Diagnostic Microbiology and Infectious Disease

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1 Diagnostic Microbiology and Infectious Disease 75 (3) 68 7 Contents lists available at SciVerse ScienceDirect Diagnostic Microbiology and Infectious Disease journal homepage: Mycobacteriology Interferon gamma mrna quantitative real-time polymerase chain reaction for the diagnosis of latent tuberculosis: a novel interferon gamma release assay Sunghyun Kim a,, Young Keun Kim b,, Hyejon Lee c, Jang-Eun Cho c,d, Hyo Youl Kim b, Young Uh e, Young Mi Kim c, Hyunjung Kim c, Sang-Nae Cho c, Bo-Young Jeon a, Hyeyoung Lee a, a Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju, South Korea b Department of Internal Medicine, Yonsei University Wonju College of Medicine, Wonju, South Korea c Department of Microbiology, College of Medicine, Yonsei University, Seoul, South Korea d Department of Biomedical Laboratory Science, Daegu Health College, Daegu, South Korea e Department of Laboratory Medicine, Yonsei University Wonju College of Medicine, Wonju, South Korea article info abstract Article history: Received 7 May Received in revised form 3 September Accepted September Available online 4 October Keywords: Tuberculosis QFT-IT IFN-γ mrna Real-time-PCR IFN-γ ELISA The interferon gamma (IFN-γ) release assay (IGRA) is widely used as a diagnostic method for latent tuberculosis infection (LTBI). The QuantiFERON-TB Gold and QuantiFERON-TB Gold In-tube (QFT-IT) tests measure plasma IFN-γ levels using enzyme-linked immunosorbent assay (ELISA), and T-SPOT.TB counts IFN-γ producing cells using enzyme-linked immunosorbent spot assay. IFN-γ mrna was evaluated as an indicator of IGRA in comparison with QFT-IT IFN-γ ELISA in 46 subjects with active TB and in 73 at low risk for TB. Significant IFN-γ mrna expression was detected from 3 min and peaked 4 h after stimulation with MTB antigens or mitogen. This was defined as the optimal time point for IFN-γ mrna real-time polymerase chain reaction (PCR). The sensitivities of IFN-γ mrna real-time PCR and IFN-γ ELISA were 84.8% (39/46) and 89.% (4/46), respectively (no significant difference). Although the specificities of IFN-γ ELISA was 4.% higher than that of IFN-γ mrna real-time PCR (6.3% versus 56.%), the difference was not statistically significant. The overall agreement between IFN-γ mrna real-time PCR and IFN-γ ELISA was 79.8% (kappa =.475). Whilst there was no difference in the performance of IFN-γ mrna real-time PCR and IFN-γ ELISA, IFN-γ mrna real-time PCR was superior to IFN-γ ELISA in terms of the time required for detection of MTB infection. 3 Elsevier Inc. All rights reserved.. Introduction Tuberculosis (TB) remains a major infectious disease, with approximately 9.3 million new TB cases annually, leading to.7 million deaths per year. Mycobacterium tuberculosis (MTB), a cause of TB, infects one-third of the world's population (Lienhardt et al., ). The situation has been worsened by combination with human immunodeficiency virus infection and the appearance of multidrugresistant or extended drug resistant MTB strains (Kumar et al., ). Control of TB depends largely on early detection and treatment of active TB cases. Smear microscopy and mycobacterial culture are the currently available diagnostic tests for active TB disease (Palomino, ). Smear microscopy is rapid, but its sensitivity is poor. Culturing the mycobacterium is the gold standard for TB diagnosis, but it takes several weeks to obtain the results. Screening and treatment of highrisk subjects with latent TB infection (LTBI) have been recognized as This study was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korean government (MEST) (no. -65). Corresponding author. Tel.: ; fax: address: hyelee@yonsei.ac.kr (H. Lee). These authors contributed equally to this work. an important control measure, especially in low-burden countries (Mazurek et al., ). Until recent years, the tuberculin skin test (TST) has been the gold standard for the diagnosis of LTBI (Pouchot et al., 997). However, it shows cross-reactivity with bacille Calmette Guérin (BCG) and many nontuberculous mycobacteria because the purified protein derivatives (PPD), a diagnostic reagent in the TST, share some antigens with BCG and several nontuberculous mycobacteria. A recently introduced diagnostic method for LTBI is the interferon gamma (IFN-γ) release assay (IGRA), which assesses the presence of MTB infection by detecting the in vitro release of IFN-γ following stimulation with MTB-specific antigens, such as early secretory antigen target 6 (ESAT-6) and culture filtrate protein, from previously sensitized T cells (Shah et al., ). Three in vitro IGRAs are now commercially licensed and are globally available: Quanti- FERON-TB Gold (QFT-G) and QuantiFERON-TB Gold In-tube (QFT-IT) (Cellestis, Carnegie, Victoria, Australia), and the T-SPOT.TB test (T-SPOT, Oxford Immunotec, Oxford, UK). All have higher specificities and sensitivities than conventional TST (Chee et al., 8). These IGRSs are similar in terms of the antigens used (ESAT-6 and CFP- for QFT-G and T-SPOT; ESAT-6, CFP-, and TB7.7 for QFT-IT) and incubation time (overnight or 6 4 h). The main differences between these tests /$ see front matter 3 Elsevier Inc. All rights reserved.

2 S. Kim et al. / Diagnostic Microbiology and Infectious Disease 75 (3) lie in the techniques used for IFN-γ detection and the specimens used. QFT-G and QFT-IT use enzyme-linked immunosorbent assay (ELISA) to detect IFN-γ produced by immune cells in whole blood after stimulation with MTB antigens (Detjen et al., 7). In contrast, T-SPOT detect IFN-γ producing cells among the mononuclear cells that respond to MTB antigens using enzyme-linked immunosorbent spot (ELISPOT) assay (Turtle et al., ). Of these assays, QFT-G and QFT-IT are widely used because sample preparation is convenient and ELISA is more commonly used. Cytokine mrna expression has also been measured and correlates well with cytokine protein level (Bibova et al., ; Turtle et al., ). Moreover, mrna expression can be measured at early time points, before synthesis of the protein has begun. To date, quantitation of IFN-γ mrna expression has not been used for the diagnosis of MTB infection. In the past few decades, with the advent of new technologies in molecular biology, new molecular methods have been introduced, such as polymerase chain reaction (PCR) and real-time PCR. Real-time PCR, in particular, has been widely used to detect bacteria, viruses, toxins, and cytokines due to its excellent sensitivity and ability to provide quantitative data (Kvach et al., ; Uyttendaele et al., 3). In the present study, we applied IFN-γ mrna quantitative realtime PCR as a novel IGRA and evaluated its usefulness for LTBI in comparison with the commercially available QTF-IT IFN-γ ELISA test.. Materials and methods.. Study participants From May to February, a prospective clinical study was undertaken at Yonsei University Wonju Christian Hospital, Wonju, Republic of Korea. All subjects provided written informed consent, and the study was approved by the Institutional Ethics Committee of Yonsei University Wonju College of Medicine (approval no. -). Patients with a high clinical suspicion of active TB were recruited and provided at least sputum specimens on separate days, which were analyzed by acid-fast bacilli (AFB) microscopy using the Ziehl Neelsen method and cultured in Löwenstein Jensen medium. The presence of MTB in positive culture samples was further confirmed using the AdvanSure TB/NTM real-time PCR kit (LG Life Science, Seoul, Korea). The presence of active TB was defined by a positive sputum smear microscopy result and/or positivity for MTB in sputum culture and/or an abnormality suggestive of TB in chest X-ray. Healthy individuals with normal chest radiographs, no known history of contact with TB patients, and no symptoms of active TB were included as a group at low risk for TB. All participants underwent chest radiography and clinical examination, and were questioned regarding their history of exposure to TB patients. It was assumed that active TB subjects were infected with M. tuberculosis according to previous studies (Mori et al., 4; Lee et al., 6), and AFB positive and/or culture-confirmed active TB subjects were used as positive control for the QFT-IT test... QuantiFERON-TB Gold In-tube IGRA was performed using the QFT-IT test (Cellestis), according to the manufacturer's instructions (Harada et al., 8; Mori et al., 4). Blood samples were collected in 3 QFT-IT collection tubes (nil, MTB antigens, and mitogen, which contain lithium heparin). The QFT-IT collection tubes were processed within h and incubated for 4 h at 37 C. Plasma was collected by centrifugation at 5 rpm for 5 min and stored at 8 C until it was assayed..3. IFN-γ ELISA assay IFN-γ concentrations in plasma samples from each subject were determined using the QFT-IT ELISA test according to the procedure outlined in the product insert. The ELISA assay was carried out by trained staff at Yonsei University Wonju Christian Hospital, Wonju, Korea. The test results were interpreted using the QFT-IT ELISA software (version no..43; Cellestis), and the cut-offs for diagnosis in the manufacturer's instructions were used. Mitogen stimulation served as an intrinsic control for blood sample quality. An IFN-γ concentration of.35 IU/mL (after subtraction of nil control IFN-γ), following exposure to MTB antigens was, considered positive for QFT-IT; a concentration of b.35 IU/mL was considered negative. If the IFN-γ response to mitogen was b.5 IU/mL higher than that for the nil control, or N8 IU/mL higher than that for the nil control, the result was deemed indeterminate (Mori et al., 4)..4. Total RNA isolation and reverse transcription For IFN-γ mrna real-time PCR, blood collection and incubation were performed as for QFT-IT, according to the manufacturer's instructions (Mannhalter et al., ). Total RNA was isolated from whole blood using at scheduled time points TRIzol Reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. Then, total RNA was incubated with deoxyribonuclease I from the Amplification Grade DNase I kit (Invitrogen) to give a final RNA concentration of ng/μl. Reverse transcription (RT) into cdna was performed using the High Capacity cdna Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, Foster City, CA, USA) with random primers, according to the supplier's instructions. No products were detected in control samples from which the reverse transcriptase was omitted..5. Real-time PCR Quantitative real-time PCR was performed using the TaqMan system (Applied Biosystem). The expression levels of IFN-γ and internal reference glyceraldehyde-3 phospate dehydrogenase (GAPDH) were measured by PCR using TaqMan expression assays. Real-time PCR was carried out with TaqMan Universal PCR Master Mix using 3 μl of cdna as the template in a total volume of μl. The thermal cycling conditions were min at 95 C, followed by 4 cycles of s at 95 C and 3 s at 6 C. All reactions were performed using an ABI 75 FAST Real-time PCR System (Applied Biosystems). The sequences of the primers ( pmol/μl) and probes (5 pmol/μl) used to amplify the desired genes were as follows: IFN-γ: sense 5 -TGA ATG TCC AAC GCA AAG CA-3, anti-sense 5 -CGA CCT CGA AAC AGC ATC TGA-3, and probe 6FAM-CGC CAG CAG CTA AAA CAG GGA AGC G- BHQ; GAPDH: sense 5 - CCA TCT TCC AGG AGC GAG ATC C-3, antisense 5 -ATG GTG GTG AAG ACG CCA GTG-3, and probe 6FAM-TCC ACG ACG TAC TCA GCG CC-BHQ. The standard curves for the target and references genes indicated the efficacy of the method (N9%). Relative quantification was performed by the -DDCT method; relative expression was calculated as the ratio between the mean CT (threshold cycle) values of the target gene and reference gene (GAPDH) in each stimulated sample in relation to a reference sample (not stimulated) (Kubista et al., 6)..6. Cut-off value determination for the study population The optimal cut-off level for IFN-γ mrna real-time PCR was calculated by receiver operator characteristic (ROC) curve analysis using data from 46 subjects with active TB and from 73 at low risk for TB. The sensitivity and specificity of the IFN-γ mrna real-time PCR were calculated from this cut-off value..7. Statistical analysis Data were analyzed using GraphPad Prism version 4. (GraphPad Software, San Diego, CA, USA). Sensitivities and specificities for MTB

3 7 S. Kim et al. / Diagnostic Microbiology and Infectious Disease 75 (3) 68 7 infection, together with 95% confidence intervals (CIs), were calculated as proportions of positive and negative cases among patients and control subjects, respectively. The values of these parameters were compared using the z test. Agreement between the test modalities was tested with McNemar's test and expressed as the kappa coefficient and overall agreement (proportion of cases giving the same result in both tests). When comparing the mean of IFN-γ measurements of both methods, and calculating the Pearson's correlation coefficient for them, the original values were log transformed. Also, nonparametric statistics (Wilcoxon's ranked sign test and Spearman's ranked correlation coefficient) were employed. When testing for differences, a value of P b.5 was used as the criterion for statistical significance. 3. Results 3.. Baseline characteristics The 9 screened subjects comprised 46 patients with active TB and 73 at low risk for TB (Table ). The median age of the enrolled subjects was 48 years (range 8 86 years) for the active TB patients and 37 years (range 8 56 years) for those at low risk for TB. The male-to-female ratios were.3: and.78: for the active TB patients and subjects at low risk for TB, respectively. Of the 46 subjects in the active TB group, 44 (86.9%) had culture- or AFB smear-confirmed TB and (4.3%) had clinical TB. Of these 46 patients, 36 (78.3%) had pulmonary TB and (.7%) had cervical tuberculous lymphadenopathy. None of the subjects at low risk for TB was reported to have any systemic disease affecting host immunity. The BCG vaccination rates were 67.4% and 78.% in the subjects with active TB and subjects at low risk for TB, respectively. 3.. Measurement of mrna expression of IFN-γ cytokine IFN-γ mr RNA expression ratio (R) IFN-γ (IU/ml) A 5 B 9 Mitogene 6 MTB antigens Non 3 min hr hr 4 hr 6 hr 8 hr hr 4 hr Incubation time 5 Mitogene 5 MTB antigens Non 3 min hr hr 4 hr 6 hr 8 hr hr 4 hr Incubation time To measure IFN-γmRNA expression, whole blood samples from 7 randomly selected TB patients (as a tentative positive control for MTB infection) were collected in 3 QFT-IT tubes and total RNA was isolated at 3 min and at,, 4, 6, 8,, and 4 h after incubation. Plasma samples were also collected at the same time points to measure IFN-γ protein levels. In the MTB antigen stimulated samples, IFN-γ mrna expression was detected from 3 min, increased up to 4 h, and was constant between 4 and 6 h (Fig. A). However, IFN-γ mrna expression decreased after this time point. In the mitogen-stimulated samples, the expression of IFN-γ was higher than that in the MTB antigen stimulated samples, but peaked at an earlier time point ( h) and then declined. Plasma IFN-γ protein levels were significantly increased from 8 h in the mitogen-stimulated samples and from h in the MTB antigen stimulated samples (Fig. B). There was no significant IFN-γ mrna or protein in the nil control samples. Based on the result, we propose that the optimal time point for measurement of IFN-γ mrna expression is at 4 h, because IFN-γmRNA expression was high in both MTB antigen stimulated samples and in the mitogen-stimulated samples at this time point. In Table Baseline characteristics of subjects. No. of subjects Median age in years (range; IQR) 48 (8 86; 37.6) 37 (8 56; 6.48) M/F ratio 6: 3:4 BCG Vaccination 3 (67.4%) 57 (78.%) TB = Tuberculosis; IQR = interquartile range; M = male; F = female. Fig.. The IFN-γ mrna expression levels (A) and IFN-γ protein levels (B) in MTB antigen and mitogen-stimulated samples of active tuberculosis (TB) patients. Blood samples from active TB patients were collected in QFT-IT tubes and stimulated with MTB antigens and mitogen, and total RNA and plasma were isolated at 3 min and at,, 4, 6, 8,, and 4 h after incubation. The IFN-γ mrna expression levels and IFN-γ protein levels were measured using IFN-γ mrna real-time PCR and IFN-γ ELISA, respectively. subsequent experiments, IFN-γ mrna expression was measured at 4 h and plasma IFN-γ protein levels was measured at 4 h IFN-γ mrna expression and its cut-off point in subjects MTB antigen stimulated samples from subjects with active TB and those at low risk for TB were analyzed by IFN-γ mrna real-time PCR. The MTB antigen dependent IFN-γmRNA expression ratio (R) was measured by subtracting the expression level of IFN-γ in nil control samples from that in the MTB antigen stimulated samples. Plasma IFN-γ levels were measured by subtracting the IFN-γ level in nil control from those in the MTB antigen stimulated samples. The median value of R ranged from.3 to 37.6 (median 3.65) in subjects with active TB and from.5 to 9. (median.8) in subjects at low risk for TB (Fig. A). IFN-γ mrna expression was significantly higher in subjects with active TB than in those at low risk for TB (P b.). IFN-γ protein levels ranged from.4 to (median.66) in subjects with active TB and from to 9. (median.) in subjects at low risk for TB (Fig. B). IFN-γ protein levels in subjects with active TB were also higher than in those at low risk for TB (P b.). There was a significant correlation between IFN-γ mrna expression and IFN-γ protein level (R =.74; Fig. 3). To assess the diagnostic performance of IFN-γ mrna real-time PCR, an ROC curve analysis was performed (Fig. 4). Patients with

4 S. Kim et al. / Diagnostic Microbiology and Infectious Disease 75 (3) A IFN-γ (IU/ml) B ratio (R) IFN-γ mrna expression Fig.. Dot plots of IFN-γ ELISA and IFN-γ mrna real-time PCR analysis. IFN-γ protein levels (A) and IFN-γ mrna expression levels (B) to MTB antigens in the subjects with active tuberculosis (TB) (n = 46) and at low risk for TB (n = 73). Dotted lines represent cut-off values. Each dot represents an individual subject. active TB were considered to be a positive control and QFT-IT negative responders were regarded as negative controls. An expression ratio of. was determined to be the optimal cut-off for measuring IFN-γ mrna expression (P b.). Use of this cut-off for IFN-γ mrna real-time PCR showed 93.7% specificity and a sensitivity of 9.8%. The area under the curve was.956 (95% CI.9.99). The selected mitogen cut-off was.3 (data not shown). Hence, IFN-γ mrna real-time PCR results were defined as follows: subjects with MTB antigen stimulated IFN-γ mrna expression ratios. (MTB antigen sample nil control) were considered positive, irrespective of mitogen response; ratios of b. for MTB antigen stimulated samples and.3 for mitogen-stimulated samples were considered negative; other values (b. for MTB antigen stimulated samples and b.3 for mitogen-stimulated samples) were considered indeterminate. The cut-off point for IFN-γ ELISA was chosen according to the manufacturer's instructions Sensitivity of IFN-γ mrna real-time PCR and IFN-γ ELISA In QFT-IT IFN-γ ELISA, 4 of the 46 TB patients gave positive results, equating to a sensitivity of 89.%. One patient had an indeterminate result due to high background levels of IFN-γ (Table ). For IFN-γ mrna real-time PCR, 39 of the 46 TB patients gave positive results, equating to a sensitivity of 84.8%. Two patients had indeterminate results due to weak responses to the mitogen. Positivity in IFN-γ ELISA was 4.3% higher than that in IFN-γ mrna real-time PCR; however, the difference was not statistically significant (P =.). Age, sex, and BCG vaccination were not significantly associated with test positivity Specificity of IFN-γ mrna real-time PCR and IFN-γ ELISA Of the 73 subjects at low risk for TB, gave positive results in IFN-γ ELISA, 44 gave negative results, and 7 gave indeterminate results (Table ). Of the 7 indeterminate results, 4 were due to high background levels and 3 to low response to the mitogen control. In IFN-γ mrna real-time analysis of the 73 subjects at low risk for TB, 7 subjects were positive and 4 were negative; 5 subjects gave indeterminate results. All 5 indeterminate results were due to weak responses to the mitogen control. The specificities were thus 6.3% for IFN-γ ELISA and 56.% for IFN-γ mrna real-time PCR Concordance/discordance between IFN-γ mrna real-time PCR and IFN-γ ELISA A total of 56 subjects (47.%) were positive by both the IFN-γ ELISA and the IFN-γ mrna real-time PCR; 6 (5.%) were positive by the IFN-γ ELISA and negative by the IFN-γ mrna real-time PCR; 7 (5.9%) were negative in the IFN-γ ELISA and positive in the IFN-γ mrna realtime PCR; and 39 (3.8%) were negative in both the IFN-γ ELISA and the IFN-γ mrna real-time PCR. The overall agreement was 79.8% (kappa =.475) (Table 3). 8 Sensitivity Specificity Fig. 3. Correlation between IFN-γ mrna expression levels and IFN-γ protein levels in subjects with active tuberculosis (TB, n = 46). The IFN-γ mrna expression levels were measured at 4 h, and IFN-γ protein levels were measured at 4 h. The correlation coefficient (R ) between IFN-γ mrna expression levels and IFN-γ protein levels was.74. Fig. 4. Receiver operator characteristic (ROC) curve analysis for IFN-γ mrna expression levels. The cut-off value for IFN-γ mrna expression ratio (R) was determined by using QFT-IT negative healthy controls as uninfected and active TB patients as MTB infected. The area under the curve was.956 (95% CI.9.99).

5 7 S. Kim et al. / Diagnostic Microbiology and Infectious Disease 75 (3) 68 7 Table Positivity of IFN-γ ELISA and IFN-γ mrna real-time PCR in subjects. No. of subjects IFN-γ ELISA Positive (%) 4 (89.) (3.) Negative (%) 4 (8.7) 44 (6.3) Indeterminate (%) (.) 7 (9.6) IFN-γ mrna real-time PCR Positive (%) 39 (84.8) 7 (35.6) Negative (%) 5 (.9) 4 (56.) Indeterminate (%) (4.3) 5 (5.8) 4. Discussion The present study reports that quantitation of IFN-γmRNA expression using real-time PCR is as sensitive and specific as commercial QFT-IT IFN-γ ELISA for the diagnosis of MTB infection. This is the first study to use IFN-γ mrna quantitative real-time PCR as an indicator of IFN-γ levels in an IGRA test. IFN-γ cytokine has been widely used as a diagnostic marker of MTB infection (Detjen et al., 7; Shah et al., ). The production of IFN-γ by immune cells has been measured using the ELISA in QFT-G and QFT-IT tests, and the number of IFN-γ producing cells has been measured using ELISPOT in T-SPOT.TB. Overall, these IGRA assays show a high sensitivity and specificity for detection of MTB infection (Chee et al., 8). In our study, significant expression of IFN-γmRNA was detected from 3 min after stimulation with MTB antigens or mitogen, and expression of IFN-γ mrna peaked at 4 h, even though there was no detectable increase in IFN-γ protein level until 6 h after incubation. This result was agreement with the study by Bibova et al. (), who reported that IFN-γ mrna was measurable. Expression of IFN-γ mrna, measured by quantitative real-time PCR, correlated well with IFN-γ protein level, which was measured by ELISA (R =.74). Interestingly, expression of IFN-γ mrna at 4 h was lower than that at 4 h (P b.). One possible explanation for this is that synthesis of IFN-γ mrna was induced by stimulation with MTB antigens or mitogen, and that its level then fell after initiation of IFN-γ protein synthesis. The sensitivities of IFN-γ mrna real-time PCR and IFN-γ ELISA in subjects with active TB were 84.8% and 89.%, respectively (P =.). This result was agreement with previous studies by Chee et al. (8) and Kabeer et al. (), who reported QFT-IT sensitivities of 83.% and 88.6%, respectively. The specificities of IFN-γ mrna real-time PCR and IFN-γ ELISA were 56.% and 6.3%, respectively, in agreement with other studies. However, the specificity in a study by Lee et al. (6) was 9.6%, higher than that in our study. This may be due to the ages of the enrolled subjects. The mean ages of the subjects at low risk for TB were 5.7 and 39.5 years in the study by Lee et al. (6) and in our study, respectively. Younger objects may have a lower chance to expose to MTB infection. The indeterminate rates in our study were 5.9% and 6.7% for the IFN-γ mrna real-time PCR and IFN-γ ELISA, respectively. Interestingly, 4 of the 7 indeterminate results in IFN-γ ELISA were due to high background levels, but all 5 indeterminate results in IFN-γ mrna real-time PCR were due to Table 3 Comparison of IFN-γ ELISA and IFN-γ mrna real-time PCR in subjects. IFN-γ ELISA IFN-γ mrna real-time PCR Total Positive Negative Indeterminate Positive 56 (47.%) 6 (5.%) (.8%) 63 (5.9%) Negative 7 (5.9%) 39 (3.8%) (.7%) 48 (4.3%) Indeterminate 3 (.5%) (.8%) 4 (3.4%) 8 (6.7%) Total 66 (55.5%) 46 (38.7%) 7 (5.9%) 9 (%) Overall agreement 79.8%; kappa =.475 (95% CI ). weak responses to the mitogen control. This suggests that IFN-γ mrna real-time PCR might not be affected with the preexisting IFN-γ in the plasma as IFN-γ ELISA because IFN-γ mrna real-time PCR detects the IFN-γ mrna expression of immune cells. The indeterminate results in our study were lower than those in other studies, which may be due to our exclusion of frail elderly individuals and severely ill patients. Overall, the sensitivity and specificity of determination of IFN-γmRNA expression were similar to those of IFN-γ ELISA, which implies that quantitation of IFN-γ gene expression might be useful for the diagnosis of MTB infection. Moreover, it is amazing that IFN-γmRNA expression could be measured after only 4 h of stimulation. This will facilitate diagnosis of MTB infection within a day. Quantitative real-time PCR was used for the measurement of IFN-γ mrna expression and is widely used to detect pathogens and other disease markers due to highly sensitive results and its automatic processing. In contrast, ELISA is tedious and takes 4 6 h to perform. The performance of IGRA involving quantitation of IFN-γ mrna may depend on epidemiologic status, ethnicity, and underlying diseases. Therefore, further studies are needed to evaluate the usefulness of IFN-γ mrna real-time PCR in clinical practice. References Bibova I, Linhartova I, Stanek O, Rusnakova V, Kubista M, Suchanek M, et al. Detection of immune cell response to M. tuberculosis-specific antigens by quantitative polymerase chain reaction. Diagn Microbiol Infect Dis ;7: Chee CB, Gan SH, Khinmar KW, Barkham TM, Koh CK, Liang S, et al. Comparison of sensitivities of two commercial gamma interferon release assays for pulmonary tuberculosis. J Clin Microbiol 8;46: Detjen AK, Keil T, Roll S, Hauer B, Mauch H, Wahn U, et al. Interferon-gamma release assays improve the diagnosis of tuberculosis and nontuberculous mycobacterial disease in children in a country with a low incidence of tuberculosis. Clin Infect Dis 7;45:3 8. Harada N, Higuchi K, Yoshiyama T, Kawabe Y, Fujita A, Sasaki Y, et al. Comparison of the sensitivity and specificity of two whole blood interferon-gamma assays for M. tuberculosis infection. J Infect 8;56: Kubista M, Andrade JM, Bengtsson M, Forootan A, Jonak J, Lind K, et al. The real-time polymerase chain reaction. Mol Aspects Med 6;7:95-5. Kumar A, Kumar AM, Gupta D, Kanchar A, Mohammed S, Srinath S, et al. Global guidelines for treatment of tuberculosis among persons living with HIV: unresolved issues. Int J Tuberc Lung Dis ;6: Kvach EJ, Ferguson D, Riska PF, Landry ML. Comparison of BD GeneOhm Cdiff real-time PCR assay with a two-step algorithm and a toxin A/B enzyme-linked immunosorbent assay for diagnosis of toxigenic Clostridium difficile infection. J Clin Microbiol ;48:9 4. Lee JY, Choi HJ, Park IN, Hong SB, Oh YM, Lim CM, et al. Comparison of two commercial interferon-gamma assays for diagnosing Mycobacterium tuberculosis infection. Eur Respir 6;J8:4 3. Lienhardt C, Glaziou P, Uplekar M, Lonnroth K, Getahun H, Raviglione M. Global tuberculosis control: lessons learnt and future prospects. Nat Rev Microbiol ;:47 6. Mannhalter C, Koizar D, Mitterbauer G. Evaluation of RNA isolation methods and reference genes for RT-PCR analyses of rare target RNA. Clin Chem Lab Med ;38:7 7. Mazurek GH, Jereb J, Vernon A, LoBue P, Goldberg S, Castro K. Updated guidelines for using interferon gamma release assays to detect Mycobacterium tuberculosis infection United States,. MMWR Recomm Rep ;59:-5. Mori T, Sakatani M, Yamagishi F, Takashima T, Kawabe Y, Nagao K, et al. Specific detection of tuberculosis infection: an interferon-gamma-based assay using new antigens. Am J Respir Crit Care Med 4;7: Palomino JC. Current developments and future perspectives for TB diagnostics. Future Microbiol ;7:59 7. Pouchot J, Grasland A, Collet C, Coste J, Esdaile JM, Vinceneux P. Reliability of tuberculin skin test measurement. Ann Intern Med 997;6: 4. Shah M, Dipietro D, Greenbaum A, Ketemepi S, Martins-Evora M, Marsiglia V, et al. Programmatic impact of QuantiFERON-TB gold in-tube implementation on latent tuberculosis diagnosis and treatment in a public health clinic. PLoS One ;7: e3655. Kabeer BSA, Raman B, Thomas A, Perumal V, Raja A. Role of QuantiFERON-TB gold, interferon gamma inducible protein- and tuberculin skin test in active tuberculosis diagnosis. PLoS One ;5:e95. Turtle L, Kemp T, Davies GR, Squire SB, Beeching NJ, Beadsworth MB. In routine UK hospital practice T-SPOT.TB is useful in some patients with a modest pre-test probability of active tuberculosis. Eur J Intern Med ;3: Uyttendaele M, Vanwildemeersch K, Debevere J. Evaluation of real-time PCR vs automated ELISA and a conventional culture method using a semi-solid medium for detection of Salmonella. Lett Appl Microbiol 3;37:386 9.

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