This study evaluates the diagnostic and prognostic utility of combined serum IFN-γ and neutrophil CD64 detection for active pulmonary tuberculosis in immunocompromised elderly patients.
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Research Article
This study evaluates the diagnostic and prognostic utility of combined serum IFN-γ and neutrophil CD64 detection for active pulmonary tuberculosis in immunocompromised elderly patients.
This study assessed the risk of active pulmonary tuberculosis (ATB) in elderly patients with weakened immunity by jointly detecting serum interferon-γ (IFN-γ) and neutrophil CD64, and analyzed the impact of this diagnostic protocol on the course of ATB. A total of 50 patients with ATB and 50 age- and sex-matched healthy controls were recruited. Subsequently, the patient's neutrophil CD64 (quantified as the percentage of CD64-positive neutrophils [CD64%]), IFN-γ (by enzyme-linked immunosorbent assay [ELISA]), C-reactive protein (CRP; by immunoturbidimetry), and erythrocyte sedimentation rate (ESR; by Westergren method). Sputum samples from patients were cultured on Löwenstein-Jensen (L-J) medium and in the MGIT 960 automated system. The diagnostic efficacy of individual and combined biomarkers was assessed with receiver operating characteristic (ROC) curve analysis, and a logistic regression model was developed for combined detection. Significantly elevated levels of both IFN-γ and CD64 were observed in ATB patients versus healthy controls (P<0.05). A diagnostic model incorporating both biomarkers had an area under the curve (AUC) of 0.838, with a sensitivity of 84.00% and a specificity of 76.00%. Both markers decreased following therapeutic intervention (P<0.05), showing the lowest values in culture-negative patients (P<0.05). Furthermore, the combined model showed predictive utility for culture conversion, attaining an AUC of 0.756 (65.71% sensitivity, 80.00% specificity; P<0.001). The combined detection of IFN-γ and CD64 can effectively diagnose the occurrence of ATB in immunocompromised elderly people, providing a reference for clinical practice.
Pulmonary tuberculosis (TB), a chronic infectious disease caused by Mycobacterium tuberculosis (MTB), causes more than 10 million new cases globally each year and is the leading cause of death from a single infectious pathogen1. Clinically, populations at high risk of ATB include immunocompromised individuals, patients with diabetes, long-term smokers, close contacts of TB cases, and elderly adults2. Among these groups, elderly adults with impaired immunity have a markedly elevated risk of ATB3. The accurate and early diagnosis of active TB (ATB) is crucial for prognosis, given its transmissibility and risk of severe progression4. Yet current strategies, reliant on symptomatology, imaging, and sputum tests, lack specific biomarkers, creating a critical diagnostic gap5. This diagnostic uncertainty is exacerbated by the lack of dynamic severity assessment indicators, often leading to empirical treatment that does not meet individual patient needs6. Consequently, identifying highly sensitive and specific biomarkers is imperative for improving the early differentiation and stratification of ATB.
Interferon-γ (IFN-γ), a central cytokine in the T-cell response to MTB-specific antigens, reflects the immune activation state in ATB, with elevated serum levels aiding in its diagnosis; moreover, it has value in differentiating between latent TB infection (LTBI) and ATB7,8. While valuable, its utility is compromised by limited specificity due to interference from Bacillus Calmette-Guerin vaccination and other infections9. Conversely, neutrophils are the first immune cell population recruited to the lungs and infiltrate ATB lesions, where their involvement in inflammatory amplification is linked to the overexpression of CD64, the high-affinity Fc-gamma receptor I10. Nevertheless, no prior study has combined IFN-γ and neutrophil CD64 to evaluate their synergistic diagnostic and prognostic utility for ATB, particularly in immunocompromised elderly adults, a cohort with unique diagnostic challenges due to blunted immune responses and atypical clinical presentations.
This study selected IFN-γ and CD64 for their complementary roles in adaptive and innate anti-MTB immunity, clinical assay accessibility, and independent diagnostic value reported in prior studies, with no combined evaluation in immunocompromised elderly patients to date. The study combines serum IFN-γ (measured via enzyme-linked immunosorbent assay [ELISA], reflecting adaptive T-cell immunity) and neutrophil CD64 (measured via flow cytometry, reflecting innate immune activation and inflammatory burden), with diagnostic performance evaluated via receiver operating characteristic (ROC) curve analysis and logistic regression modeling. The utility of this combined assay for the diagnosis and monitoring of ATB disease progression in immunocompromised elderly adults was systematically evaluated.
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The study protocol received approval from the Institutional Review Board of Ganzhou Fifth People's Hospital, Ganzhou, Jiangxi, China. Written informed consent was obtained from all individuals who participated in the study. All procedures involving viable Mycobacterium tuberculosis (MTB) were performed in a biosafety level 3 (BSL-3) containment facility, in full compliance with national biosafety guidelines for handling infectious pathogens. For NaOH-mediated sputum liquefaction, operators wore full personal protective equipment (N95 respirators, nitrile gloves, fluid-resistant gowns, and face shields) to prevent chemical and aerosol exposure. The reagents and the equipment used are listed in the Table of Materials.
1. Research participants
The study protocol begins with enrollment and informed consent of eligible participants and ends with the completion of statistical analysis of all primary and secondary endpoints. This retrospective single-center study enrolled ATB patients (ATB group) and healthy controls (control group) from our hospital between May 2023 and May 2025, with all study procedures completed before data analysis. The flow chart of the study is shown in Figure 1. The ATB and control groups were comparable at baseline, with no significant differences in demographic characteristics and common chronic comorbidities (P>0.05, Table 1), confirming the homogeneity of the two groups.
2. Sample size estimation
The sample size was estimated using preliminary data from 10 subjects, which showed median IFN-γ levels of 20 pg/mL in ATB patients versus 10 pg/mL in healthy controls. To detect a significant difference (assuming significance level [α]=0.05, two-tailed; test power [1-β]=80%; medium effect size [Cohen's d≈1.2]), an analysis with PASS 15.0 software yielded a minimum requirement of 42 participants per group (actual power=81.2%, α=0.05, two-tailed). Factoring in a 10%–15% dropout rate, we ultimately recruited a total of 100 subjects (50 per group) after screening against the study criteria.
3. Collection of test samples
3–5 mL of fasting venous blood were collected from each patient. The blood was drawn directly into an EDTA tube for CD64 analysis by flow cytometry and a clot activator tube for serum separation. The serum was then assayed for IFN-γ, C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR) levels.
4. Flow cytometry
Whole blood (100 µL) was incubated with a pre-mixed antibody cocktail (5 µL each of CD16-FITC, CD14-APC, and CD64-PE) for 20 min at room temperature in the dark. Erythrocytes were subsequently lysed with 1 mL of BD FACS Lysing Solution for 10 min, protected from light. Following centrifugation (centrifugation at 300 × g for 5 min at room temperature), the supernatant was discarded. The remaining cell pellet was washed twice with phosphate-buffered saline (PBS) and finally resuspended in 500 µL of PBS for immediate acquisition on the flow cytometer. For analysis, we first gated the leukocyte population using forward scatter (FSC) and side scatter (SSC) parameters. Neutrophils were then selectively identified within this gate as events negative for CD14-APC (excluding monocytes) and positive for CD16-FITC. The CD64-positive population was defined using an isotype-matched control, with the gate set to include <1% of isotype control events; the percentage of CD64-positive neutrophils (CD64%) was calculated as the proportion of gated neutrophils falling within this positive gate. For quality control purposes, the study included a healthy control sample in every experiment to establish a baseline MFI range (15–30). Any sample MFI persistently above 30 was noted. Daily instrument calibration was also performed using BD CS&T beads to maintain consistent fluorescence compensation and voltage settings. Instrument acquisition parameters: Flow cytometric acquisition was performed on a flow cytometer, with a minimum of 10,000 gated neutrophil events collected per sample. Forward scatter (FSC) and side scatter (SSC) voltages were set to 350 V and 280 V, respectively. Fluorescence channel voltages were optimized daily using BD CS&T beads to ensure consistent detection and compensation. Visual Quality Checkpoints: (1) The leukocyte population was clearly resolved on FSC/SSC plots with no significant debris contamination; (2) The neutrophil gate was confirmed to be CD14-negative and CD16-positive, with <5% monocyte contamination; (3) The CD64 fluorescence histogram was unimodal with no significant non-specific staining.
5. ELISA
Serum was separated by centrifugation, and the assay was performed according to the manufacturer's instructions of the ELISA kit. Briefly, 50 µL of standard or test serum samples were dispensed into antibody-coated wells and incubated at 37 °C for 90 min. Subsequent steps involved the sequential addition of biotinylated anti-human IFN-γ antibody (100 µL), streptavidin-horseradish peroxidase (HRP) complex (100 µL), chromogenic substrate, and stop solution. A four-parameter fitting algorithm was applied to the standard curve to calculate final IFN-γ concentrations in pg/mL. Quality control: Assay validity required that low/medium/high QC values (15.6, 62.5, 250 pg/mL) fall within the specified ± 2 SD range, with intra- and inter-assay coefficients of variation (CVs) maintained below 8% and 10%, respectively.
6. Immunoturbidimetric assay
Following centrifugation, serum CRP levels were determined using an automated biochemical analyzer with a matched latex particle-enhanced immunoturbidimetric kit, according to the manufacturer's standardized protocol. The assay was based on the agglutination of CRP with specific antibodies bound to latex particles, which increased turbidity in direct proportion to the CRP concentration and was quantified photometrically. Quality control: Each assay batch included low (<3 mg/L) and high-concentration (>100 mg/L) quality control sera (Beckman). The obtained values were required to fall within the manufacturer's specified acceptable range (±3 SD). The intra-assay CV was maintained at ≤5%, and the inter-assay CV at ≤8%.
7. Westergren method
The venous blood in the clot activator tube was allowed to stand for 1 h. The plasma was then aspirated into a 1.6 mL Westergren tube and placed vertically in an ESR analyzer at 37 °C for 1 h. The result was automatically calculated and reported as the sedimentation height in mm/h. Quality control: The analyzer was calibrated daily using a standard sedimentation tube for zero-point calibration.
8. MTB culture
Sputum specimens were liquefied with 4% NaOH, neutralized, and centrifuged (centrifugation at 1200 × g for 15 min at room temperature), followed by supernatant removal. The resulting sediment was inoculated onto Löwenstein-Jensen (L-J) medium (2-3 drops per tube). The cultures were incubated at 37℃ in a constant-temperature incubator and examined weekly for colony growth. A positive result was defined by the appearance of typical cauliflower-like colonies within ≥2 weeks. Quality control: Quality control measures comprised concurrent negative (sterile saline) and positive (H37Rv) controls in each batch, along with monthly sterility testing of the culture media.
9. Treatment and follow-up
All ATB patients received standardized first-line anti-tuberculosis therapy (2HRZE/4HR regimen: 2-month intensive phase with isoniazid, rifampicin, pyrazinamide, ethambutol; 4-month continuation phase with isoniazid and rifampicin). Sputum mycobacterial culture was performed at baseline and 2 months after treatment initiation to evaluate culture conversion.
10. Waste disposal
Biohazardous waste (including residual blood samples, sputum specimens, and MTB culture materials) was decontaminated via autoclaving at 121 °C for 30 min prior to final disposal. Chemical waste (including NaOH solution, lysis buffer, and fluorescent staining reagents) was collected in sealed, designated hazardous waste containers and disposed of in accordance with institutional chemical waste management protocols.
11. Statistical methods
The study analysis used SPSS 30.0. Categorical data (e.g., gender) were compared by chi-square tests. Normality of continuous data was verified via the Shapiro-Wilk test, and homogeneity of variance via the Levene test. Normally distributed continuous data with homogeneous variance were compared using independent-samples t-tests; non-normally distributed data were compared using the Mann-Whitney U test. ROC curve analysis was performed using the "ROC Curve" module, with binary diagnostic status (ATB vs. control) as the state variable and biomarker levels as the test variable. The combined diagnostic model was constructed via binary logistic regression, with IFN-γ and CD64 entered as continuous independent variables and diagnostic status (ATB=1, control=0) as the dependent variable. The regression formula was: Logit(P) = -3.274 + 0.082×IFN-γ (pg/mL) + 0.115×CD64%. The cut-off value for combined detection was based on the model-predicted probability, with the optimal threshold determined by the Youden index. Model-predicted probabilities were used to generate the combined ROC curve. Correlations were calculated using Pearson's coefficient. A P-value<0.05 defines statistical significance.
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The diagnostic efficacy of the combined detection of IFN-γ and CD64 in ATB
Compared with the control group, the mean serum IFN-γ level in the ATB group increased by 22.57% (21.84 ± 4.23 vs. 16.91 ± 4.41 pg/mL, P<0.05), and the mean CD64% increased by 24.97% (14.90 ± 4.04 vs. 11.18 ± 3.56, P<0.05). ROC curve analysis indicated that IFN-γ possessed a diagnostic sensitivity of 64.00% and specificity of 86.00% for ATB (AUC=0.803, P<0.001), whereas CD64 exhibited a sensitivity of 84.00% and speci...
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This study proposes an integrated dual-biomarker diagnostic and prognostic framework combining serum IFN-γ (a marker of adaptive T-cell immunity) and neutrophil CD64 (a marker of innate immune activation) for ATB in immunocompromised elderly patients, a high-risk population with unmet clinical needs for accurate, non-invasive TB testing. The management of ATB is complicated by its infectious nature and potential for severe disease, underscoring the need for early, precise diagnosis and monitoring12...
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The authors declare no conflicts of interest.
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| AU5800 automatic biochemical analyzer | Beckman Coulter | B04077 | An instrument used to detect and analyze the chemicals of life. |
| CD14-APC | Agilent | 8730057 | A fluorescently labeled antibody for flow cytometry that specifically recognizes the CD14 antigen. |
| CD16-FITC | Agilent | 8920064 | A fluorescently labeled antibody for flow cytometry that specifically recognizes the CD16 antigen. |
| CD64-PE | Agilent | 8730119 | A fluorescently labeled antibody for flow cytometry that specifically recognizes the CD64 antigen. |
| CytoFLEX LX flow cytometry | Beckman Coulter | C00445 | A cell analyzer that measures the fluorescence intensity of stained cell markers. |
| IFN-γ ELISA kit | Wuhan Jilid Biotechnology Co., LTD | J20272 | A tool based on the double antibody sandwich assay for the detection of IFN-γ concentrations. |
| L-J medium | Shandong Topu Biological Engineering Co., LTD | M1607 | A medium used for the isolation and culture of mycobacteria such as Mycobacterium tuberculosis. |
| PASS | NCSS | v.15.0 | A software to support power analyses and sample-size calculations. |
| SPSS | IBM | v.30.0 | A software for data processing, data analysis, and data visualization. |
| XS-1000i erythrocyte sedimentation analyzer | Sysmex | V147451 | A medical device used for clinical blood testing. |