Sixty patients diagnosed with active pulmonary TB who completed treatment using first-line anti-TB agents at Kosin University Gospel Hospital from January to December 2010 and 30 age- and sex-matched healthy controls were enrolled in this retrospective study. The diagnosis of active pulmonary TB was based on a positive respiratory specimen culture and/or a positive result in a TB-polymerase chain reaction assay. Treatment regimens for TB have an initial phase of 2 months, followed by a continuation phase of either 4 or 7 months (total of 6 or 9). For initial treatment of TB, patients were treated with a 4-drug regimen including isoniazid, rifampin, ethambutol, and pyrazinamide. After 2 months, pyrazinamide was discontinued.

Sputum smear for acid-fast bacilli (AFB), mycobacterial culture for M. tuberculosis, and chest radiography were examined every 1 or 2 months during treatment. For patients who did not expectorate sputum, bronchoscopy was conducted for mycobacterial culture of M. tuberculosis. Patients with malignancy or recurrent TB infection were excluded based on medical history evaluation.

The objective of this study was to compare the serum NSE concentrations of TB patients with those of age-and sex-matched healthy controls. The study further aimed to determine whether NSE concentration changes after anti-TB treatment and whether NSE concentration could be used to monitor TB activity and treatment response. Patients underwent blood sampling to determine serum NSE concentration before and after completion of anti-TB treatment. The group of patients with TB was composed of 25 men and 35 women with a mean age of 64.2 years (range, 18 to 78). All patients underwent clinical, laboratory, and radiologic evaluations and were categorized into either a focal segmental infiltration group (35 patients total, including 10 tuberculoma, 15 segmental infiltration, and 10 small nodular infiltration patients) or an extensive infiltration group (25 patients, including five miliary and 20 lobar infiltration patients) (Table 1). Both serum and biopsy samples were obtained from the Department of Pathology at Kosin University College of Medicine. This study was approved by the Institutional Review Board and Ethics Committee of Kosin University Gospel Hospital (KUGH IRB No. 13-085) and was conducted in accordance with the Declaration of Helsinki.

Serum concentrations of NSE and hs-CRP were determined by radioimmunoassay. Normal serum NSE and hs-CRP concentrations were set at less than 15 ng/mL and 3 mg/L, respectively. Hemolytic specimens were excluded because blood cell lysis influences the measured concentrations of both biomarkers.

Immunohistochemical staining of lung biopsy specimens of 10 TB-infected patients was performed using antibodies against NSE and a human macrophage marker, CD68. Specimens were obtained from the Department of Pathology at Kosin University College of Medicine. Immunohistochemistry was performed on 4-μm-thick sections of paraffin blocks using the Bond-Max Autostainer (Leica Microsystems, Bannockburn, IL, USA). Tissue sections were deparaffinized and rehydrated following standard procedures. Heat-induced antigen retrieval was carried out, and sections were incubated with primary antibodies for 32 minutes at 37oC at a dilution of 1:3 polyclonal rabbit anti-NSE and 1:6,000 anti-CD68 antibodies (Dako, Carpinteria, CA, USA). Primary antibody binding was detected using the Bond Polymer Refine Detection kit (Leica Microsystems) according to the manufacturer’s instructions.

Tissue microarrays consisting of 100 tissue cores (80 TB-infected, eight lung cancer, and 12 normal) were purchased from US Biomax (Rockville, MD, USA). No clinical information except age, sex, and pathological status of each patient was available for the tissues in these arrays. Patient characteristics and NSE and CD68 staining intensities in the tissue microarray are described in Supplementary Table 1. For immunofluorescence analysis, tissue slides were deparaffinized and hydrated. For antigen retrieval, slides were immersed in citrate buffer (0.01 M, pH 6.0) and heated twice in a microwave (700 W or higher) for 5 minutes each. Slides were permeabilized by incubation in phosphate-buffered saline (PBS) containing 0.1% Triton X-100 for 5 minutes and then incubated with 10% normal serum in PBS for 1 hour to block nonspecific antibody binding. Slides were then incubated with a mixture of antibody to NSE (1:200 dilution; Dako) and CD68 (1:100 dilution; Dako) overnight at 4°C. After primary antibody incubation, slides were washed three times in PBS each for 5 minutes and incubated with secondary antibodies (Alexa Fluor 546-conjugated anti-mouse antibody and Alexa Fluor 488-conjugated anti-rabbit antibody, Invitrogen, Carlsbad, CA, USA) for 1 hour. Specimen epifluorescence was examined using confocal laser-scanning microscopy. The specificity of each antibody was confirmed by staining with rabbit or mouse polyclonal immunoglobulin G. A well-trained pathologist read the slides and scored the signals of NSE and CD68 blinded to clinical information.

Statistical analyses were conducted using SPSS version 20.0 (IBM Co., Armonk, NY, USA). Patient characteristics were summarized descriptively. A two-tailed p value less than 0.05 was considered statistically significant. Correlation analyses were performed to generate the Pearson correlation coefficients to determine the relationships between variables. Paired t tests were conducted to compare the differences in NSE and hs-CRP concentrations before and after anti-TB treatment.

NSE serum concentration was significantly higher in pulmonary TB patients (mean [range], 24.59 ng/mL [15.20 to 40.00]) than in matched healthy controls (5.05 ng/mL [2.00 to 8.00]; p < 0.05). In addition, hs-CRP was also significantly higher in pulmonary TB patients (4.24 mg/L [0.61 to 8.64]) than in controls (0.80 mg/L [0.10 to 4.30]; p < 0.001) (Table 1). The mean serum concentrations of NSE and hs-CRP in the extensive type group (25.12 ng/mL and 4.12 mg/L, respectively) were significantly higher than those in the focal segmental type group (20.23 ng/mL and 2.12 mg/L, respectively; p = 0.04).

Serum NSE concentration positively correlated with hsCRP concentration in active pulmonary TB patients (p = 0.002, r² = 0.741) (Fig. 1). After anti-TB therapy (i.e., at 6 or 9 months after the start of anti-TB treatment), the concentrations of both biomarkers were considerably lower than before anti-TB therapy; NSE decreased from 24.59 (15.20 to 40.00) to 7.23 (2.2 to 11.4) ng/mL and hs-CRP from 4.24 (0.61 to 8.64) to 1.62 (0.40 to 3.82) mg/L (Fig. 2).

Immunohistochemical staining revealed a large number of both NSE- (green arrows) and CD68-positive (red arrows) macrophages around caseating necrosis in granulomatous lesions, indicating that NSE is produced by CD68-positive macrophages within granulomatous lesions (Fig. 3).

NSE and CD68 were examined in patient tissue microarrays containing normal lung, lung cancer, TB-infected, and necrotic TB-infected tissues. Staining of both NSE and CD68 was notably higher in TB tissues with nascent and necrotic lesions than in normal tissue (Fig. 4). Immunofluorescence analysis showed that both NSE and CD68 staining were strong at the cores of granulomas in both nascent and necrotic granulomatous lung tissues. Double staining of NSE and CD68 showed that NSE co-localized with CD68 in nascent and necrotic granulomas (Figs. 5 and 6). Information on the integrated density values of CD68 and NSE staining from immunofluorescence analysis of the tissue microarray are described in Supplementary Table 2.