The present comparative study was approved by The Health Research Ethics Committee, Faculty of Medicine, Universitas Padjadjaran (approval no. 834/UN6.KEP/EC/2018; Bandung, Indonesia) and conducted in accordance with the Declaration of Helsinki. The recruitment of study participants was conducted at Hasan Sadikin Central Hospital, Bandung between August 2018 and August 2019. A total of 70 participants were recruited with mean age of 35±8 years; 56 males and 14 females. Written informed consent was obtained from all study participants.

Patients aged ≥18 years with a positive diagnosis of HIV according to the national algorithm testing sequence by The Ministry of Health of the Republic of Indonesia were included as participants (28). Patients with pregnancy, malignancy and/or autoimmune conditions were excluded. The serum of participants was collected and stored at -80˚C before analysis at The Immunology Research Unit, Faculty of Medicine, Universitas Padjadjaran.

TB status was determined based on the presence of classical clinical symptoms of TB (chronic cough, weight loss, and night sweats) as well as the result of the acid-fast staining test by Ziehl-Neelsen method, MTB culture by using Lowenstein-Jensen medium and IFN-γ release assay (IGRA). LTB was defined as a positive IGRA result without clinical or radiographic findings of ATB. Subsequently, 70 participants were divided into three categories: i) HIV-ATB (n=19), ii) HIV-LTB n=21) and iii) HIV-alone (n=30).

Samples were stained with 0.3% Carbol-fuchsin for 5 min. The stain was washed off with running water. Next, the slide was covered with 3% acid alcohol for 1 min and rinsed with running water. For counterstaining step, the slide was covered with 0.1% methylene blue stain for 20 sec followed by washing with running water and air-drying. Finally, the slide was examined microscopically under a high power objective. A positive result from the acid-fast staining test was determined if a minimum of one acid-fast bacili in 100 fields was found.

The sputum sample was mixed with an equal volume of 4% NaOH solution in a centrifuge tube, vortexed, and incubated for 15 min at room temperature. After adding 14 ml of sterile phosphate-buffered saline (PBS) at pH 6.8, the mixture was centrifuged at 3,000 g/min for 15 min at room temperature. To re-suspend the sediment, the supernatant was discarded, followed by adding 1 ml of PBS (pH 6.8). The sediment was inoculated into vials containing Lowenstein-Jensen (L-J) medium and incubated at 37˚C for 8 weeks and inspected weekly. A positive result from MTB culture was determined if at least one colony was detected.

IGRA using QFT-Plus (Qiagen GmbH) was performed according to the manufacturer's protocols as previously described (29). In brief, 1-ml-heparinized blood samples were incubated at room temperature (17-25˚C) before transfer to four QFT-Plus Blood Collection Tubes as follows: Nil, containing heparin; TB Antigen Tube 1 (TB1), comprising early secreted antigenic target (ESAT)-6 and culture filtrate protein (CFP)-10 peptide; TB2, containing ESAT-6 and 10 kDa CFP-10 peptide and mitogen, contained phytohemagglutinin, a non-specific stimulant of T-cells. The aliquoted tubes were inverted to mix 10 times before 16-24 h incubation at 37˚C, followed by centrifugation at 3,000 x g for 15 min at room temperature. Furthermore, 50 µl working strength conjugate, 50 µl plasma samples and 50 µl standards were added to the appropriate ELISA well plate. The ELISA plate was incubated at room temperature for 120 min, followed by washing using wash buffer and incubation at room temperature for 30 min. A total of 50 µl enzyme-stopping solution was added to each well. The optical density was measured using the BioTek ELx800™ Absorbance Microplate Reader (BioTek® Instruments, Inc.) at 450 and 650 nm and analyzed using the QFT Plus analysis software (ver.2.71; Qiagen GmbH). A positive test for IGRA was defined as concentration of TB1 and/or TB2 minus Nil ≥0.35 IU/ml or ≥25% of the Nil value.

IL-17A, IFN-γ, TNF, IL-10, IL-6, IL-4 and IL-2 levels were quantified in plasma samples using the BD CBA Human Th1/Th2/Th17 Cytokine kit (BD Biosciences) based on the manufacturer's protocols. Briefly, 50 µl each mixed captured beads, standard solutions, unknown samples and detection reagent were added to each assay tube, followed by 3 h incubation at room temperature with protection from light. The samples were washed with 1 ml wash buffer and centrifuged at 200 x g for 5 min at room temperature. The bead pellet of each assay tube was resuspended with 300 µl wash buffer after aspirating the supernatant. The cytokine concentration in each sample was measured using the BD FACSLyric™ Flow Cytometry System (BD Biosciences) and analyzed using the FCAP Array™ Software (ver.3.0; BD Biosciences). The theoretical detection limits were 18.9, 3.7, 3.8, 4.5, 2.4, 4.9 and 2.6 pg/ml for IL-17A, IFN-γ, TNF, IL-10, IL-6, IL-4 and IL-2, respectively.

CD4 cell count was performed using the PIMA™ CD4 assay (Abbott Pharmaceutical Co. Ltd.). Briefly, ~25 µl venous blood sample was added to the PIMA™ CD4 cartridge. In the case of finger prick, the sample was directly collected onto the cartridge. PIMA™ CD4 cartridge was inserted immediately into the Alere PIMA CD4 analyzer (Alere, Inc.) and CD4⁺ cell count was automatically calculated.

Kolmogorov-Smirnov normality test was performed using GraphPad Prism (ver.9.5.1; Dotmatics). Kruskall-Wallis and Dunn's post hoc analyses were used to compare the cytokine levels between >2 groups. Data are presented as the mean ± standard deviation. The result of cytokine analyses were presented as median (range). P<0.05 was considered to indicate a statistically significant difference.

The characteristics of the study participants are presented in Table I. Among 70 participants in the present study, 56 (80%) were male and 14 (20%) were female. The mean age of participants was 35 (25-43) years. The total participants with HIV-ATB, HIV-LTB, and HIV-alone that had a CD4 count <350 cells/µl were 15 (78.9%), 5 (23.8%) and 11 (36.7%), respectively.

The comparison of IL-17A, IFN-γ, TNF, IL-10, IL-6, IL-4 and IL-2 levels in patients with HIV-ATB, HIV-LTB and HIV is presented in Table II. In the HIV-ATB group, the median level of all cytokines was higher compared with that in the HIV-LTB and HIV groups. Furthermore, a significantly higher median expression of IFN-γ, TNF, IL-10, IL-6 and IL-2 was observed in the HIV-ATB compared to HIV-LTB and HIV groups.

IFN-γ, TNF, IL-10, IL-6 and IL-2 were significantly higher in patients with HIV-ATB compared with those with HIV-LTB. (Fig. 1). Significant difference in IFN-γ, IL-10, IL-6 and IL-2 levels between patients in the HIV-ATB group and those in the HIV group was observed.

Cytokine levels in patients with CD4 count <350 cells/µl was next assessed (Table III). Median concentration of IFN-γ, IL-10, IL-6 and IL-2 was significantly different between HIV-ATB, HIV-LTB and HIV groups. IFN-γ, IL-10, IL-6, and IL-2 levels were also significantly higher in HIV-ATB compared to HIV-LTB and HIV groups. By contrast, only TNF and IL-10 levels differed significantly between the HIV-ATB, HIV-LTB, and HIV groups in patients with with CD4 count ≥350 cells/µl (Table IV). However, post hoc analysis revealed no statistical difference (data not shown). For CD4 count <350 cells/µl, IFN-γ, IL-10 and IL-6 levels were significantly higher in HIV-ATB compared with HIV-LTB and HIV groups (Fig. 2).