ratio (OR) were computed to estimate the effect size of NLR on ICU admission. The diagnostic performance was examined by calculating the area under the curve (AUC) derived from ROC curve analysis.

Results

In Figure 1, NLR was significantly elevated (p <0.0001) in ICU compared to non-ICU patients of both genders (Figure 1A), in males (Figure 1B), and in females (Figure 1C). Stratified by age, young (Figure 1D), adults (Figure 1E), and elderly subjects (Figure 1F) in the ICU all had significantly increased NLR than those not admitted to the ICU (p <0.0001).

Figure 1 Elevated NLR in ICU patients. NLR values for (A) all subjects, (B) males, (C) females, (D) young, (E) adults, and (F) elderlies. Data are shown as medians + IQR. ****(p <0.0001).

Table 1 separates patients into normal NLR and high NLR groups and compares demographic and clinical parameters between the two cohorts. It was revealed that patients with increased NLR were significantly older (p = 0.0127), had lower SBP (p = 0.0098) and DBP (p <0.0001), increased creatinine (p = 0.0129), electrolyte imbalance with diminished Ca²⁺ (p = 0.0036) and elevated Mg²⁺ (p = 0.0101), abnormal liver markers, worse RBC indices, and depleted basophils (p = 0.0029) and eosinophils (p <0.0001) than those with normal NLR.

Table 1 Patient Characteristics Stratified by NLR

The distribution of subjects based on ICU status is shown in Table 2. The percentage of ICU patients with high NLR was significantly higher than those with normal NLR (X² = 33.69, p <0.0001) which persisted when either gender was analyzed alone.

Table 2 Distribution of Study Subjects Based on NLR and ICU Status

Spearman’s rank correlation identified several factors associated with NLR (Table 3). Markers that were significantly correlated with NLR when both sets of cohorts were analyzed together included SBP, ALT, AST, unconjugated bilirubin, RBCs, hemoglobin, and basophils. However, the statistical significance was lost upon separation of subjects by ICU status. Monocyte count was the only exclusively significant marker in non-ICU patients, whereas weigh, BMI, Cl⁻, and HCO₃⁻ were significantly correlated with NLR only in ICU patients. Notably, age, Ca²⁺, Mg²⁺, CO₂, albumin, and ALP showed significant correlations with NLR when both cohorts and when non-ICU subjects were analyzed, but not in the critically ill. In contrast, DBP, hematocrit, MCHC, and platelets were significantly correlated with NLR in all subjects and in ICU patients, but not in non-ICU individuals. Creatinine, total bilirubin, and conjugated bilirubin were the only markers whose significant correlations persisted in all patient groups.

Table 3 Correlation of NLR with Clinical Parameters

The regression analysis (Table 4) demonstrated consistent significant association between ICU admission and elevated NLR. In the unadjusted model, ICU was a significant predictor of NLR (B = 0.014, p <0.0001); an effect likely undermined by covariates unaccounted for. When corrected for age and gender, the association became stronger (B = 8.971, p <0.0001) and age emerged as a significant but modest predictor of NLR (B = 0.0715, p = 0.0475). In the fully adjusted model, the association of ICU and NLR was further reinforced (B = 9.651, p = 0.0005) and BMI (B = −0.392, p = 0.0340) and direct bilirubin (B = 0.056, p = 0.0051) were identified as negative and positive predictors, respectively.

Table 4 Regression Analysis of NLR

Table 5 outlines the risk measures of ICU status and NLR. ICU requirement was 2.49 and 2.54 times more common in males and females with elevated NLR, respectively (p = 0.0002). Also, ICU admission was 4.25 and 4.40 times more likely when NLR was elevated in males and females, respectively (p = 0.0001). Specifically, there was a 78% and 76% chance of ICU admission when NLR was elevated as revealed by the positive predictive value (PPV) of 0.78 for males and 0.76 for females. Nonetheless, the negative predictive value (NPV) was moderate (0.54 for males and 0.55 for females) indicating that normal NLR does not reliably rule out the need for intensive monitoring.

Table 5 Risk Measures of ICU Status by NLR

In Table 6, the diagnostic accuracy of NLR for ICU requirement was evaluated. The AUC was significant and comparable between both genders and among all age groups ranging from 0.7186 to 0.7838, reflecting a good discriminatory ability. Figure 2 shows the ROC curves for all patients stratified by gender and by age.

Table 6 Diagnostic Accuracy of NLR for ICU Admission

Figure 2 Diagnostic accuracy of NLR for ICU admission. ROC curves for (A) all subjects, (B) males, (C) females, (D) young, (E) adults, and (F) elderlies.

Discussion

VAN is widely used for serious Gram-positive infections and a substantial portion of patients often require intensive monitoring. Sensitive markers that could reliably identify those at risk of ICU admission are, however, lacking. This study presents the first evidence of the association between elevated NLR and the requirement for ICU admission in adult patients receiving VAN. Altogether, the findings enclosed herein may be invaluable to incorporate NLR in the routine management of patients with infection-related critical illness.

It is demonstrated that increased NLR reliably segregates ICU and non-ICU patients receiving VAN (Figure 1) which is comprehensible considering the pivotal role of neutrophils as first responders to bacterial infections. Naess et al²⁹ reported that increased NLR was observed in hospitalized patients with septicemia which also outperformed traditional markers including leukocytes, neutrophils, and C-reactive protein. However, procalcitonin was better than NLR in predicting bacterial infection.³⁰ We were unable to assess procalcitonin levels in our cohort due to missing data although it would have provided valuable insights particularly in relation to NLR and under VAN exposure; a context which has not yet been studied.

Certain characteristics were significantly different between the two patient groups. Subjects with increased NLR were significantly older which is consistent with accumulating inflammation during aging.³¹ In neutrophils, aging attenuates receptor sensitivity and downstream functions including chemotaxis and phagocytosis which increases susceptibility to infection.³² Neutrophils may also exhibit delayed egress from the site of infection which further exacerbates inflammation.³³ In contrast, experimental studies have shown that aged neutrophils challenged with bacterial products released more free radicals and extracellular traps and displayed stronger phagocytosis.³⁴ Therefore, neutrophil subpopulation analysis may be valuable to accurately assess the contribution of aged cells to the inflammatory damage associated with bacterial infection.

Individuals with increased NLR also had significantly lower BP (Table 1) which was negatively correlated with NLR in ICU patients (Table 3). Hypotension is commonly observed in critically ill patients especially those with septic shock due to various mechanisms including vasodilation by nitric oxide and prostaglandins, increased endothelial permeability, and reduced cardiac output. This eventually leads to impaired tissue perfusion and multi-organ failure if fluid resuscitation and vasoactive agents are not promptly instituted.³⁵

BMI was also a negative independent predictor of NLR under VAN therapy (Table 4). Obese individuals often have chronic, low-grade inflammation which might mask acute NLR elevations. Additionally, leptin and adiponectin released by adipose tissue differentially regulate neutrophil function,³⁶ potentially altering NLR values. Indeed, the nutritional status of ICU patients and the altered VAN distribution and clearance relative to the BMI deserve further exploration. Although our regression model is adjusted for major confounders, it is important to mention that concurrent infection and medication use, especially steroids and immunosuppressants, may potentially impact NLR. Controlling for these variables is likely to strengthen the association between NLR and critical illness.

Importantly, significant correlations with elevated NLR were observed in ICU patients including increased creatinine and diminished Cl⁻ and HCO₃⁻ (Table 3). Also, lower Ca²⁺ and higher Mg²⁺ were seen in patients with elevated NLR (Table 1). Cytokines released due to systemic inflammation may lead to vasoconstriction and compromised renal perfusion which complicates the nephrotoxicity caused by VAN exposure leading to electrolyte imbalance. Specifically, hypocalcemia, an extremely common finding in the critically ill,³⁷ is caused by parathyroid suppression or resistance, increased cellular uptake, and compromised Mg²⁺ and calcitriol metabolism.³⁸ Notably, Steele et al³⁹ reported that hypocalcemia was associated with a longer ICU stay and mortality and a similar finding was also observed in pediatrics.⁴⁰ Interestingly, it has been suggested that hypocalcemia may rather play a protective role in sepsis⁴¹ and corrective treatment may in fact worsen patient outcomes.⁴² Collectively, these findings strongly suggest that NLR may serve as a novel systemic marker of impaired kidney function and acid-base dysregulation in critically ill patients on VAN therapy. In particular, monitoring NLR trends relative to acute kidney injury before, during, and after administration of VAN may help mitigate potential renal derangement and ionic dysregulation.

Liver markers showed significant differences in subjects with increased NLR (Table 1) and regression analysis revealed that conjugated bilirubin was an independent predictor of NLR in ICU settings (Table 4). In particular, divided by NLR, we found that patients with high values had significantly lower albumin levels. It is fairly established that albumin is a negative acute-phase reactant and inflammation-related hypoalbuminemia often develops secondary to increased capillary permeability, impaired hepatic synthesis, accelerated albumin catabolism, and increased urinary and gastrointestinal loss. Alarmingly, careful attention must be given to albumin monitoring as previous studies have found a significant association between reduced levels and worse patient outcomes including mortality.⁴³

Increased AST, ALT, and ALP activities were also noted in the high NLR group which is consistent with liver dysfunction encountered in critical illness and sepsis. Transaminases reflect hypoxic hepatitis whereas ALP and conjugated bilirubin are highly suggestive of hepatobiliary obstruction. In fact, jaundice is a significant risk factor for complications and death in ICU patients.⁴⁴ Nonetheless, it must be noted that, similar to hypocalcemia, mild hyperbilirubinemia in sepsis may rather be protective through mitigating oxidative stress.⁴⁵ Interestingly, NLR has been shown to be a predictor of complications following hepaticojejunostomy for bile duct injury repair⁴⁶ and a prognostic marker of liver transplantation patients.⁴⁷

ROC curve analysis revealed that NLR has a good diagnostic power for ICU admission (Table 6 and Figure 2) which is in line with Riché et al who reported that NLR predicts mortality due septic shock.⁴⁸ It has been demonstrated that neutrophil mobilization from the bone marrow is under the regulation of two opposing mediators, C-X-C chemokine receptor type 2 (CXCR2) and CXCR4,³⁴ which could offer mechanistic insights into the observed increase in NLR. Importantly, since it had a PPV of 78% and 76% in males and females, respectively (Table 5), NLR may be used to risk-stratify patients undergoing VAN therapy either as a standalone test or alongside other markers. However, longitudinal studies in diverse populations are urgently needed to confirm these findings.

In conclusion, this report shows that NLR is correlated with critical illness under VAN therapy. Although the cross-sectional design and the single-center setting preclude establishment of causality between elevated NLR and ICU admission and limit generalizability to other settings or populations, the current study benefits from a well-defined cohort, uniform data collection and clinical protocols, and a comprehensive dataset. Further prospective, multi-center studies in diverse populations are likely to reveal the prognostic implications of NLR, in addition to established markers such as procalcitonin, as a novel index of infection severity and responsiveness to antimicrobial therapy. Additionally, studies tracking dynamic changes in NLR during VAN therapy may clarify its utility in guiding real-time clinical decisions.

Institutional Review Board Statement

This study was approved by the Ethics Committee of King Saud University (approval number: E-25-9553; approval date: February 12, 2025).

Data Sharing Statement

The data that support the findings of this study are available upon reasonable request from the corresponding author (M.A.A.) and with permission from King Saud University Medical City.

Informed Consent Statement

Informed consent was waived due to the retrospective design of the study. The waiver was obtained by the Ethics Committee of King Saud University.

Acknowledgment

The authors are thankful to the Ongoing Research Funding Program, King Saud University, Riyadh, Saudi Arabia for funding this research project through grant number ORF-2025-554.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Funding

This work was funded by the Ongoing Research Funding Program, King Saud University, Riyadh, Saudi Arabia under grant number ORF-2025-554.

Disclosure

The authors declare no conflicts of interest.

References

1. Patel S, Preuss CV, Bernice F. Vancomycin. Treasure Island (FL): StatPearls; 2025. ineligible companies. Disclosure: Charles Preuss declares no relevant financial relationships with ineligible companies. Disclosure: Fidelia Bernice declares no relevant financial relationships with ineligible companies.

2. Dincel S, Demirpolat E. Evaluation of the appropriateness of vancomycin therapeutic drug monitoring in the intensive care unit with a clinical pharmacy approach, a cross-sectional study. Eur J Hosp Pharm. 2024. doi:10.1136/ejhpharm-2023-004073

3. Kobayashi SD, Malachowa N, DeLeo FR. Neutrophils and bacterial immune evasion. J Innate Immun. 2018;10(5–6):432–441. doi:10.1159/000487756

4. Kobayashi SD, Malachowa N, DeLeo FR. Influence of microbes on neutrophil life and death. Front Cell Infect Microbiol. 2017;7:159. doi:10.3389/fcimb.2017.00159

5. Richardson IM, Calo CJ, Ginter EL, et al. Diverse bacteria elicit distinct neutrophil responses in a physiologically relevant model of infection. iScience. 2024;27(1):108627. doi:10.1016/j.isci.2023.108627

6. Li Y, Karlin A, Loike JD, et al. A critical concentration of neutrophils is required for effective bacterial killing in suspension. Proc Natl Acad Sci USA. 2002;99(12):8289–8294. doi:10.1073/pnas.122244799

7. Tian L, Zhou W, Wu X, et al. CTLs: killers of intracellular bacteria. Front Cell Infect Microbiol. 2022;12:967679. doi:10.3389/fcimb.2022.967679

8. Holub M, Klučková Z, Helcl M, et al. Lymphocyte subset numbers depend on the bacterial origin of sepsis. Clin Microbiol Infect. 2003;9(3):202–211. doi:10.1046/j.1469-0691.2003.00518.x

9. Lai Y, Fan J, Lv N, et al. Neutrophil-lymphocyte ratio as predictor for acute infection after primary total joint arthroplasty in rheumatoid arthritis patients. Orthop Surg. 2025;17(5):1314–1321. doi:10.1111/os.70002

10. Chen J, Luan T, Zhang Y, et al. Analysis of pathogen distribution, drug sensitivity and inflammatory indicators related to pelvic infection after hysterectomy. Eur J Obstet Gynecol Reprod Biol. 2025;306:210–218. doi:10.1016/j.ejogrb.2025.01.024

11. Deng H, Wu X, Peng B. Early predictive value of infectious markers for ventilator-associated pneumonia after Stanford type a aortic dissection surgery. Rev Cardiovasc Med. 2025;26(2):26002. doi:10.31083/RCM26002

12. Beech AS, Lea S, Kolsum U, et al. Bacteria and sputum inflammatory cell counts; a COPD cohort analysis. Respir Res. 2020;21(1):289. doi:10.1186/s12931-020-01552-4

13. Chen Y, Ye L-J, Wu Y, et al. Neutrophil-lymphocyte ratio in predicting infective endocarditis: a case-control retrospective study. Mediators Inflamm. 2020;2020:8586418. doi:10.1155/2020/8586418

14. Shi GQ, Yang L, Shan L-Y, et al. Investigation of the clinical significance of detecting PTX3 for community-acquired pneumonia. Eur Rev Med Pharmacol Sci. 2020;24(16):8477–8482. doi:10.26355/eurrev_202008_22645

15. Calis AG, Karaboga B, Uzer F, et al. Correlation of pneumonia severity index and curb-65 score with neutrophil/lymphocyte ratio, platelet/lymphocyte ratio, and monocyte/lymphocyte ratio in predicting in-hospital mortality for community-acquired pneumonia: observational study. J Clin Med. 2025;14(3):728. doi:10.3390/jcm14030728

16. Dhakal OP, Dhakal M, Dhakal N. Evaluation of the relationship between procalcitonin and total leukocyte count, neutrophil and neutrophil/lymphocyte ratio in patients with systemic inflammatory response syndrome and sepsis: a hospital-based observational study. J Assoc Physicians India. 2025;73(2):31–34. doi:10.59556/japi.73.0791

17. Niu D, Huang Q, Yang F, et al. Serum biomarkers to differentiate gram-negative, gram-positive and fungal infection in febrile patients. J Med Microbiol. 2021;70(7). doi:10.1099/jmm.0.001360

18. Cao C, Xie H, Guo R, et al. First insight into eosinophils as a biomarker for the early distinction of COVID-19 from influenza A in outpatients. Exp Ther Med. 2025;29(3):56. doi:10.3892/etm.2025.12806

19. Batur LK, Koc S. Association between vitamin D status and secondary infections in patients with severe COVID-19 admitted in the intensive care unit of a tertiary-level hospital in Turkey. Diagnostics. 2022;13(1).

20. Qiu C, Yu C, Yang L, et al. The neutrophil-lymphocyte ratio as a risk factor for all-cause mortality among individuals with resolved HBV infection: evidence from the NHANES 1999-2018. Front Public Health. 2024;12:1493439. doi:10.3389/fpubh.2024.1493439

21. Liu Y, Zhao W, Hu C, et al. Predictive value of the neutrophil-to-lymphocyte ratio/serum albumin for all-cause mortality in critically ill patients suffering from COPD. Int J Chron Obstruct Pulmon Dis. 2025;20:659–683. doi:10.2147/COPD.S497829

22. Sezak N, Karaca B, Balik R, et al. Prognostic value of neutrophil/lymphocyte, lymphocyte/c-reactive protein, platelet/ lymphocyte rates in Covid-19 cases monitored in the intensive care unit. Angiology;2025. 33197251318094. doi:10.1177/00033197251318094

23. Zeba S, Surbatovic M, Udovicic I, et al. Immune cell-based versus albumin-based ratios as outcome predictors in critically ill COVID-19 patients. J Inflamm Res. 2025;18:73–90. doi:10.2147/JIR.S488972

24. Roldgaard M, Benfield T, Tingsgard S. Blood neutrophil to lymphocyte ratio is associated with 90-day mortality and 60-day readmission in gram negative bacteremia: a multi-center cohort study. BMC Infect Dis. 2024;24(1):255. doi:10.1186/s12879-024-09127-0

25. Koyuncu H, Oflu AT, Güngör A, et al. Platelet mass index, systemic immune-inflammation index, and neutrophil-lymphocyte ratio as practical markers in childhood brucellosis. Rev Paul Pediatr. 2025;43:e2024123. doi:10.1590/1984-0462/2025/43/2024123

26. Soyaltin E, Erfidan G, Kavruk M, et al. Predictors of febrile urinary tract infection caused by extended-spectrum beta-lactamase-producing bacteria. Turk J Pediatr. 2022;64(2):265–273. doi:10.24953/turkjped.2020.2371

27. Majka G, Mazurek H, Strus M, et al. Chronic bacterial pulmonary infections in advanced cystic fibrosis differently affect the level of sputum neutrophil elastase, IL-8 and IL-6. Clin Exp Immunol. 2021;205(3):391–405. doi:10.1111/cei.13624

28. Gurol G, Ciftci IH, Terzi HA, et al. Are there standardized cutoff values for neutrophil-lymphocyte ratios in bacteremia or sepsis? J Microbiol Biotechnol. 2015;25(4):521–525. doi:10.4014/jmb.1408.08060

29. Naess A, Nilssen SS, Mo R, et al. Role of neutrophil to lymphocyte and monocyte to lymphocyte ratios in the diagnosis of bacterial infection in patients with fever. Infection. 2017;45(3):299–307. doi:10.1007/s15010-016-0972-1

30. Tanriverdi H, Örnek T, Erboy F, et al. Comparison of diagnostic values of procalcitonin, C-reactive protein and blood neutrophil/lymphocyte ratio levels in predicting bacterial infection in hospitalized patients with acute exacerbations of COPD. Wien Klin Wochenschr. 2015;127(19–20):756–763. doi:10.1007/s00508-014-0690-6

31. Bandaranayake T, Shaw AC. Host Resistance and Immune Aging. Clin Geriatr Med. 2016;32(3):415–432. doi:10.1016/j.cger.2016.02.007

32. Fulop T, Larbi A, Pawelec G, et al. Immunology of aging: the birth of inflammaging. Clin Rev Allergy Immunol. 2023;64(2):109–122. doi:10.1007/s12016-021-08899-6

33. Nomellini V, Brubaker AL, Mahbub S, et al. Dysregulation of neutrophil CXCR2 and pulmonary endothelial icam-1 promotes age-related pulmonary inflammation. Aging Dis. 2012;3(3):234–247. doi:10.1089/jir.2011.0058

34. Drew W, Wilson DV, Sapey E. Inflammation and neutrophil immunosenescence in health and disease: targeted treatments to improve clinical outcomes in the elderly. Exp Gerontol. 2018;105:70–77. doi:10.1016/j.exger.2017.12.020

35. Cinel I, Kasapoglu US, Gul F, et al. The initial resuscitation of septic shock. J Crit Care. 2020;57:108–117. doi:10.1016/j.jcrc.2020.02.004

36. Gomez-Casado G, Jimenez‐Gonzalez A, Rodriguez‐Muñoz A, et al. Neutrophils as indicators of obesity-associated inflammation: a systematic review and meta-analysis. Obes Rev. 2025;26(3):e13868. doi:10.1111/obr.13868

37. Fernandes C, Pereira L. Hypocalcemia in critical care settings, from its clinical relevance to its treatment: a narrative review. Anaesth Crit Care Pain Med. 2024;43(6):101438.

38. Kelly A, Levine MA. Hypocalcemia in the critically ill patient. J Intensive Care Med. 2013;28(3):166–177. doi:10.1177/0885066611411543

39. Steele T, Kolamunnage-Dona R, Downey C, et al. Assessment and clinical course of hypocalcemia in critical illness. Crit Care. 2013;17(3):R106. doi:10.1186/cc12756

40. Haghbin S, Serati Z, Sheibani N, et al. Correlation of hypocalcemia with serum parathyroid hormone and calcitonin levels in pediatric intensive care unit. Indian J Pediatr. 2015;82(3):217–220. doi:10.1007/s12098-014-1536-y

41. He W, Huang L, Luo H, et al. The positive and negative effects of calcium supplementation on mortality in septic ICU patients depend on disease severity: a retrospective study from the MIMIC-III. Crit Care Res Pract. 2022;2022:2520695. doi:10.1155/2022/2520695

42. Collage RD, Howell GM, Zhang X, et al. Calcium supplementation during sepsis exacerbates organ failure and mortality via calcium/calmodulin-dependent protein kinase kinase signaling. Crit Care Med. 2013;41(11):e352–60. doi:10.1097/CCM.0b013e31828cf436

43. Mirsaeidi M, Omar HR, Sweiss N. Hypoalbuminemia is related to inflammation rather than malnutrition in sarcoidosis. Eur J Intern Med. 2018;53:e14–e16. doi:10.1016/j.ejim.2018.04.016

44. Bernal W. The liver in systemic disease: sepsis and critical illness. Clin Liver Dis (Hoboken. 2016;7(4):88–91. doi:10.1002/cld.543

45. Jenniskens M, Langouche L, Vanwijngaerden Y-M, et al. Cholestatic liver (dys)function during sepsis and other critical illnesses. Intensive Care Med. 2016;42(1):16–27. doi:10.1007/s00134-015-4054-0

46. Martinez-Mier G, Carbajal-Hernández R, López-García M, et al. Prospective analysis of preoperative C-reactive protein and neutrophil-to-lymphocyte ratio as predictors of postoperative complications in bile duct injury repair. Updates Surg. 2025;77(1):47–56. doi:10.1007/s13304-024-02054-4

47. Abrol S, Tandon M, Raghu AM, et al. Role of preoperative neutrophil-lymphocyte ratio in predicting prognosis after liver transplantation for chronic liver failure. Cureus. 2025;17(3):e80749. doi:10.7759/cureus.80749

48. Riche F, Gayat E, Barthélémy R, et al. Reversal of neutrophil-to-lymphocyte count ratio in early versus late death from septic shock. Crit Care. 2015;19(1):439. doi:10.1186/s13054-015-1144-x