The study was conducted at GF Jooste Hospital, a community-based referral hospital in Cape Town, South Africa. Most patients are commenced on TB treatment and ART in community primary care clinics, but are referred to GF Jooste Hospital when complications occur for investigations and/or admission. Patients with a first episode of TB are treated with rifampicin, isoniazid, ethambutol, and pyrazinamide for 2 months followed by rifampicin and isoniazid for 4 months. For subsequent episodes the treatment duration is 8 months, including streptomycin for the first 2 months. At the time of the study the preferred ART regimen for patients on TB treatment was stavudine, lamivudine, and efavirenz.
Between June 2, 2005 and December 20, 2007 we enrolled participants into a randomized placebo-controlled clinical trial of prednisone for the treatment of paradoxical TB-IRIS. The methods have been described in detail (11
), but are summarized here. Consecutive patients with suspected paradoxical TB-IRIS referred to the hospital were screened using standardized case definitions for paradoxical TB-IRIS (3
). We limited enrollment to four TB-IRIS manifestations. Only patients with new or recurrent tuberculosis symptoms and one or more of the following manifestations were enrolled: (1
) infiltrate on chest radiograph, (2
) enlarging lymph node(s), (3
) serous effusion(s), or (4
) cold abscess(es). Patients with immediately life-threatening TB-IRIS were excluded. TB diagnosis was made on the basis of culture, smear microscopy, or clinicoradiological diagnosis (17
Participants were randomized to receive either prednisone at 1.5 mg/kg/day for 2 weeks followed by 0.75 mg/kg/day for 2 weeks or placebo administered identically. If significant clinical deterioration occurred after 2 weeks of follow-up, the study protocol allowed participants to be switched to open-label prednisone or earlier if life-threatening deterioration occurred. Follow-up was for 12 weeks. The primary end point was the cumulative number of days hospitalized, and number of outpatient therapeutic procedures performed counted as one additional hospital day (11
Timing of Blood Samples
Blood samples were taken from participants enrolled in the trial before starting study medication (Week 0) and then at Weeks 2 and 4. Placebo-treated participants who were switched to open-label prednisone were not excluded from this immunology study, but any sample taken after the switch to prednisone was excluded.
One hundred and ten patients were enrolled in the clinical trial (55 received placebo and 55 prednisone). Blood samples were taken from the first 73 participants in the clinical trial for this immunology study provided that laboratory staff were available to process specimens during working hours. If at least one repeat specimen (at 2 or 4 wk) was available then the participant was included in these analyses. The inclusion and exclusion of participants were not subject to systematic bias because the treatment allocation was randomized and unknown to clinical or laboratory staff at the time of specimen processing. To analyze results by treatment response, participants were classified as improvers (symptoms improving at Week 2 and no new symptoms) or nonimprovers (symptoms unchanged or worsened at Week 2 by comparison with Week 0 symptoms) irrespective of treatment allocation.
Ethical Permission and Trial Registration
The University of Cape Town Human Research Ethics Committee approved the study (337/2004). Written informed consent was obtained from all participants. The trial was registered with the International Standard Randomized Controlled Trial Number register (ISRCTN 21322548).
Peripheral Blood Mononuclear Cell Isolation
Peripheral blood mononuclear cells (PBMCs) were isolated from 30 ml of blood collected in sodium-heparin BD Vacutainers (Preanalytical Solutions, BD Diagnostics, Franklin Lakes, NJ), using the Ficoll separation method (Ficoll-Paque PLUS, GE Healthcare Bio-Sciences AB, Uppsala, Sweden) on the day of collection.
ELISPOT analysis was performed as previously described (14
) on the same day. Antigenic stimuli were endotoxin free and were assayed in duplicate wells. Concentrations used were as follows: early secreted antigen target (ESAT)-6, 5 μg/ml; α-crystallin 1, 10 μg/ml; α-crystallin 2, 20 μg/ml; and 38-kD antigen, 2.5 μg/ml. Purified protein derivative (PPD) was used at 200 IU/ml (4.6 μg/ml) and heat-killed H37Rv Mycobacterium tuberculosis
(MTB) was used at a multiplicity of infection (MOI) of 1:1 (200,000 H37Rv:200,000 PBMCs). Control wells included phytohemagglutinin (5 μg/ml) and no antigenic stimulus. The number of IFN-γ spot-forming cells/106
PBMCs on ELISPOT plates was counted on an ImmunoSpot series 3B analyzer (Cellular Technology Ltd, Cleveland, OH). Forty-one participants were included in the ELISPOT analysis (16 placebo-treated and 25 prednisone-treated).
RNA Isolation and Quantitative Reverse Transcription-Polymerase Chain Reaction after Mycobacterium tuberculosis Stimulation of PBMCs
After PBMC isolation, cells at 2.5 × 106/ml in RPMI–10% fetal calf serum were rested overnight in an incubator at 37°C in 5% CO2. Thereafter the PBMCs were restimulated with heat-killed H37Rv Mycobacterium tuberculosis for 24 hours at an MOI of 1:1 and unstimulated cultures were also incubated. After restimulation, PBMCs were harvested and lysed in 350 μl of buffer RLT for lysis (Qiagen, Valencia, CA). Lysates were collected for RNA analysis and tissue culture supernatants were preserved at –80°C until used in the Luminex multiplex experiments.
RNA was extracted from PBMC lysates according to the RNeasy mini kit spin protocol for isolation of total RNA from animal cells (Qiagen) as per the manufacturer’s instructions and stored at –80°C until further use. Primers and probes for reverse transcription-polymerase chain reaction (RT-PCR) were purchased from Applied Biosystems (Foster City, CA) as predesigned inventoried assay reagents. We used the following TaqMan gene expression assays: IL-1β, Hs00174097_m1 (catalogue number); IL-2, Hs00174114_m1; IL-4, Hs00174122_m1; IL-6, Hs00985639_m1; IL-8, Hs01038788_m1; IL-10, Hs00174086_m1; IL-12 p40, Hs01011518_m1; IL-13, Hs00174379_m1; IL-15, Hs00542562_m1; IL-17A, Hs00174383_m1; IL-22, Hs00220924_m1; IL-27, Hs00377399_m1; TNF-α, Hs00174128_m1; IFN-γ, Hs00174143_m1; granulocyte-macrophage colony-stimulating factor (GM-CSF), Hs00171266_m1; CCL3, Hs00234142_m1; CCL4, Hs99999148_m1; CCL5, Hs00174575_m1. RNA concentration was determined by NanoDrop ND 1000 (Thermo Scientific, Wilmington, DE) and samples were diluted to give an RNA working solution concentration of approximately 10 ng/μl. RT-PCR was performed according to the TaqMan RNA-to-CT
1-Step kit protocol (Applied Biosystems, Foster City, CA). The reaction mixture was prepared using the following outlined procedure: 1 μl of TaqMan gene expression assay, 10 μl of 2× buffer, 0.5 μl of RT enzyme, and 8.5 μl of diluted mRNA for each reaction. β-Actin was used as an endogenous control throughout. RT-PCR was performed on an ABI PRISM 7000 platform under the following universal thermal cycling conditions: reverse transcription at 48°C for 15 minutes, enzyme activation at 95°C for 15 seconds (40 cycles), annealing/primer extension at 60°C for 1 minute (40 cycles). Transcript abundance was calculated by subtracting the cycle threshold (CT) of β-actin from the CT of the gene of interest to derive a ΔCT value. Fold induction of genes in response to heat-killed H37Rv stimulation was calculated by the ΔΔCT method: the ΔCT of the unstimulated sample was subtracted from the ΔCT of the stimulated sample and 2 was then raised to the power of –ΔΔCT. Values obtained were normalized by log10 transformation and these values are reported. RT-PCR was performed in 25 participants (9 placebo-treated and 16 prednisone-treated), who had cells available for RNA extraction at Week 0 and at least one additional time point.
Luminex Multiplex Assay for Cytokine/Chemokine Concentrations in Supernatants and Serum
Supernatants from stimulated and unstimulated 24-hour cultures (see above) were later assayed for the following cytokines and chemokines: IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 p40, IL-13, IL-15, TNF-α, IFN-γ, IP-10, GM-CSF, CCL3, CCL4, and CCL5. These assays were performed in 96-well filter plates on the Bio-Plex platform (Bio-Rad Laboratories, Hercules, CA) using customized Milliplex kits (Millipore, St. Charles, MO) according to the manufacturer’s instructions. Assays were performed in 29 participants (12 placebo-treated and 17 prednisone-treated). The change in concentrations, subtracting background from stimulated, was used for comparisons.
Serum was stored at –80°C and, using the same protocols, the following cytokines/chemokines were assayed in serum samples at a later time point: IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12 p40, TNF-α, IFN-γ, IP-10, GM-CSF, CCL3, and CCL4. Assays were performed in 58 participants (27 placebo-treated and 31 prednisone-treated). In all Luminex multiplex experiments samples from the same patient were included on the same 96-well plate.
IL-8 Sandwich ELISA
A human IL-8 ELISA core kit (Koma Biotechnology, Seoul, South Korea) was used and the assay was performed according to the manufacturer’s instructions.
Medians are presented with the interquartile range (IQR) and means are presented with the standard deviation. Categorical variables were compared by chi-square or Fisher exact test and continuous variables by Wilcoxon signed-rank test. Unpaired and paired normally distributed variables were compared by Student unpaired and paired t tests. Paired nonparametric variables were analyzed by paired Wilcoxon signed-rank test. To factor multiple comparisons, P values were multiplied by n – 1 (Bonferroni correction) where n = number of comparisons. STATA 10.1 (StataCorp, College Station, TX) and GraphPad Prism version 5.0a software (GraphPad Software, San Diego, CA) were used.