aDepartment of Immunology, Tuberculosis Research Centre (ICMR), India
bGovernment Hospital of Thoracic Medicine, Tambaram Sanatorium, Chennai, Tamil Nadu, India.
Received 5 August, 2009
Revised 14 October, 2009
Accepted 23 October, 2009
The accelerated progression of tuberculosis (TB) in HIV infection [1,2] and the higher risk of mortality [3,4] demands diagnostic tests that detect Mycobacterium tuberculosis infection and disease at an early stage. Recently developed T-cell-based interferon gamma (IFNγ) release assays (IGRAs) have shown promise in the diagnosis of latent TB infection (LTBI). These assays are also being evaluated for diagnosis of active disease. Unlike tuberculin skin test (TST), they are least affected by prior Bacillus Calmette–Guerin vaccination and environmental mycobacteria . IGRAs are commercially available in two formats: QuantiFERON-TB Gold in-tube (QFT-IT; Cellestis Inc., Valencia, California, USA) and T-SPOT.TB (Oxford Immunotec, Oxford, UK). Although IGRAs perform better than TST in immunocompromised individuals, they have suboptimal sensitivity in those individuals [6–8]. Particularly, QFT-IT gives more indeterminate results (3–17%) than T.SPOT.TB due to the paucity of the source of IFNγ, namely the CD4+ T cells .
Recently, IFNγ-inducible protein-10 was suggested as a better diagnostic marker for TB infection [10–14]. A study  with very few individuals reported higher sensitivity of IFNγ-inducible protein-10 over IGRAs. Nonetheless, the utility of IFNγ-inducible protein-10 among HIV-infected individuals has not been evaluated so far. The present study compared the levels of IFNγ and IFNγ-inducible protein-10 in the culture of whole blood samples from HIV–TB patients.
A total of 50 newly diagnosed, smear-positive HIV–TB patients were recruited for this study. Six sputum samples were collected from each patient. Concentrated sputum samples were used for smear microscopy, and the smear-positive sputum samples were cultured in Lowenstein Jensen (Biomerieux Inc., Marcy I'Etoile, France) and also in liquid MP BacT medium (Biomerieux Inc.). Study participants' age ranged from 18 to 54 (median 38) years, and 72% of them were men. Forty-five individuals were HIV-1 positive and five were dually infected. Blood was drawn for CD3, CD4 and CD8 cell counts, QFT-IT and inducible protein-10 assays.
QFT-IT was carried out as per the manufacturer's instructions (Cellestis Ltd., Carnegie, Victoria, Australia), and the results were interpreted using the software supplied. IFNγ-inducible protein-10 levels were measured in the supernatants from QFT-IT tubes using BD Opt-EIA kits (BD Biosciences, San Jose, California, USA). Briefly, capture antibody (mouse antihuman inducible protein-10 mAb) in carbonate–bicarbonate buffer (pH 9.6) at the recommended concentration was coated in the 96-well polystyrene plates (NUNC MaxiSorp; NUNC AB, Roskilde, Denmark). After overnight incubation at 4°C, the excess antibodies were washed off using PBS, 0.1% Tween 20 (Himedia Laboratories, Mumbai, India). The samples were added and incubated for 2 h at 37°C room temperature (RT). After washing, secondary antibody (biotinylated antihuman inducible protein-10 mAb) conjugated with peroxidase was incubated for 1 h at RT. Excess antibodies were washed, and tetramethylbenzidine (Sigma-Aldrich Corporation, St Louis, Missouri, USA) was added and incubated for 30 min at RT. The reaction was arrested by the addition of 2 N H2SO4.
Due to the lack of gold standard for latent TB diagnosis and TB being highly endemic in our setting, we selected those individuals who were HIV seronegative, apparently free of TB symptoms, did not have close family contact of TB, were negative for both QFT-IT and TST and defined them as healthy individuals. To determine the cut-off point for IFNγ-inducible protein-10, we measured the IFNγ-inducible protein-10 levels in 50 healthy individuals (median 32; age range 21–45 years) and found that IFNγ-inducible protein-10 response to TB antigens ranged from 0 to 300 pg/ml. Hence, we chose 300 pg/ml as the cut-off point for IFNγ-inducible protein-10 for TB antigens. For mitogen, we chose 200 pg/ml as the cut-off point based on the earlier study results . The individuals with at least 300 pg/ml for TB antigens (TB antigen-nil), irrespective of mitogen response, were considered as positive; individuals with less than 300 pg/ml for TB antigens and at least 200 pg/ml for mitogen were considered as negative; others (<300 pg/ml for TB antigen and <200 pg/ml for mitogen) were considered as indeterminate.
All 50 HIV–TB patients were positive by culture, and the presence of M. tuberculosis was confirmed by Gen-Probe method (Gen-Probe Inc., San Diego, California, USA). The median CD4 cell count was 86 (interquartile range = 38 188) cells/μl, with a range of 11–502 cells/μl (available only for 36 individuals). The IFNγ and IFNγ-inducible protein-10 secretion to TB antigen was in the range 0–26 IU/ml (median 1.375 IU/ml) and 0–14 630 pg/ml (median 1309 pg/ml), respectively (Fig. 1). Of 50 HIV–TB patients, 35 [70%, 95% confidence interval (CI) = 57–83] were positive, five (10%, 95% CI = 2–18) were negative and 10 (20%, 95% CI = 9–31) were indeterminate for QFT-IT. The IFNγ-inducible protein-10 assay showed 43 positive (86%, 95% CI = 76–96), two negative (4%, 95% CI = 0–9) and five indeterminate (10%, 95% CI = 2–18) results.
Among the 10 QFT-IT indeterminate individuals, five (50%) became positive for IFNγ-inducible protein-10 and the other five remained indeterminate. Of the five QFT-IT-negative individuals, three became positive for IFNγ-inducible protein-10. One IFNγ-inducible protein-10-negative individual was positive for QFT-IT. When indeterminate results were considered as negative, QFT-IT and IFNγ-inducible protein-10 yielded 70% and 86% sensitivities, respectively. The sensitivity of IFNγ-inducible protein-10 was significantly higher than QFT-IT (P = 0.045). No significant difference in the CD4 cell count was observed between QFT-IT/IFNγ-inducible protein-10-positive and negative individuals. The CD4 cell counts did not significantly differ between QFT-IT-negative/indeterminate and IFNγ-inducible protein-10-negative/indeterminate individuals. However, IFNγ-inducible protein-10 yielded indeterminate results only when CD4 cell count was less than 50 cells/μl, whereas QFT-IT yielded indeterminate results when CD4 cell count was less than 200 cells/μl.
Our results show that IFNγ-inducible protein-10 detected more HIV–TB cases and particularly yielded 50% less indeterminate results when compared with QFT-IT. This is the first study to compare IFNγ and IFNγ-inducible protein-10 levels in HIV–TB patients recruited from a setting, endemic for both HIV and TB. We speculate the following reasons for higher sensitivity of IFNγ-inducible protein-10. First, IFNγ-inducible protein-10 is mainly secreted by monocytes/macrophages  in contrast to IFNγ, which is secreted mainly by CD4 cells and hence less affected by HIV infection and less influenced by low CD4 cell counts when compared with QFT-IT. Second, as IFNγ-inducible protein-10 is an amplified signal of IFNγ [16,17], even small number of IFNγ-secreting cells can induce large quantity of IFNγ-inducible protein-10 secretion.
This study was conducted with a small sample size, and CD4 cell counts were available for 36 individuals only, which emphasizes the requirement for further studies on this issue. In addition, the specificity of IFNγ-inducible protein-10 also has to be assessed in HIV-positive, TB-negative patients to validate the cut-off point. In conclusion, our preliminary study provides an interesting hypothesis that IFNγ-inducible protein-10 may be evaluated as a better alternative marker for LTBI diagnosis in immunocompromised individuals.
The authors thank the project consultant Dr Lee W. Riley, Division of Infectious Diseases, School of Public Health, University of California, Berkeley, California, USA, for fruitful discussions. The statistical assistance provided by Dr Venkatesan Perumal, Head, Department of Statistics, Tuberculosis Research Centre, is kindly acknowledged. Basirudeen Syed Ahamed Kabeer is the recipient of Senior Research Fellowship from Indian Council of Medical Research (ICMR), New Delhi, India. This project is financially supported by National Institutes of Health (NIH) (R03) grant (AI064055).
The funding agency had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
This article was accepted for poster presentation in the 2nd Global symposium on IGRA, held at Dubrovnik, Croatia, 30 May to 1 June 2009.
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