Abstract
In this prospective, longitudinal study on 948 HIV-1-infected patients, subjects with an indeterminate IFN-γ (gamma interferon) release assay (IGRA) result at baseline were at significantly higher risk of developing AIDS-defining manifestations other than tuberculosis (TB) irrespective of CD4+ T cell count. Thus, in HIV-1-infected patients with advanced quantitative CD4+ T cell depletion, an indeterminate IGRA might indicate an additional loss of global T cell function, warranting detailed clinical evaluation and careful follow-up.
TEXT
Mycobacterium tuberculosis-specific IFN-γ (gamma interferon) release assays (IGRAs) are widely used screening tools for the detection of tuberculosis (TB) infection, and their implementation has been incorporated in several national TB guidelines (10).
Indeterminate results of the QuantiFERON-TB Gold In-Tube assay (QFT-GIT) (Cellestis, Carnegie, Australia) (8), i.e., results neither positive nor negative, have been reported with a frequency of up to 14% in individuals infected with human immunodeficiency virus type 1 (HIV-1) (1).
Among HIV-1-uninfected individuals, indeterminate QFT-GIT results have been associated with advanced age and underlying disease in patients receiving immunosuppressive treatment (6). In a study carried out among HIV-1-infected patients in Tanzania, both male patients and patients aged ≥33 years were more likely to have an indeterminate QFT-GIT result (1). However, the prognostic value of indeterminate QFT-GIT results in terms of the subsequent development of AIDS-defining manifestations and/or death is unknown.
This study is part of a project initiated in 2006 to evaluate the QFT-GIT assay for routine use in HIV-1-infected individuals in a low-incidence country (2).
(This work was presented in part at the 6th International AIDS Society Conference on HIV Pathogenesis, Treatment and Prevention, 17 to 20 July 2011, Rome, Italy [abstract TUPE158].)
Venous blood samples were drawn in three QFT-GIT evacuated tubes, precoated with either M. tuberculosis-specific antigens (ESAT-6, CFP-10, and TB7.7), phytohemagglutinin (PHA) for the positive control, or no antigen (nil) for the negative control. The QFT-GIT assay was performed as recommended by the manufacturers (3). The CD4+ T cell count and HIV-1 RNA viral load were measured at the time of blood draw for the QFT-GIT assay.
Statistical analyses were conducted with SPSS, version 16.0 (SPSS Inc., Chicago, IL). Median values were compared by the nonparametric Wilcoxon-Mann-Whitney U rank-sum test. The χ2 or Fisher's exact test was used to compare proportions. Multivariate analyses were performed by general linear and Cox proportional hazard regression models. All P values were two-tailed, and P < 0.05 was considered to denote statistical significance.
Independent predictors for indeterminate QFT-GIT results.
Baseline characteristics of the study participants are shown in Table 1. The QFT-GIT assay yielded indeterminate results in 5.8% (55/948) of subjects. All indeterminate QFT-GIT results were caused by inadequate IFN-γ response in the positive control.
Table 1.
Baseline characteristics of HIV-1-infected subjects undergoing QFT-GIT testing
| Characteristic | n | QFT-GIT determinate | QFT-GIT indeterminate | Pa |
|---|---|---|---|---|
| No. of subjects | 948 | 893 | 55 | |
| Demographic category | ||||
| Sex, no. (%) | ||||
| Female | 284 | 262 (29) | 22 (40) | |
| Male | 664 | 631 (71) | 33 (60) | 0.094 |
| Median age, yr (IQR) | 39 (31–46) | 39 (31–46) | 35 (29–44) | 0.066 |
| Age in yr by stratum, no. (%) | ||||
| <30 | 190 | 174 (19) | 16 (29) | |
| 30–40 | 333 | 313 (35) | 20 (37) | |
| 41–50 | 281 | 266 (30) | 15 (27) | 0.084 |
| >50 | 144 | 140 (16) | 4 (7) | |
| Ethnicity, no. (%) | ||||
| White | 829 | 784 (88) | 45 (82) | 0.194 |
| Black | 92 | 86 (10) | 6 (11) | 0.756 |
| Asian | 18 | 15 (1.5) | 3 (5) | 0.081 |
| Latino | 9 | 8 (0.5) | 1 (2) | 0.417 |
| HIV-1-related factors | ||||
| Mode of infection with HIV-1, no. (%) | ||||
| Heterosexual contact | 376 | 356 (40) | 20 (36) | 0.606 |
| Homosexual contact | 352 | 338 (38) | 14 (25) | 0.561 |
| Injection-drug use | 187 | 169 (19) | 18 (33) | 0.013 |
| Other or unknown | 33 | 30 (3) | 3 (6) | 0.433 |
| Median CD4+ T cell count, at enrollment (IQR) | 385 (254.3–553.8) | 393 (269.5–565.5) | 188 (75–363) | <0.001 |
| CD4+ T cell count/mm3 by stratum, no. (%) | ||||
| <100 | 71 | 56 (6) | 15 (27) | <0.001 |
| 100–200 | 98 | 83 (9) | 15 (27) | <0.001 |
| 201–350 | 234 | 223 (25) | 11 (20) | 0.407 |
| 351–500 | 237 | 231 (26) | 6 (11) | 0.033 |
| >500 | 308 | 300 (34) | 8 (15) | 0.013 |
| Median CD4+ T cell nadir (IQR) | 201 (89–314.8) | 208 (94–316.5) | 107 (18–249) | 0.001 |
| Median log10 HIV RNA (IQR) | 1.99 (1.7–4.5) | 1.76 (1.7–4.5) | 3.74 (1.7–5.2) | 0.002 |
| HIV-1 RNA copies/ml by stratum, no. (%) | ||||
| <50 | 460 | 442 (50) | 18 (33) | 0.087 |
| >100,000 | 135 | 117 (13) | 18 (33) | <0.001 |
| On ART, at enrollment, no. (%) | 526 | 500 (56) | 26 (47) | 0.657 |
| Prior AIDS manifestation, no. (%) | 206 | 184 (21) | 22 (40) | <0.001 |
| Prior active TB, no. (%) | 30 | 30 (3) | 0 (0) | 0.412 |
Values in boldface are statistically significant.
A total of 55.5% (526/948) of the study population was on antiretroviral therapy (ART) at study enrollment. Although a greater proportion of patients with determinate than with indeterminate QFT-GIT results were receiving ART, this difference was not statistically significant (56.0% versus 47.3%, P = 0.657) (Table 1).
Patients with an indeterminate QFT-GIT result were more likely to have acquired HIV-1 infection by intravenous drug abuse than were subjects with determinate QFT-GIT results (odds ratio [OR], 2.1; P < 0.05; Table 2). Among patients with indeterminate QFT-GIT results, both median actual (188 versus 393 cells/mm3; P < 0.001) and nadir (107 versus 208 cells/mm3; P = 0.001) CD4+ T cell counts were significantly lower while median HIV-1 RNA levels were significantly higher (3.7 versus 1.8 log10 copies/ml, P = 0.002) than in subjects with interpretable results. Forty percent versus 20.6% of subjects with an indeterminate or determinate QFT-GIT result, respectively, had previously been diagnosed with an AIDS-defining disease according to the CDC classification (4) (OR, 2.0; P = 0.079). Male sex and age of >30 years were not associated with an indeterminate QFT-GIT result (Table 2).
Table 2.
Multivariate analysis of risk factors for indeterminate QFT-GIT results
| Risk factor | Total no. of subjects with QFT-GIT results (n = 948) | Total no. of subjects with indeterminate QFT-GIT results (n = 55) | OR (95% CI) | Pa | Adjusted OR (95% CI) | Pa |
|---|---|---|---|---|---|---|
| Male sex | 664 | 33 | 0.6 (0.4–1.1) | 0.094 | 0.8 (0.5–1.8) | 0.476 |
| Age of >30 yr | 719 | 39 | 0.6 (0.3–1.1) | 0.084 | 0.5 (0.4–1.2) | 0.384 |
| Injection drug use | 187 | 18 | 2.1 (1.2–3.8) | 0.013 | 2.1 (1.4–3.4) | 0.038 |
| CD4+ T cell count of <100 cells/mm3 | 71 | 15 | 5.6 (2.9–10.8) | <0.001 | 4.9 (2.5–7.9) | <0.001 |
| HIV-1 RNA level of >100,000 copies/ml | 135 | 18 | 3.6 (2.0–6.6) | <0.001 | 3.2 (2.0–4.8) | 0.016 |
| On ART, at enrollment | 526 | 26 | 0.9 (0.5–1.5) | 0.657 | ||
| Prior AIDS manifestation | 206 | 22 | 2.9 (1.7–5.1) | <0.001 | 2.0 (1.2–3.5) | 0.079 |
Values in boldface are statistically significant.
Development of AIDS-defining illnesses and/or death during follow-up.
The median follow-up time for the 948 subjects was 33 months (interquartile range [IQR], 27 to 39). Eighty-one percent (768/948) or 94.9% (900/948) of patients had a follow-up time of at least 24 or 12 months, respectively. During the observational period, 37 individuals (3.9%; 37/948) were newly diagnosed with an AIDS-defining manifestation with a median delay of 403.5 days (IQR, 313.25 to 439.75) from inclusion. The most frequently observed AIDS-defining illnesses were esophageal candidiasis, Pneumocystis jirovecii pneumonia, and wasting syndrome. Active TB was exclusively observed in subjects with determinate (positive) QFT-GIT results. At baseline, 14.4% (8/55) and 3.2% (29/893) of the 37 patients who progressed to AIDS had been tested as QFT-GIT indeterminate and determinate, respectively (OR, 5.1; 95% confidence interval [CI], 2.2 to 11.7; P < 0.001). Provision of ART at enrollment and at follow-up as well as virological suppression was equally distributed between the two QFT-GIT groups.
Overall, 41 patients died during follow-up. The most frequent cause of death was bacterial and/or fungal infection followed by acute respiratory distress syndrome (ARDS).
The rate of progression to AIDS or death was 21.8% (12/55) among QFT-GIT-indeterminate subjects and 6.5% (58/893) among patients with positive or negative QFT-GIT results (P < 0.001). Multivariate analysis revealed the following independent risk factors for progression to AIDS and/or death: HIV-1 RNA level of >100,000 copies/ml (HR, 6.3; P = 0.001) and indeterminate QFT-GIT result (HR, 3.8; P < 0.05) (Table 3).
Table 3.
Risk factors for the development of AIDS-defining manifestations and/or death
| Risk factor | No. (%) of subjects with AIDS and/or death (n = 70) | HRa (95% CI) | Pb |
|---|---|---|---|
| Age of >30 yr | 60 (86) | 0.72 (0.31–1.68) | 0.446 |
| Male sex | 48 (69) | 1.10 (0.40–3.02) | 0.851 |
| CD4+ T cell count of <100 cells/mm3 | 24 (34) | 2.76 (0.88–8.66) | 0.083 |
| HIV-1 RNA level of >100,000 copies/ml | 25 (36) | 6.33 (2.08–19.22) | 0.001 |
| On ART at baseline | 36 (51) | 0.87 (0.40–1.91) | 0.089 |
| QFT-GIT indeterminate result | 12 (17) | 3.75 (1.18–11.87) | 0.025 |
HR, hazard ratio.
Values in boldface are statistically significant.
In this study, a total of 168 subjects (17.7%) had a CD4+ T cell count of <200 cells/mm3 at baseline. The QFT-GIT assay was determinate in 82.1% (138/168) and indeterminate in 17.9% (30/168) of subjects. There was no difference between QFT-GIT-determinate and -indeterminate subjects in terms of median CD4+ T cell count, HIV-1 RNA level, and ART provision as well as virological suppression at baseline and follow-up.
Within the follow-up period, however, AIDS-defining manifestations occurred in 7 QFT-GIT-indeterminate compared to 11 QFT-GIT-determinate individuals (23.3% versus 8.0%; OR, 3.5; P < 0.05). Of these 18 patients, 8 had been on ART at baseline and 10 were started on ART during follow-up, with three patients not being virologically suppressed at the time of AIDS.
In this prospective, longitudinal study on HIV-1-infected individuals followed for 3 years, the QFT-GIT assay yielded indeterminate results in 5.8% of the 948 subjects tested. In patients with indeterminate QFT-GIT results, median CD4+ T cell counts were significantly lower than those in subjects with a determinate QFT-GIT result, consistent with previously published data (2, 7). In terms of risk factors for indeterminate QFT-GIT results, we found that in addition to low actual and nadir CD4+ T cell counts, both injection-drug use and high HIV-1 RNA load were associated with indeterminate results.
So far, no longitudinal and prospective study on the value of new-generation IGRAs, focusing on TB-related and unrelated clinical outcomes of a larger cohort of HIV-1-infected subjects, has been published (9). To the best of our knowledge, this part of our single-center study is the first to assess the prognostic value of indeterminate and determinate QFT-GIT results in HIV-1 infection. None of the subjects tested as QFT-GIT indeterminate developed active TB disease within the follow-up period. Despite the fact that ART was provided to all subjects according to the European AIDS Clinical Society (EACS) guidelines (5) and that the majority of patients had been on ART at study inclusion, the rate of progression to AIDS-defining illnesses was significantly higher among individuals with an indeterminate QFT-GIT result than among patients with a determinate QFT-GIT result. Furthermore, in patients with a CD4+ T cell count of <200 cells/mm3, an indeterminate QFT-GIT result indicated a significantly increased risk for the development of AIDS-defining diseases.
The limitations of this study include its single-center nature and the fact that individuals with positive and/or indeterminate QFT-GIT results may have been monitored more closely than were those with negative test results. However, at least one chest X-ray and/or computed tomography of the chest and abdomen was performed in all subjects every year to mitigate this limitation.
Collectively, there is now growing evidence that IGRAs perform well both in immunocompetent individuals and in patients with moderate immunodeficiency. However, among patients with advanced immunodeficiency, those with an indeterminate QFT-GIT result are at increased risk of serious clinical events other than TB. Thus, in HIV-1-infected patients with advanced quantitative CD4+ T cell depletion, an indeterminate QFT-GIT might indicate an additional loss of global T cell function warranting detailed clinical evaluation and careful follow-up.
ACKNOWLEDGMENTS
We thank Regina Aichwalder, Silvia Reichholf, and Alexander Riegler for excellent technical assistance and the patients who participated in this study.
M.C.A., A.R., and N.K. designed the study and enrolled patients. M.C.A., J.T., F.B., and N.K. contributed to data collection. T.R. performed statistical analysis. M.C.A., J.T., F.B., T.R., A.R., and N.K. participated in data interpretation. M.C.A. wrote the article.
All authors have read and approved the final manuscript.
There were no conflicts of interest for any author and no financial support.
Footnotes
Published ahead of print 16 May 2012
REFERENCES
- 1. Aabye MG, et al. 2009. The impact of HIV infection and CD4 cell count on the performance of an interferon gamma release assay in patients with pulmonary tuberculosis. PLoS One 4:e4220 doi:10.1371/journal.pone.0004220 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. Aichelburg MC, et al. 2009. Detection and prediction of active tuberculosis disease by a whole-blood interferon-gamma release assay in HIV-1-infected individuals. Clin. Infect. Dis. 48:954–962 [DOI] [PubMed] [Google Scholar]
- 3. Cellestis 2003. QuantiFERON-CMI. Package insert. Cellestis, Carnegie, Victoria, Australia [Google Scholar]
- 4. Centers for Disease Control and Prevention 1992. 1993 revised classification system for HIV infection and expanded surveillance case definition for AIDS among adolescents and adults. MMWR Recomm. Rep. 41(RR-17):1–19 [PubMed] [Google Scholar]
- 5. Clumeck N, Pozniak A, Raffi F. 2008. European AIDS Clinical Society (EACS) guidelines for the clinical management and treatment of HIV-infected adults. HIV Med. 9:65–71 [DOI] [PubMed] [Google Scholar]
- 6. Kobashi Y, et al. 2009. Indeterminate results of QuantiFERON TB-2G test performed in routine clinical practice. Eur. Respir. J. 33:812–815 [DOI] [PubMed] [Google Scholar]
- 7. Luetkemeyer AF, et al. 2007. Comparison of an interferon-gamma release assay with tuberculin skin testing in HIV-infected individuals. Am. J. Respir. Crit. Care Med. 175:737–742 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. Mori T, et al. 2004. Specific detection of tuberculosis infection: an interferon-gamma-based assay using new antigens. Am. J. Respir. Crit. Care Med. 170:59–64 [DOI] [PubMed] [Google Scholar]
- 9. Rangaka MX, et al. 2012. Predictive value of interferon-gamma release assays for incident active tuberculosis: a systematic review and meta-analysis. Lancet Infect. Dis. 12:45–55 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. Richeldi L. 2006. An update on the diagnosis of tuberculosis infection. Am. J. Respir. Crit. Care Med. 174:736–742 [DOI] [PubMed] [Google Scholar]