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Performance of a T-cell-based diagnostic test for tuberculosis infection in HIV-infected individuals is independent of CD4 cell count

Dheda, Keertana,e; Lalvani, Ajitc; Miller, Robert Fb; Scott, Geoffd; Booth, Helend; Johnson, Margaret Ae; Zumla, Alimuddina; Rook, Graham AWa

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doi: 10.1097/01.aids.0000191923.08938.5b
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Abstract

Patients co-infected with HIV and tuberculosis have substantial mortality [1], morbidity [2] and a high rate of progression of latent tuberculosis infection (LTBI) to active disease (∼10% per year) [3]. In the absence of HAART, the treatment of LTBI reduces morbidity in developing countries [4] and in developed countries is a key element of tuberculosis prevention and control [5,6]. The diagnosis of LTBI in HIV-infected individuals is challenging, because of false-negative tuberculin skin test (TST) results (between 26 and 41%), as a result of cutaneous anergy [7–10].

New blood tests for diagnosing LTBI measure the in-vitro response of T helper type 1 IFN-γ-secreting T cells to Mycobacterium tuberculosis-specific antigens [11–13]; like the TST, they are likely to be adversely affected in patients with impaired cellular immunity. Unlike the TST, which has no internal positive control, the new tests use mitogen-stimulated IFN-γ secretion by T cells as a positive control to signal whether test results are technically evaluable. This permits the discrimination between evaluable negative test results and indeterminate test results caused by technical failure of the positive control. The proportion of evaluable versus indeterminate results will be an important determinant of the clinical utility of these new tests.

There are two commercially available assays [11]. A whole-blood enzyme-linked-immunosorbent assay that measures IFN-γ concentrations in supernatants (QuantiFERON-TB GOLD; Cellestis Ltd., Australia) was recently evaluated in a routine hospital setting. Notably, over 20% of results were indeterminate as a result of the failure of the mitogen-driven positive control, and this was significantly associated with iatrogenic immunosuppression [14]. This assay has not been evaluated in HIV-infected individuals.

An enzyme-linked immunospot assay (ELISPOT) that detects individual IFN-γ-producing T cells (T-SPOT TB; Oxford Immunotec, Oxford, UK) had low technical failure rates and high diagnostic sensitivity in HIV-infected adults and children with active tuberculosis [15–18]. However, an in-house ELISPOT assay found that approximately 25% of HIV-infected patients being evaluated for active tuberculosis had indeterminate results [19]. It is possible that assay failure rates are related to the severity of immunosuppression; however, no studies have reported patients’ CD4 T-cell counts. We investigated the performance of T-SPOT TB in HIV-infected individuals with varying levels of immunosuppression and CD4 T-cell counts. To determine whether outcomes were related to T helper cell profiles we measured IL-4, IL-4δ2 and IFN-γ messenger RNA levels in whole blood.

Forty-eight subjects (44% black African; 52% male; median age 35 years), 19 HIV-uninfected healthy controls and 29 asymptomatic HIV-infected individuals [median (range) CD4 cell count 361 (15–784) cells/μl] were recruited. Most HIV-negative controls but not HIV-positive individuals had a history of low-risk exposure to M. tuberculosis. A chest radiograph, clinical examination and relevant investigations were performed to exclude subclinical tuberculosis and concomitant infections. Plasma HIV viral load, total peripheral blood CD3+, CD4 and CD8 cell counts were performed in all HIV-infected individuals.

To exclude LTBI, IFN-γ ELISPOT responses to ESAT-6/CFP-10 peptide pools were performed according to the manufacturer's instructions (Oxford Immunotec) using peripheral blood mononuclear cells (PBMC; 200 000 cells per well). The number of IFN-γ spot-forming T cells per million PBMC was enumerated by a ‘blinded’ operator using an ELISPOT reader. A failed positive control was defined, according to the manufacturer's instructions, as less than 20 spots in the phytohaemagglutinin-positive control wells (∼100 spots/106 PBMC).

RNA was extracted from whole blood, reverse transcribed and real-time quantitative reverse transcriptase–polymerase chain reaction for IL-4, IL-4δ2 (splice variant and natural antagonist of IL-4 [20]) and IFN-γ was performed as previously described [21].

In-vitro anergy (< 20 spot-forming cells/200 000 PBMC) occurred in one out of 29 HIV-infected individuals (∼3%) and none of 19 HIV-negative controls. The median (interquartile range) phytohaemagglutinin responses were similar in those with and without HIV infection [670 (447–1055) versus 850 (707–1015); P = 0.5, Fig. 1a]. HIV-infected individuals with CD4 cell counts less than 200 cells/μl had significantly lower phytohaemagglutinin responses compared with those with CD4 cell counts greater than 200 cells/μl [765 (585–877) versus 947 (785–1055); P = 0.04, Fig. 1b]. The anergic patient had a CD4 cell count greater than 200 cells/μl. In HIV-positive individuals there was a correlation between phytohaemagglutinin responses and total peripheral CD3+ counts (P = 0.03, Fig. 1b) but not CD4 cell counts, CD8 cell counts, the CD4/CD8 cell ratio, or plasma viral load.

F1-16
Fig. 1:
Phytohaemagglutinin responses (spot-forming cells per million peripheral mononuclear cells) in HIV-negative and HIV-infected subjects stratified by CD4 cell count. (a) CD4 cell count less than 200 cells/μl versus greater than 200 cells/μl; and (b) correlation between IFN-γ phytohaemagglutinin responses and total peripheral CD3+ cell counts (× 103/ml). Responses were not significantly different in HIV-negative compared with HIV-positive donors, although subjects with CD4 cell counts of less than 200 cells/μl had significantly lower phytohaemagglutinin (PHA) responses compared with those with CD4 cell counts greater than 200 cells/μl; PHA responses correlated with peripheral CD3+ counts. Groups were compared using the Mann–Whitney U test and Spearman rank-sum correlation. PBMC, Peripheral blood mononuclear cells; SFC, spot-forming cells.

LTBI was diagnosed in one out of 19 HIV-negative controls (∼5%; recent immigrant with high-risk exposure) and two out of 29 HIV-infected individuals (7%; both immigrants with previous exposure). In HIV-positive individuals there was no correlation between phytohaemagglutinin responses and IL-4 mRNA levels, T helper type 2/1 (IL-4/IFN-γ) or IL-4/IL-4δ2 ratios.

This is the first evaluation of the impact of varying levels of HIV-associated immunosuppression and CD4 cell counts on T-SPOT TB performance. We found that indeterminate results as a result of the failure of the positive control are infrequent (∼3%), even at low CD4 cell counts; none of the patients with CD4 cell counts less than 200 cells/μl had indeterminate test results. By contrast, almost a third of HIV-infected individuals showed cutaneous anergy to TST [7–10].

We believe there are two reasons why T-SPOT TB performs well even in patients with advanced immunosuppression. First, ELISPOT technology is the most sensitive means for detecting IFN-γ-secreting antigen-specific T cells derived from blood [22,23]. Therefore, although the magnitude of the mitogen response was slightly lower in HIV-infected individuals with CD4 cell counts less than 200 cells/μl, responses were still well above the threshold for the positive control. Second, mitogen-stimulated T-cell responses are preserved until late in the course of HIV infection [24], and effector memory cells, which are phytohaemagglutinin responsive, were prominent in HIV-positive individuals [25].

The correlation between the magnitude of the positive control response and CD3+ T-cell counts is interesting and may relate to phytohaemagglutinin binding to cell surface CD3 molecules evoking potent IFN-γ responses [26,27]. The lack of correlation with unstimulated T helper type 2/1 cytokine profiles may reflect the lack of T helper type 2 responses in healthy HIV-infected individuals [28].

It remains to be determined to what extent responses to tuberculosis-specific antigens in HIV-infected individuals with LTBI will be affected at low CD4 cell counts. The only published study in healthy HIV-positive individuals suggested that ELISPOT may detect LTBI in individuals with cutaneous anergy [15]. We detected LTBI in two HIV-positive individuals in this study. Having established that the technical performance of T-SPOT TB is independent of the degree of HIV-associated immunosuppression, the next priority is to determine prospectively its diagnostic sensitivity and specificity in HIV-positive individuals with LTBI.

Acknowledgements

The authors are grateful for the assistance of Drs M.C. Lipman, R.A.M. Breen, I.A. Cropley, S. Bhagani, M. Tyrer and the clinic staff at the Royal Free and Middlesex Hospitals NHS trust in facilitating sample collection. They would also like to thank Mr I. Durrant from Oxford Immunotec for his technical assistance.

Sponsorship: This work was supported by a grant from the British Lung Foundation.

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