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Clinical utility of QuantiFERON-TB GOLD In-Tube and tuberculin skin test in patients with tuberculous pleural effusions
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Diagnostic Microbiology and Infectious Disease 71 (2011) 263 – 266 www.elsevier.com/locate/diagmicrobio
Mycobacteriology
Clinical utility of QuantiFERON-TB GOLD In-Tube and tuberculin skin test in patients with tuberculous pleural effusions Jae Ho Chunga , Chang Hoon Hanb,⁎, Chong Ju Kimb , Sun Min Leeb a
Department of Internal Medicine, Myongji Hospital, Kwandong University College of Medicine, Koyang 412-270, Korea b Department of Internal Medicine, National Health Insurance Corporation Ilsan Hospital, Koyang 410-719, Korea Received 4 March 2011; accepted 27 June 2011
Abstract Interferon gamma release assays are used for diagnosing latent tuberculosis (TB); however, their role in diagnosing tuberculous pleural effusion (TPE) is not defined. The aim of this study was to evaluate the usefulness of the QuantiFERON-TB Gold In-Tube assay (QFT-IT) and compare this assay with the tuberculin skin test (TST) for diagnosing TPE in settings where tuberculosis is endemic and bacillus Calmette–Guérin vaccination is mandatory. The TST and QFT-IT test were conducted prospectively with 101 patients presenting with clinically suspected TPE. Of the 97 evaluable subjects, 54 had TPE. The sensitivity, specificity, positive predictive value, and negative predictive value were, respectively, 76.9%, 61.1%, 74.1%, and 64.7% for QFT-IT; 72.5%, 71.7%, 77.1%, and 66.7% for TST; and 83.7%, 45.7%, 68.3%, and 66.7% for QFT-IT plus TST. Thus, the QFT-IT test may be more useful than the TST for diagnosing TPE. Although the combination of QFT-IT and TST had higher sensitivity, it had poor specificity owing to the high prevalence of latent TB in our setting. © 2011 Elsevier Inc. All rights reserved. Keywords: Tuberculous pleural effusion; Interferon gamma release assay
1. Introduction Tuberculous pleural effusion (TPE) is a common manifestation in extrapulmonary tuberculosis (TB) and is the most common type of pleural effusion in many countries (Valdés et al., 2003). Conventional diagnostic tests for TPE include microscopic examination of the pleural fluid for acid-fast bacilli (AFB); mycobacterial culture of pleural fluid, sputum, or pleural tissue; and histopathologic examination of pleural tissue for granulomatous inflammation. TPE usually contains a low number of mycobacteria, making the diagnostic sensitivity of both direct microscopy and pleural fluid cultures relatively low (Harada et al., 2008). Only 5% of effusions develop smear positive results for AFB, and only 25–37% grow Mycobacterium tuberculosis on cultures (Bueno et al., 1990; Light 1999; Seibert et al., 1991). Other surrogate markers, such as adenosine deaminase (ADA), are now widely used. However, the capability of measuring ADA is not widely available in clinical ⁎ Corresponding author. Tel.: +82-31-900-0263; fax: +82-31-900-0343. E-mail address: [email protected] (C.H. Han). 0732-8893/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2011.06.015
laboratories and ADA assays can be nonspecific even in high-burden settings (Trajman et al., 2008). Biopsy of pleural tissue for combined histologic examination and mycobacterial culturing of pleural fluid and tissue is the most sensitive of the currently available diagnostic methods, but it may still be falsely negative in 15–20% of cases (Light 1999; Palomino et al., 2007). In addition, pleural biopsy is invasive, and because of its technical difficulties, the yield as well as the complication rates of pleural biopsy depends on the skill of the operator. TB proteins encoded by the region of difference-1 gene of M. tuberculosis are used in commercially available interferon-gamma release assay (IGRA) blood tests. Three commercial kits are available: T-SPOT.TB (Oxford Immunotec, Oxford, UK), QuantiFERON-TB Gold (QFT-G, Cellestis, Victoria, Australia), and QuantiFERON-TB Gold In-Tube (QFT-IT, Cellestis, Victoria, Australia). The available data suggest that IGRAs are less influenced by prior bacillus Calmette–Guérin (BCG) vaccination and environmental mycobacteria. Although IGRAs are used for latent TB diagnosis, studies to define their role in diagnosing TPE are limited, especially in endemic settings. In South Korea, the
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J.H. Chung et al. / Diagnostic Microbiology and Infectious Disease 71 (2011) 263–266
incidence of active TB is intermediate (70–90/100,000 per year) and BCG vaccination is mandatory. In this study, we evaluated the utility of the QFT-IT and the tuberculin skin test (TST) for diagnosing TPE in clinical practice in South Korea.
as AFB-negative and culture-negative pleural fluid, adenosine deaminase activity ≥40 U/L, and/or strong clinical evidence consistent with active TB, followed by a clinical decision to treat with TB chemotherapy. Patients with alternative diagnoses made on histology or pleural fluid aspiration and with a 6-month follow-up with no features to suggest TPE were classified as non-TPE.
2. Materials and methods 2.1. Study population and diagnostic evaluation
2.2. Tuberculin skin test
Over a 12-month period, 101 patients with suspected TPE were prospectively enrolled from the Myongji Hospital and the National Health Insurance Corporation Ilsan Hospital in Koyang, South Korea, after obtaining informed consent. For this study, only diagnostic thoracentesis was considered, and for cases in which multiple procedures were performed, only the initial data were used. Of the 101 patients, 4 were excluded: 2 patients had unverifiable patient details, 1 sample coagulated on arrival, and 1 sample was incorrectly harvested (ascites instead of pleural fluid). Thus, 97 patients had evaluable results. Patients who were under 18 years of age, pregnant, or refused consent were not recruited. The study protocol was approved by the respective institutional ethics committees (Myongji Hospital IRB number: 08-034; Ilsan Hospital IRB number: SUYON 2008-43). Patient history was reviewed, followed by a detailed physical examination and routine hematologic investigations. Evaluations included HIV testing and sputum examination by fluorescence microscopy for AFB and mycobacterial culturing. In addition, aspirated pleural fluids were evaluated using biochemical assays for protein and glucose, cytologic observation for malignant cells, and microbiological testing for bacterial pathogens, including M. tuberculosis, using fluorescence microscopy, Gram staining, and culturing. When the TPE or malignant pleural effusion was suspected, multiple (N4) closed pleural biopsies were performed using an Abraham's needle under local anesthesia. Malignant pleural effusion was diagnosed when the pleural fluid cytology or pleural biopsy findings were positive, or when a patient with a known malignancy had a persistent pleural effusion and alternative diagnoses had been excluded. Parapneumonic pleural effusion was defined as the clinical and radiologic diagnosis of pneumonia. Other diagnoses were established based on clinical and laboratory data. Patients with recent (b1 year) tuberculosis therapy or corticosteroid, immunosuppressive, or antiretroviral therapy; effusions of undetermined origin or with more than 1 possible cause; empyema; or hemothorax were excluded. The patients were categorized as confirmed TPE, probable TPE, or non-TPE. Confirmed TPE was defined as positive M. tuberculosis culture (sputum, pleural fluid, or tissue) and/or histology keeping with TB (caseous necrosis or AFB with or without granuloma formation) and a clinicoradiologic picture consistent with TPE, including a clinical response to anti-TB treatment. Probable TPE was defined
The TST involved an intradermal injection of 0.1 mL (2 tuberculin units) of the purified protein derivative RT23 (Statens Serum Institute, Copenhagen, Denmark) into the anterior surface of the forearm, using a standard tuberculin syringe. The reaction was evaluated 48–72 h after the injection, and the transverse diameter (in millimeters) of the area of induration was recorded. A positive TST result was defined as an induration with a transverse diameter ≥10 mm. 2.3. QuantiFERON-TB Gold In-Tube assay The QFT-IT test was performed following the manufacturer's instructions (Cellestis). First, the blood was directly collected in three 1-mL heparin-containing tubes. One tube contained only heparin as negative control, and the second tube contained overlapping peptides representing the entire sequences of CFP-10 and ESAT-6, and another peptide representing a portion of TB7.7. The third tube contained phytohemagglutinin as positive control. Three tubes with whole blood were incubated for 16 to 24 h at 37 °C. The test was considered positive when the difference between the TB antigen value and the nil value was ≥0.35 IU/mL; the difference had to be ≥25% of the nil value to be valid. The TST was performed after the QFT-IT, because the QFT-IT results are influenced by the TST. 2.4. Statistical analysis Statistical analyses were performed using SPSS version 12.0 (SPSS, Chicago, IL, USA) and MedCalc version 9.5.2.0 software (MedCalc Software, Mariakerke, Belgium). The McNemar chi-squared test was used to compare the sensitivity and specificity between the diagnostic tests. Values of P b 0.05 were considered to indicate statistical significance.
3. Results The data of 97 patients were analyzed in this study. Their demographics and clinical characteristics are shown in Table 1. Forty patients had confirmed TPE, 14 were classified as probable TPE, and 43 were classified as non-TPE (28 with parapneumonic effusions and 15 with malignant pleural effusions). All patients were HIV negative. Thirteen (13%) patients had a history of pulmonary TB. Nine (9%) of the 97 patients had an indeterminate QFT-IT test result.
J.H. Chung et al. / Diagnostic Microbiology and Infectious Disease 71 (2011) 263–266 Table 1 Demographic and clinical characteristics of the 97 patients with suspected TPE
Age, median (range) Male/female Presence of TB scar on chest radiographs in patients without previous history of TB treatment History of previous TB treatment
TPE, n = 54
Non-TPE, n = 43
43 (19–92) 37/17 4 (7%)
69 (20–94) 33/10 5 (11%)
6 (11%)
7 (16%)
Data are presented as numbers (percentages) unless otherwise indicated. TPE = tuberculous pleural effusions; TB = tuberculosis.
With the QFT-IT test, 40 (74%) of the 54 TPE and probable TPE cases were positive and 12 (22%) were negative. A comparison of the TST and QFT-IT test results for the TPE and non-TPE groups is shown in Table 2 and Table 3. Excluding the indeterminate results, the QFT-IT yielded a diagnostic sensitivity of 76.9% (95% CI: 62.8– 87.0%), which was significantly higher (P = 0.003) than the sensitivity of the TST (72.5%; 95% CI: 57.0–83.7%). We further assessed whether combining the 2 tests could improve the sensitivity. Among 90 subjects tested with the combination of both methods, 33 (36.7%) were negative and 6 (6.7%) were indeterminate. Excluding the indeterminate results, the sensitivity of the QFT-IT plus TST was 83.7% (95% CI: 69.8–92.2%) with a specificity of 45.7% (95% CI: 29.2–63.1%).
4. Discussion Accumulating evidence supports the superior sensitivity and specificity of IGRAs compared with the TST (Mantoux) for the detection of M. tuberculosis infection (Diel et al., 2010; Pai et al., 2008). The QuantiFERON-TB Gold (QFTG) assay detects latent TB infection with high specificity and has been approved as a diagnostic test for M. tuberculosis infection. Nevertheless, it has limitations. A major logistic limitation is that blood must be stimulated with M. tuberculosis–specific antigens within 12 h of collection, which restricts the use of the QFT-G assay to laboratories or institutions that are close to a blood collection facility. To
265
overcome this limitation, the QFT-IT assay has been developed. Blood is collected directly into a collection tube containing the M. tuberculosis–specific antigens ESAT-6 and CFP-10, and a peptide from the M. tuberculosis–specific antigen TB7.7 (Rv2654). With the immediate stimulation of lymphocytes with antigen and the addition of 3 antigens in one tube, the QFT-IT assay exhibits enhanced sensitivity and equivalently high specificity for the detection for M. tuberculosis, compared with the QFT-G assay (Arend et al., 2007; Mahomed et al., 2006). The diagnostic value of IGRAs in TPE has been examined in several studies (Ariga et al., 2007; Baba et al., 2008; Chegou et al., 2008; Losi et al., 2007; Wilkinson et al., 2005). Losi et al. enrolled 41 European patients, 10 of whom had been classified with definite pleural TB (by culture or polymerase chain reaction), 10 with presumptive TB (positive treatment response), and 21 with nontuberculous effusions. With the use of the T-SPOT.TB assay in these patients, the overall sensitivity and specificity for blood were 90% and 67%, respectively. In Tokyo, Ariga et al. prospectively recruited 28 HIV-negative patients with TPE, as demonstrated by a positive fluid or tissue culture. Stimulated interferon release from both blood and effusion mononuclear cell samples was quantified with the QFT-G assay, yielding a sensitivity of 78% and specificity of 70% at the optimal cut-off for the study population. Baba et al. categorized South African patients presenting with pleural effusion and TB symptoms as having definite (n = 12), presumptive (n = 16), or nontuberculous (n = 6) pleural effusions. In these patients, the QFT-IT assay achieved an overall sensitivity of 71% and specificity of 100%. The TSPOT.TB test has been used in 2 studies in areas with low TB prevalence (Losi et al., 2007; Wilkinson et al., 2005), whereas the QFT-IT test has been used in 3 studies in areas with high prevalence (Ariga et al., 2007; Baba et al., 2008; Chegou et al., 2008). HIV patients were included in 2 of the QFT-IT studies (Baba et al., 2008; Chegou et al., 2008), and our study used the QFT-IT in non-HIV patients. False-positive test results are common when IGRAs are applied to blood and pleural fluid because of the inability of the test to distinguish between active and latent TB. This significantly limits the usefulness of IGRAs in the diagnosis of TPE, especially in endemic regions. Studies (Losi et al.,
Table 2 Sensitivity and specificity of QFT-IT and TST in the diagnosis of tuberculous pleural effusions TPE
Non-TPE
QFT-IT (n = 54)
Positive Negative Indeterminate % of Sensitivitya % of Specificitya
Total
Confirmed
Probable
40 12 2 76.9
31 8 1
9 4 1
TST (n = 51)
QFT-IT (n = 43)
TST (n = 39)
37 14 0 72.5
14 22 7
11 28 0
61.1
71.7
Data are presented as numbers unless otherwise indicated. QFT-IT = QuantiFERON TB Gold In-Tube assay; TST = tuberculin skin test. a Calculated after excluding indeterminate results.
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Table 3 Diagnostic validity of the TST, QFT-IT, and TST + QFT-IT
TST QFT-IT TST+QFT-IT
Sensitivitya
Specificitya
PPV
NPV
LR+
LR−
72.5 (58–83.7) 76.9 (62.8–87.0) 83.7 (69.8–92.2)
71.7 (54.9–84.5) 61.1 (43.5–76.4) 45.7 (29.2–63.1)
77.1 (62.3–87.5) 74.1 (60.1–84.6) 68.3 (54.9–79.4)
66.7 (50.4–79.9) 64.7 (46.5–79.7) 66.7 (44.7–83.6)
1.85 (1.19–2.85) 1.98 (1.28–3.06) 1.54 (1.11–2.14)
0.47 (0.29–0.74) 0.48 (0.22–0.64) 0.36 (0.18–0.72)
Data are presented as percentage (95% confidence interval). PPV = Positive predictive value; NPV = negative predictive value; LR+ = likelihood ratio of a positive test result; LR− = likelihood ratio of a negative test result; TST = tuberculin skin test; QFT-IT = QuantiFERON TB Gold In-Tube assay. a Calculated after excluding indeterminate results.
2007; Wilkinson et al., 2005) conducted in low TB– prevalence settings with largely immunocompetent patient populations have demonstrated a negative predictive value for active pleural TB using IGRAs applied to blood; the pleural fluid appeared to produce fewer false-negative results, probably due to the compartmentalization of sensitized T cells in the pleural space. In areas where TB and HIV are common, the sensitivity of IGRAs has been shown to be poor regardless of the sample type. The diagnostic value of the test is significantly diminished by high proportions of indeterminate results in HIV-positive individuals. The existing studies, which had been performed in the area with a high TB prevalence rate, had all been conducted in the HIV-endemic area. But this study has been performed on HIV-negative patients and has excluded immunocompromised host. Furthermore, it has an advantage that the sample size in this study is relatively larger than that in other studies. It seems to be meaningful as a study performed in unannounced Asia. Commercially available IGRAs were designed for use with peripheral blood and have not been validated for use with pleural fluid. Larger prospective studies are required to fine tune technical aspects such as the optimal volume of pleural fluid, optimal antigen concentration, and optimal cutoff levels required for performance. Our results show that the QFT-IT test may be more useful than the TST for diagnosing TPE in HIV-seronegative individuals. The combination of the TST and QFT-IT test showed higher sensitivity (83.7%), suggesting that the 2 tests in combination may be useful in diagnosing TPE as a supplement marker along with existing diagnostic tools. However, their role was limited by poor specificity in our setting because of the high prevalence of latent TB infection. References Arend SM, Thijsen SF, Leyten EM, Bouwman JJ, Franken WP, Koster BF, Cobelens FG, van Houte AJ, Bossink AW (2007) Comparison of two interferon-gamma assays and tuberculin skin test for tracing tuberculosis contacts. Am J Respir Crit Care Med 175:618–627. Ariga H, Kawabe Y, Nagai H, Kurashima A, Masuda K, Matsui H, Tamura A, Nagayama N, Akagawa S, Machida K, Hebisawa A, Nakajima Y, Yotsumoto H, Mori T (2007) Diagnosis of active tuberculous serositis
by antigen-specific interferon-gamma response of cavity fluid cells. Clin Infect Dis 45:1559–1567. Baba K, Sornes S, Hoosen AA, Lekebe JM, Mpe MJ, Langeland N, DyrholRiise AM (2008) Evaluation of immune responses in HIV infected patients with pleural tuberculosis by the QuantiFERON TB-Gold interferon-gamma assay. BMC Infect Dis 8:35. Bueno EC, Clemente GM, Castro CB, Martin ML, Ramos RS, Panizo GA, Glez-Rio MJ (1990) Cytologic and bacteriologic analysis of fluid and pleural biopsy specimens with Cope's needle. Study of 414 patients. Arch Intern Med 150:1190–1194. Chegou NN, Walzl G, Bolliger CT, Diacon AH, van den Heuvel MM (2008) Evaluation of adapted whole-blood interferon-gamma release assays for the diagnosis of pleural tuberculosis. Respiration 76:131–138. Diel R, Loddenkemper R, Nienhaus A (2010) Evidence-based comparison of commercial interferon-gamma release assays for detecting active TB: a meta-analysis. Chest 137:952–968. Harada N, Higuchi K, Yoshiyama T, Kawabe Y, Fujita A, Sasaki Y, Horiba M, Mitarai S, Yonemaru M, Ogata H, Ariga H, Kurashima A, Wada A, Takamori M, Yamagishi F, Suzuki K, Mori T, Ishikawa N (2008) Comparison of the sensitivity and specificity of two whole blood interferon-gamma assays for M. tuberculosis infection. J Infect 56:348–353. Light RW (1999) Useful tests on the pleural fluid in the management of patients with pleural effusions. Curr Opin Pulm Med 5:245–249. Losi M, Bossink A, Codecasa L, Jafari C, Ernst M, Thijsen S, Cirillo D, Ferrarese M, Greinert U, Fabbri LM, Richeldi L, Lange C (2007) Use of a T-cell interferon-gamma release assay for the diagnosis of tuberculous pleurisy. Eur Respir J 30:1173–1179. Mahomed H, Hughes EJ, Hawkridge T, Minnies D, Simon E, Little F, Hanekom WA, Geiter L, Hussey GD (2006) Comparison of Mantoux skin test with three generations of a whole blood IFN-gamma assay for tuberculosis infection. Int J Tuberc Lung Dis 10(3):310–316. Pai M, Zwerling A, Menzies D (2008) Systematic review: T-cell-based assays for the diagnosis of latent tuberculosis infection: an update. Ann Intern Med 149:177–184. Palomino JC, Leão SC, Ritacco V, Eds (2007) Tuberculosis 2007 From basic science to patient care. 1st ed. pp. 498–500. www.tuberculosistextbook. com/tuberculosis2007.pdf. Accessed: January 22, 2011. Seibert AF, Haynes J Jr, Middleton R, Bass JB Jr (1991) Tuberculous pleural effusion. Twenty-year experience. Chest 99:883–886. Trajman A, Pai M, Dheda K, van Zyl Smit R, Zwerling AA, Joshi R, Kalantri S, Daley P, Menzies D (2008) Novel tests for diagnosing tuberculous pleural effusion: what works and what does not? Eur Respir J 31:1098–1106. Valdés L, Pose A, San José E, Martinez Vázquez JM (2003) Tuberculous pleural effusions. Eur J Intern Med 14:77–88. Wilkinson KA, Wilkinson RJ, Pathan A, Ewer K, Prakash M, Klenerman P, Maskell N, Davies R, Pasvol G, Lalvani A (2005) Ex vivo characterization of early secretory antigenic target 6-specific T cells at sites of active disease in pleural tuberculosis. Clin Infect Dis 40:184–187.
Diagnostic Microbiology and Infectious Disease 71 (2011) 263 – 266 www.elsevier.com/locate/diagmicrobio
Mycobacteriology
Clinical utility of QuantiFERON-TB GOLD In-Tube and tuberculin skin test in patients with tuberculous pleural effusions Jae Ho Chunga , Chang Hoon Hanb,⁎, Chong Ju Kimb , Sun Min Leeb a
Department of Internal Medicine, Myongji Hospital, Kwandong University College of Medicine, Koyang 412-270, Korea b Department of Internal Medicine, National Health Insurance Corporation Ilsan Hospital, Koyang 410-719, Korea Received 4 March 2011; accepted 27 June 2011
Abstract Interferon gamma release assays are used for diagnosing latent tuberculosis (TB); however, their role in diagnosing tuberculous pleural effusion (TPE) is not defined. The aim of this study was to evaluate the usefulness of the QuantiFERON-TB Gold In-Tube assay (QFT-IT) and compare this assay with the tuberculin skin test (TST) for diagnosing TPE in settings where tuberculosis is endemic and bacillus Calmette–Guérin vaccination is mandatory. The TST and QFT-IT test were conducted prospectively with 101 patients presenting with clinically suspected TPE. Of the 97 evaluable subjects, 54 had TPE. The sensitivity, specificity, positive predictive value, and negative predictive value were, respectively, 76.9%, 61.1%, 74.1%, and 64.7% for QFT-IT; 72.5%, 71.7%, 77.1%, and 66.7% for TST; and 83.7%, 45.7%, 68.3%, and 66.7% for QFT-IT plus TST. Thus, the QFT-IT test may be more useful than the TST for diagnosing TPE. Although the combination of QFT-IT and TST had higher sensitivity, it had poor specificity owing to the high prevalence of latent TB in our setting. © 2011 Elsevier Inc. All rights reserved. Keywords: Tuberculous pleural effusion; Interferon gamma release assay
1. Introduction Tuberculous pleural effusion (TPE) is a common manifestation in extrapulmonary tuberculosis (TB) and is the most common type of pleural effusion in many countries (Valdés et al., 2003). Conventional diagnostic tests for TPE include microscopic examination of the pleural fluid for acid-fast bacilli (AFB); mycobacterial culture of pleural fluid, sputum, or pleural tissue; and histopathologic examination of pleural tissue for granulomatous inflammation. TPE usually contains a low number of mycobacteria, making the diagnostic sensitivity of both direct microscopy and pleural fluid cultures relatively low (Harada et al., 2008). Only 5% of effusions develop smear positive results for AFB, and only 25–37% grow Mycobacterium tuberculosis on cultures (Bueno et al., 1990; Light 1999; Seibert et al., 1991). Other surrogate markers, such as adenosine deaminase (ADA), are now widely used. However, the capability of measuring ADA is not widely available in clinical ⁎ Corresponding author. Tel.: +82-31-900-0263; fax: +82-31-900-0343. E-mail address: [email protected] (C.H. Han). 0732-8893/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2011.06.015
laboratories and ADA assays can be nonspecific even in high-burden settings (Trajman et al., 2008). Biopsy of pleural tissue for combined histologic examination and mycobacterial culturing of pleural fluid and tissue is the most sensitive of the currently available diagnostic methods, but it may still be falsely negative in 15–20% of cases (Light 1999; Palomino et al., 2007). In addition, pleural biopsy is invasive, and because of its technical difficulties, the yield as well as the complication rates of pleural biopsy depends on the skill of the operator. TB proteins encoded by the region of difference-1 gene of M. tuberculosis are used in commercially available interferon-gamma release assay (IGRA) blood tests. Three commercial kits are available: T-SPOT.TB (Oxford Immunotec, Oxford, UK), QuantiFERON-TB Gold (QFT-G, Cellestis, Victoria, Australia), and QuantiFERON-TB Gold In-Tube (QFT-IT, Cellestis, Victoria, Australia). The available data suggest that IGRAs are less influenced by prior bacillus Calmette–Guérin (BCG) vaccination and environmental mycobacteria. Although IGRAs are used for latent TB diagnosis, studies to define their role in diagnosing TPE are limited, especially in endemic settings. In South Korea, the
264
J.H. Chung et al. / Diagnostic Microbiology and Infectious Disease 71 (2011) 263–266
incidence of active TB is intermediate (70–90/100,000 per year) and BCG vaccination is mandatory. In this study, we evaluated the utility of the QFT-IT and the tuberculin skin test (TST) for diagnosing TPE in clinical practice in South Korea.
as AFB-negative and culture-negative pleural fluid, adenosine deaminase activity ≥40 U/L, and/or strong clinical evidence consistent with active TB, followed by a clinical decision to treat with TB chemotherapy. Patients with alternative diagnoses made on histology or pleural fluid aspiration and with a 6-month follow-up with no features to suggest TPE were classified as non-TPE.
2. Materials and methods 2.1. Study population and diagnostic evaluation
2.2. Tuberculin skin test
Over a 12-month period, 101 patients with suspected TPE were prospectively enrolled from the Myongji Hospital and the National Health Insurance Corporation Ilsan Hospital in Koyang, South Korea, after obtaining informed consent. For this study, only diagnostic thoracentesis was considered, and for cases in which multiple procedures were performed, only the initial data were used. Of the 101 patients, 4 were excluded: 2 patients had unverifiable patient details, 1 sample coagulated on arrival, and 1 sample was incorrectly harvested (ascites instead of pleural fluid). Thus, 97 patients had evaluable results. Patients who were under 18 years of age, pregnant, or refused consent were not recruited. The study protocol was approved by the respective institutional ethics committees (Myongji Hospital IRB number: 08-034; Ilsan Hospital IRB number: SUYON 2008-43). Patient history was reviewed, followed by a detailed physical examination and routine hematologic investigations. Evaluations included HIV testing and sputum examination by fluorescence microscopy for AFB and mycobacterial culturing. In addition, aspirated pleural fluids were evaluated using biochemical assays for protein and glucose, cytologic observation for malignant cells, and microbiological testing for bacterial pathogens, including M. tuberculosis, using fluorescence microscopy, Gram staining, and culturing. When the TPE or malignant pleural effusion was suspected, multiple (N4) closed pleural biopsies were performed using an Abraham's needle under local anesthesia. Malignant pleural effusion was diagnosed when the pleural fluid cytology or pleural biopsy findings were positive, or when a patient with a known malignancy had a persistent pleural effusion and alternative diagnoses had been excluded. Parapneumonic pleural effusion was defined as the clinical and radiologic diagnosis of pneumonia. Other diagnoses were established based on clinical and laboratory data. Patients with recent (b1 year) tuberculosis therapy or corticosteroid, immunosuppressive, or antiretroviral therapy; effusions of undetermined origin or with more than 1 possible cause; empyema; or hemothorax were excluded. The patients were categorized as confirmed TPE, probable TPE, or non-TPE. Confirmed TPE was defined as positive M. tuberculosis culture (sputum, pleural fluid, or tissue) and/or histology keeping with TB (caseous necrosis or AFB with or without granuloma formation) and a clinicoradiologic picture consistent with TPE, including a clinical response to anti-TB treatment. Probable TPE was defined
The TST involved an intradermal injection of 0.1 mL (2 tuberculin units) of the purified protein derivative RT23 (Statens Serum Institute, Copenhagen, Denmark) into the anterior surface of the forearm, using a standard tuberculin syringe. The reaction was evaluated 48–72 h after the injection, and the transverse diameter (in millimeters) of the area of induration was recorded. A positive TST result was defined as an induration with a transverse diameter ≥10 mm. 2.3. QuantiFERON-TB Gold In-Tube assay The QFT-IT test was performed following the manufacturer's instructions (Cellestis). First, the blood was directly collected in three 1-mL heparin-containing tubes. One tube contained only heparin as negative control, and the second tube contained overlapping peptides representing the entire sequences of CFP-10 and ESAT-6, and another peptide representing a portion of TB7.7. The third tube contained phytohemagglutinin as positive control. Three tubes with whole blood were incubated for 16 to 24 h at 37 °C. The test was considered positive when the difference between the TB antigen value and the nil value was ≥0.35 IU/mL; the difference had to be ≥25% of the nil value to be valid. The TST was performed after the QFT-IT, because the QFT-IT results are influenced by the TST. 2.4. Statistical analysis Statistical analyses were performed using SPSS version 12.0 (SPSS, Chicago, IL, USA) and MedCalc version 9.5.2.0 software (MedCalc Software, Mariakerke, Belgium). The McNemar chi-squared test was used to compare the sensitivity and specificity between the diagnostic tests. Values of P b 0.05 were considered to indicate statistical significance.
3. Results The data of 97 patients were analyzed in this study. Their demographics and clinical characteristics are shown in Table 1. Forty patients had confirmed TPE, 14 were classified as probable TPE, and 43 were classified as non-TPE (28 with parapneumonic effusions and 15 with malignant pleural effusions). All patients were HIV negative. Thirteen (13%) patients had a history of pulmonary TB. Nine (9%) of the 97 patients had an indeterminate QFT-IT test result.
J.H. Chung et al. / Diagnostic Microbiology and Infectious Disease 71 (2011) 263–266 Table 1 Demographic and clinical characteristics of the 97 patients with suspected TPE
Age, median (range) Male/female Presence of TB scar on chest radiographs in patients without previous history of TB treatment History of previous TB treatment
TPE, n = 54
Non-TPE, n = 43
43 (19–92) 37/17 4 (7%)
69 (20–94) 33/10 5 (11%)
6 (11%)
7 (16%)
Data are presented as numbers (percentages) unless otherwise indicated. TPE = tuberculous pleural effusions; TB = tuberculosis.
With the QFT-IT test, 40 (74%) of the 54 TPE and probable TPE cases were positive and 12 (22%) were negative. A comparison of the TST and QFT-IT test results for the TPE and non-TPE groups is shown in Table 2 and Table 3. Excluding the indeterminate results, the QFT-IT yielded a diagnostic sensitivity of 76.9% (95% CI: 62.8– 87.0%), which was significantly higher (P = 0.003) than the sensitivity of the TST (72.5%; 95% CI: 57.0–83.7%). We further assessed whether combining the 2 tests could improve the sensitivity. Among 90 subjects tested with the combination of both methods, 33 (36.7%) were negative and 6 (6.7%) were indeterminate. Excluding the indeterminate results, the sensitivity of the QFT-IT plus TST was 83.7% (95% CI: 69.8–92.2%) with a specificity of 45.7% (95% CI: 29.2–63.1%).
4. Discussion Accumulating evidence supports the superior sensitivity and specificity of IGRAs compared with the TST (Mantoux) for the detection of M. tuberculosis infection (Diel et al., 2010; Pai et al., 2008). The QuantiFERON-TB Gold (QFTG) assay detects latent TB infection with high specificity and has been approved as a diagnostic test for M. tuberculosis infection. Nevertheless, it has limitations. A major logistic limitation is that blood must be stimulated with M. tuberculosis–specific antigens within 12 h of collection, which restricts the use of the QFT-G assay to laboratories or institutions that are close to a blood collection facility. To
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overcome this limitation, the QFT-IT assay has been developed. Blood is collected directly into a collection tube containing the M. tuberculosis–specific antigens ESAT-6 and CFP-10, and a peptide from the M. tuberculosis–specific antigen TB7.7 (Rv2654). With the immediate stimulation of lymphocytes with antigen and the addition of 3 antigens in one tube, the QFT-IT assay exhibits enhanced sensitivity and equivalently high specificity for the detection for M. tuberculosis, compared with the QFT-G assay (Arend et al., 2007; Mahomed et al., 2006). The diagnostic value of IGRAs in TPE has been examined in several studies (Ariga et al., 2007; Baba et al., 2008; Chegou et al., 2008; Losi et al., 2007; Wilkinson et al., 2005). Losi et al. enrolled 41 European patients, 10 of whom had been classified with definite pleural TB (by culture or polymerase chain reaction), 10 with presumptive TB (positive treatment response), and 21 with nontuberculous effusions. With the use of the T-SPOT.TB assay in these patients, the overall sensitivity and specificity for blood were 90% and 67%, respectively. In Tokyo, Ariga et al. prospectively recruited 28 HIV-negative patients with TPE, as demonstrated by a positive fluid or tissue culture. Stimulated interferon release from both blood and effusion mononuclear cell samples was quantified with the QFT-G assay, yielding a sensitivity of 78% and specificity of 70% at the optimal cut-off for the study population. Baba et al. categorized South African patients presenting with pleural effusion and TB symptoms as having definite (n = 12), presumptive (n = 16), or nontuberculous (n = 6) pleural effusions. In these patients, the QFT-IT assay achieved an overall sensitivity of 71% and specificity of 100%. The TSPOT.TB test has been used in 2 studies in areas with low TB prevalence (Losi et al., 2007; Wilkinson et al., 2005), whereas the QFT-IT test has been used in 3 studies in areas with high prevalence (Ariga et al., 2007; Baba et al., 2008; Chegou et al., 2008). HIV patients were included in 2 of the QFT-IT studies (Baba et al., 2008; Chegou et al., 2008), and our study used the QFT-IT in non-HIV patients. False-positive test results are common when IGRAs are applied to blood and pleural fluid because of the inability of the test to distinguish between active and latent TB. This significantly limits the usefulness of IGRAs in the diagnosis of TPE, especially in endemic regions. Studies (Losi et al.,
Table 2 Sensitivity and specificity of QFT-IT and TST in the diagnosis of tuberculous pleural effusions TPE
Non-TPE
QFT-IT (n = 54)
Positive Negative Indeterminate % of Sensitivitya % of Specificitya
Total
Confirmed
Probable
40 12 2 76.9
31 8 1
9 4 1
TST (n = 51)
QFT-IT (n = 43)
TST (n = 39)
37 14 0 72.5
14 22 7
11 28 0
61.1
71.7
Data are presented as numbers unless otherwise indicated. QFT-IT = QuantiFERON TB Gold In-Tube assay; TST = tuberculin skin test. a Calculated after excluding indeterminate results.
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Table 3 Diagnostic validity of the TST, QFT-IT, and TST + QFT-IT
TST QFT-IT TST+QFT-IT
Sensitivitya
Specificitya
PPV
NPV
LR+
LR−
72.5 (58–83.7) 76.9 (62.8–87.0) 83.7 (69.8–92.2)
71.7 (54.9–84.5) 61.1 (43.5–76.4) 45.7 (29.2–63.1)
77.1 (62.3–87.5) 74.1 (60.1–84.6) 68.3 (54.9–79.4)
66.7 (50.4–79.9) 64.7 (46.5–79.7) 66.7 (44.7–83.6)
1.85 (1.19–2.85) 1.98 (1.28–3.06) 1.54 (1.11–2.14)
0.47 (0.29–0.74) 0.48 (0.22–0.64) 0.36 (0.18–0.72)
Data are presented as percentage (95% confidence interval). PPV = Positive predictive value; NPV = negative predictive value; LR+ = likelihood ratio of a positive test result; LR− = likelihood ratio of a negative test result; TST = tuberculin skin test; QFT-IT = QuantiFERON TB Gold In-Tube assay. a Calculated after excluding indeterminate results.
2007; Wilkinson et al., 2005) conducted in low TB– prevalence settings with largely immunocompetent patient populations have demonstrated a negative predictive value for active pleural TB using IGRAs applied to blood; the pleural fluid appeared to produce fewer false-negative results, probably due to the compartmentalization of sensitized T cells in the pleural space. In areas where TB and HIV are common, the sensitivity of IGRAs has been shown to be poor regardless of the sample type. The diagnostic value of the test is significantly diminished by high proportions of indeterminate results in HIV-positive individuals. The existing studies, which had been performed in the area with a high TB prevalence rate, had all been conducted in the HIV-endemic area. But this study has been performed on HIV-negative patients and has excluded immunocompromised host. Furthermore, it has an advantage that the sample size in this study is relatively larger than that in other studies. It seems to be meaningful as a study performed in unannounced Asia. Commercially available IGRAs were designed for use with peripheral blood and have not been validated for use with pleural fluid. Larger prospective studies are required to fine tune technical aspects such as the optimal volume of pleural fluid, optimal antigen concentration, and optimal cutoff levels required for performance. Our results show that the QFT-IT test may be more useful than the TST for diagnosing TPE in HIV-seronegative individuals. The combination of the TST and QFT-IT test showed higher sensitivity (83.7%), suggesting that the 2 tests in combination may be useful in diagnosing TPE as a supplement marker along with existing diagnostic tools. However, their role was limited by poor specificity in our setting because of the high prevalence of latent TB infection. References Arend SM, Thijsen SF, Leyten EM, Bouwman JJ, Franken WP, Koster BF, Cobelens FG, van Houte AJ, Bossink AW (2007) Comparison of two interferon-gamma assays and tuberculin skin test for tracing tuberculosis contacts. Am J Respir Crit Care Med 175:618–627. Ariga H, Kawabe Y, Nagai H, Kurashima A, Masuda K, Matsui H, Tamura A, Nagayama N, Akagawa S, Machida K, Hebisawa A, Nakajima Y, Yotsumoto H, Mori T (2007) Diagnosis of active tuberculous serositis
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