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Interferon (IFN)-γ release assays (IGRAs) are probably the most accurate tests for the detection of latent Mycobacterium tuberculosis infection, but IGRAs are labour intensive and the transport of samples over longer distances is difficult. IFN-γ-induced protein (IP)-10 is expressed at 100-fold higher levels than IFN-γ, and IP-10 release assays have comparable performance to IGRAs. The aim of this study was to explore the diagnostic potential of a novel IP-10 release assay based on dried plasma spots (DPS). The presence of IP-10 and IFN-γ was determined in plasma and in DPS by ELISA. Diagnostic algorithms for plasma and DPS tests for IP-10 were developed on a training cohort comprising 60 tuberculosis (TB) patients and 59 healthy controls. Diagnostic accuracy was assessed in a validation cohort comprising 78 TB patients and 98 healthy controls. Plasma was measured in Spain and DPS samples were sent to Denmark using the conventional postal service for analysis. IP-10 was readily detectable in both plasma and DPS, and correlation was excellent (r(2) = 0.95). QuantiFERON-TB Gold In-Tube (QFT-TB) and IP-10 in DPS and plasma rendered comparable sensitivity (78%, 82% and 84%, respectively), specificity (100%, 97% and 97%, respectively) and indeterminate rates (p>0.55). The DPS-based IP-10 test has comparable diagnostic accuracy to the QFT-TB and samples can be sent via conventional mail over long distances for analysis without affecting the results.

In this clinical audit, we assessed retrospectively the current practice of respiratory physicians with respect to interferon gamma (IFNγ) release assay (IGRA) testing for tuberculosis (TB), as recommended by the 2011 National Institute of Health and Care Excellence (NICE) guidelines for the diagnosis and management of TB. All IGRAs requested by respiratory physicians over a 3-year period were identified retrospectively, and both results and clinical indications analysed. Of the total number of IGRAs carried out, 90% formed part of investigations of suspected active TB. However, 89% of the patients had not had a documented Mantoux test and human immunodeficiency virus (HIV) status was unclear in the 35.2% of patients treated for active TB. Of patients with chest X-rays suggestive of TB, 92.3% were treated for active TB. Of the patients under the age of 35 with reactive IGRAs, 84.6% were treated for active or latent TB and 15.4% had justifiable reasons for not receiving chemoprophylaxis. Based on the results of our audit, IGRAs are commonly being utilised for the investigation of active TB, which is contrary to current guidance.

The enzyme-linked immunospot (ELISpot) assay is a widely used method for immune monitoring in cancer immunotherapy trials. In the ELISpot assay, peripheral blood mononuclear cells (PBMC) are stimulated with specific antigens, and cytokines of interest produced by individual cells are detected. In the standard procedure, T cells rely for antigen presentation on other cells like the monocyte/macrophage population present among the PBMC. Whereas oligopeptides can be added directly to the ELISpot assay without the necessity of a pre-incubation step, protein antigens must be internalized and processed by antigen-presenting cells to accomplish efficient presentation via HLA class I or II. We have studied the impact of sources for different antigen-presenting cell (i.e. PBMC-resident monocytes and monocyte-derived dendritic cells maturated with Poly I:C and PGE-2 based cocktails) on ELISpot assay performance and defined an optimized dendritic cell-based ELISpot protocol. This protocol is suitable for monitoring immune responses directed to protein antigens with higher sensitivity than the standard procedure., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Development of Interferon-Gamma Release Assays (IGRAs) and implementation of their use in clinical practice almost 10 years ago has revolutionised diagnosis of latent tuberculosis (TB) infection (LTBI). The commercially available IGRAs, TSPOT.TB (Oxford Immunotech, Oxford, UK) and QuantiFERON Gold In-Tube (Cellestis, Victoria, Australia), allow detection of TB infection with greater specificity and sensitivity than the tuberculin skin test (TST) and are now recommended for diagnosis of LTBI. The TSPOT.TB assay is a simplified enzyme-linked immunospot assay (ELISpot) that enumerates TB-specific T lymphocytes (T cells) secreting interferon-gamma (IFNγ). In comparison, the QuantiFERON Gold In-Tube assay constitutes an enzyme-linked immunosorbent assay (ELISA) to quantify IFNγ released into blood plasma after incubation of whole blood with TB antigens. Release of IFNγ, as a result of antigen stimulation of TB-specific T cells within blood, is indicative of TB infection. Although IGRAs have significant advantages over the TST in diagnosis of latent TB, they have significant limitations. Discovery of new antigens and advances in methodology for measuring cellular immunity have recently paved the way for novel tests that overcome these limitations. By establishing for the first time technological platforms for T cell based diagnosis in diagnostic service laboratories, IGRAs provide a bridgehead to clinical application of T cell based diagnosis in routine practice., (Copyright © 2013 Elsevier Inc. All rights reserved.)

An understanding of the diagnostic performance of interferon-gamma release assays (in terms of parameters such as specificity, positive predictive value, and likelihood ratio) is important in the diagnosis of active tuberculosis in elderly Japanese patients because the high proportion of a prior history of tuberculosis among these patients can lead to misleading results. To elucidate the diagnostic performance of such assays, we examined the results of the QuantiFERON-TB Gold In-Tube test (QFT-GIT) in 65 patients in a younger group, and 52 patients in an elderly group who were suspected of having active tuberculosis and who had received the QFT-GIT. The median ages of the younger patients and elderly patients were 54 and 78 years, respectively. Among patients with active tuberculosis, the number of those with positive results on the QFT-GIT was similar in the two age groups [15 out of 17 (88.2 %) in the younger patients compared with 7 out of 8 (87.5 %) in the elderly patients]. However, in the patients with other diseases the number of those with positive QFT-GIT results was significantly different in the younger and elderly groups, being 6.3 and 27.3 %, respectively (P = 0.01). Although the sensitivity, negative predictive value, and negative likelihood ratio were similar in the two groups, the specificity, positive predictive value, and positive likelihood ratio were significantly lower in the elderly patients, being 72.7% (95 % confidence interval [CI], 57.2-85.0) compared with 93.8% (95 % CI, 82.8-98.7), 36.8% (95 % CI, 16.3-61.6) compared with 83.8% (95 % CI, 58.6-96.4), and 3.21 (95 % CI, 1.85-5.56) compared with 14.12 (95 % CI, 4.66-42.81), respectively. In the elderly patients with positive results on the test, a comparison of clinical data between those with active tuberculosis and those with other diseases demonstrated that the only clinical parameter showing a significant difference between these two groups was the radiological finding of small nodules in the patients with active tuberculosis (P < 0.01). The QFT-GIT may be less accurate in elderly patients, and radiological findings can be helpful in the clinical evaluation of patients with positive results on the test.

Healthcare workers (HCW) are a risk group for TB. Even in countries with low TB incidence, the risk of TB in HCW is elevated for a wide range of tasks in healthcare, and the prevention of nosocomial infection of HCW remains as a challenge. IFN-γ release assays (IGRA) facilitate the screening of HCW for latent TB infection. In comparison with the tuberculin skin test, the IGRA reduces the number of x-rays and the amount of chemoprevention needed. However, a borderline zone should be introduced for the interpretation of IGRA results in the serial testing of HCW. More data on disease progression depending on conversion and reversion in IGRA is needed and a better test, which is able to distinguish recent from remote latent TB infection, would be desirable in the future.

Background: Clinical data with use of serial interferon-γ release assay (IGRA) testing in US health-care workers (HCWs) are limited., Methods: A single-center, retrospective chart review was done from 2007 to 2010 of HCWs who underwent preemployment QuantiFERON-TB Gold In-Tube testing. Demographic data, bacille Calmette-Guérin history, prior tuberculin skin test result if done, and baseline and serial IGRA values were obtained. The number of IGRA converters and reverters and their subsequent management by infectious disease physicians were reviewed. Quantitative IGRA-negative values were not available., Results: A total of 7,374 IGRAs were performed on newly hired HCWs. Of these tests, 486 (6.6%) were positive at baseline, 305 (4.1%) were indeterminate, and 6,583 (89.3%) were negative. From 2007 to 2010, 52 of 1,857 HCWs (2.8%) with serial IGRA tests were identified as converters, with a serial IGRA median value of 0.63 IU/mL. Seventy-one percent of HCWs with IGRA conversion had values ≤ 1 IU/mL. None of the converters had active TB or were part of an outbreak investigation., Conclusions: Clinical significance of most QuantiFERON-TB Gold In-Tube conversions in serial testing remains a challenging task for clinicians. The use of a single cutoff point criterion for IGRA may lead to overdiagnosis of new TB infections. Clinical assessment and evaluation may help to prevent unnecessary therapy in these cases. The criteria for defining conversions and reversions by establishing new cutoffs needs to be evaluated further, especially in HCWs.

Background: Given the current lack of effective vaccines against TB, the accuracy of screening tests for determining or excluding latent TB infection (LTBI) is decisive in effective TB control. This meta-analysis critically appraises studies investigating the positive and the negative predictive value (PPV and NPV, respectively) from a test-determined LTBI state for progression to active TB of interferon-γ release assays (IGRAs) and the tuberculin skin test (TST)., Methods: We searched MEDLINE, EMBASE, and Cochrane bibliographies for relevant articles. After qualitative evaluation, the PPV and NPV for progression of commercial and “in-house” IGRAs and the TST for persons not receiving preventive treatment in the context of the respective IGRA studies were pooled using both a fixed and a random-effect model. Weighted rates were calculated for all study populations and for groups solely at high risk of TB development., Results: The pooled PPV for progression for all studies using commercial IGRAs was 2.7% (95% CI, 2.3%-3.2%) compared with 1.5% (95% CI, 1.2%-1.7%) for the TST (P < .0001). PPV increased to 6.8% (95% CI, 5.6%-8.3%) and 2.4% (95% CI, 1.9%-2.9%) for the IGRAs and the TST, respectively, when only high-risk groups were considered (P < .0001). Pooled values of NPV for progression for both IGRAs and the TST were very high, at 99.7% (95% CI, 99.5%-99.8%) and 99.4% (95% CI, 99.2%-99.5%), respectively, although they were significantly higher for IGRAs (P < .01)., Conclusions: Commercial IGRAs have a higher PPV and NPV for progression to active TB compared with those of the TST, especially when performed in high-risk persons.

Rationale: There is uncertainty regarding how to interpret discordance between tests for latent tuberculosis infection., Objectives: The objective of this study was to assess discordance between commercially available tests for latent tuberculosis in a low-prevalence population, including the impact of nontuberculous mycobacteria., Methods: This was a cross-sectional comparison study among 2,017 military recruits at Fort Jackson, South Carolina, from April to June 2009. Several tests were performed simultaneously with a risk factor questionnaire, including (1) QuantiFERON-TB Gold In-Tube test, (2) T-SPOT.TB test, (3) tuberculin skin test, and (4) Battey skin test using purified protein derivative from the Battey bacillus., Measurements and Main Results: In this low-prevalence population, the specificities of the three commercially available diagnostic tests were not significantly different. Of the 88 subjects with a positive test, only 10 (11.4%) were positive to all three tests; 20 (22.7%) were positive to at least two tests. Bacille Calmette-Guérin vaccination, tuberculosis prevalence in country of birth, and Battey skin test reaction size were associated with tuberculin skin test-positive, IFN-γ release assay-negative test discordance. Increasing agreement between the three tests was associated with epidemiologic criteria indicating risk of infection and with quantitative test results., Conclusions: For most positive results the three tests identified different people, suggesting that in low-prevalence populations most discordant results are caused by false-positives. False-positive tuberculin skin test reactions associated with reactivity to nontuberculous mycobacteria and bacille Calmette-Guérin vaccination may account for a proportion of test discordance observed.

Background: We determined the consistency of positive interferon-gamma (IFN-γ) release assays (IGRAs) to detect latent TB infection (LTBI) over one-year postpartum in HIV-1-infected women., Methods: Women with positive IGRAs during pregnancy had four 3-monthly postpartum IGRAs. Postpartum change in magnitude of IFN-γ response was determined using linear mixed models., Results: Among 18 women with positive pregnancy IGRA, 15 (83%) had a subsequent positive IGRA; 9 (50%) were always positive, 3 (17%) were always negative, and 6 (33%) fluctuated between positive and negative IGRAs. Women with pregnancy IGRA IFN-γ>8 spot forming cells (SFCs)/well were more likely to have consistent postpartum IGRA response (odds ratio: 10.0; 95% confidence interval (CI): 0.9-117.0). Change in IFN-γ response over postpartum was 10.2 SFCs/well (95% CI: -1.5-21.8 SFCs/well)., Conclusion: Pregnancy positive IGRAs were often maintained postpartum with increased consistency in women with higher baseline responses. There were modest increases in magnitude of IGRA responses postpartum.

Although interferon-γ release assays (IGRAs) are intended for diagnosing latent tuberculosis (TB), we hypothesised that in a high-burden setting: 1) the magnitude of the response when using IGRAs can distinguish active TB from other diagnoses; 2) IGRAs may aid in the diagnosis of smear-negative TB; and 3) IGRAs could be useful as rule-out tests for active TB. We evaluated the accuracy of two IGRAs (QuantiFERON®-TB Gold In-tube (QFT-GIT) and T-SPOT®.TB) in 395 patients (27% HIV-infected) with suspected TB in Cape Town, South Africa. IGRA sensitivity and specificity (95% CI) were 76% (68-83%) and 42% (36-49%) for QFT-GIT and 84% (77-90%) and 47% (40-53%) for T-SPOT®.TB, respectively. Although interferon-γ responses were significantly higher in the TB versus non-TB groups (p<0.0001), varying the cut-offs did not improve discriminatory ability. In culture-negative patients, depending on whether those with clinically diagnosed TB were included or excluded from the analysis, the negative predictive value (NPV) of QFT-GIT, T-SPOT®.TB and chest radiograph in smear-negative patients varied between 85 and 89, 87 and 92, and 98% (for chest radiograph), respectively. Overall accuracy was independent of HIV status and CD4 count. In a high-burden setting, IGRAs alone do not have value as rule-in or -out tests for active TB. In smear-negative patients, chest radiography had better NPV even in HIV-infected patients.

Background: Imperfect sensitivity of interferon-γ release assay (IGRA) is a potential problem to detect tuberculosis. We made a thorough investigation of the factors that can lead to false negativity of IGRA., Methods: We recruited 543 patients with new smear-positive pulmonary tuberculosis in Hanoi, Viet Nam. At diagnosis, peripheral blood was collected and IGRA (QuantiFERON-TB Gold In-Tube) was performed. Clinical and epidemiological information of the host and pathogen was collected. The test sensitivity was calculated and factors negatively influencing IGRA results were evaluated using a logistic regression model in 504 patients with culture-confirmed pulmonary tuberculosis., Results: The overall sensitivity of IGRA was 92.3% (95% CI, 89.6%-94.4%). The proportions of IGRA-negative and -indeterminate results were 4.8% (95% CI, 3.1%-7.0%) and 3.0% (95% CI, 1.7%-4.9%). Age increased by year, body mass index <16.0, HIV co-infection and the increased number of HLA-DRB1*0701 allele that patients bear showed significant associations with IGRA negativity (OR = 1.04 [95% CI, 1.01-1.07], 5.42 [1.48-19.79], 6.38 [1.78-22.92] and 5.09 [2.31-11.22], respectively). HIV co-infection and the same HLA allele were also associated with indeterminate results (OR = 99.59 [95% CI, 15.58-625.61] and 4.25 [1.27-14.16])., Conclusions: Aging, emaciation, HIV co-infection and HLA genotype affected IGRA results. Assessment of these factors might contribute to a better understanding of the assay.

The purpose of this prospective, cross-sectional observational study was to compare the tuberculin skin testing (TST) with QuantiFERON-TB Gold-In Tube (QTF-GIT) for the detection of latent tuberculosis infection in healthcare workers (HCWs). The study included 78 volunteers who are HCWs at the same tertiary care teaching hospital for chest diseases and tuberculosis. Participants with active tuberculosis, immunodefficiency or malnutrition were not included. The TST was administered by the Mantoux method. Peptides representing ESAT-6, CFP-10 and TB7-7 were used as TB-specific antigens in the whole-blood Interferon-gamma (IFN-g) assay (QTF-GIT). There was a statistically significant relation between the number of Bacillus Calmette-Guerin (BCG) scars and the diameter of TST (p< 0.01). QTF results according to previous BCG vaccinations did not significantly differ (p> 0.05). There was an intermediate concordance between two tests (k: 0.346). QTF-GIT has a sensitivity of 56.14% (both TST and QTF-GIT are positive), specificity of 90.48% (both TST and QTF-GIT are negative); positive predictive value of 94.12% and negative predictive value of 43.18% and accuracy is 65.38%. There was a statistically significant relation between TST diameter and QTF result (p< 0.01). Latent tuberculosis infection prevalance of our study population was 43% according to QTF-GIT test, 73% according to TST and BCG vaccination rate was 87%. In conclusion, TST is affected by previous BCG vaccinations, QTF-GIT is not. We can recommend QTF-GIT test for the detection of latent tuberculosis infection as an alternative to TST in populations with routine BCG vaccination programme.