Indian Journal of Pathology and Microbiology

ORIGINAL ARTICLE
Year
: 2013  |  Volume : 56  |  Issue : 2  |  Page : 125--128

Evaluation of a new tuberculosis-related interferon gamma release assay for tuberculosis infection diagnosis in Huzhou, eastern China


Fuchu Qian1, Weihong Wang2, Zhihong Qiu3, Yiqun Shen2, Jiangfang He4, Dongli Li1, Jing Zhong1, Licheng Dai1,  
1 Huzhou Key Laboratory of Molecular Medicine, Huzhou Central Hospital, Huzhou, China
2 Department of Infectious diseases, Huzhou Central Hospital, Huzhou, China
3 Center for Tuberculosis Disease Control and Prevention, Huzhou Center for Disease Control and Prevention, Huzhou, China
4 Department of Laboratory Medicine, Huzhou Central Hospital, Huzhou, China

Correspondence Address:
Licheng Dai
Huzhou Key Laboratory of Molecular Medicine, Huzhou Central Hospital, 198 HongQi Road, Huzhou, Zhejiang Province 313 000
China

Abstract

Objective: To compare the performance of a new tuberculosis-related interferon gamma release assay (TB-IGRA) with that of QuantiFERON-TB Gold In-Tube (QFT-GIT) for TB infection diagnosis in China. Materials and Methods: A total of 458 active TB patients and 378 healthy individuals were enrolled. Among the 458 active TB patients, 395 had pulmonary TB and 63 had extra-pulmonary TB. The blood samples were collected from the active TB patients and health controls; then TB-IGRA and QFT-GIT were used to detect interferon gamma (IFN-γ) levels. Results: The sensitivity, specificity, positive predictive value, and negative predictive value in TB infection diagnosis for active TB by the TB-IGRA were 83.4%, 94.2%, 94.5%, and 82.4%, respectively. For QFT-GIT, the sensitivity, specificity, positive predictive value, and negative predictive value in TB infection diagnosis for active TB were 81.4%, 97.1%, 97.1%, and 81.2%, respectively. Conclusions: TB-IGRA had a high sensitivity and specificity for TB infection; it could be comparable with the QFT-GIT assay. It might be a powerful assisting tool for TB infection diagnosis in the Chinese clinical setting.



How to cite this article:
Qian F, Wang W, Qiu Z, Shen Y, He J, Li D, Zhong J, Dai L. Evaluation of a new tuberculosis-related interferon gamma release assay for tuberculosis infection diagnosis in Huzhou, eastern China.Indian J Pathol Microbiol 2013;56:125-128


How to cite this URL:
Qian F, Wang W, Qiu Z, Shen Y, He J, Li D, Zhong J, Dai L. Evaluation of a new tuberculosis-related interferon gamma release assay for tuberculosis infection diagnosis in Huzhou, eastern China. Indian J Pathol Microbiol [serial online] 2013 [cited 2020 Jul 6 ];56:125-128
Available from: http://www.ijpmonline.org/text.asp?2013/56/2/125/118694


Full Text

 Introduction



Tuberculosis (TB) remains a major health problem in the world. About one-third of population is infected with Mycobacterium tuberculosis (MTB). [1] China is one of the TB high-burden countries, where the infection rate of MTB is 44.5%, ranking second in the world in terms of the number of patients with TB. The active pulmonary TB prevalence rate is 367/100,000, and it is estimated that there are 4.51 million active pulmonary TB all over the country. [2] The situation of TB prevention and control for china is very serious.

The tuberculin skin test (TST) has been used for TB infection diagnosis for many years, but the main drawback of this method is poor specificity, especially in TB high-epidemic areas, due to the wide vaccination of Bacille Calmette Guerin (BCG) and exposure to nontuberculous mycobacteria (NTM). [3],[4]

Interferon gamma (IFN-γ) release assays (IGRAs) have been developed for diagnosis of MTB infection. [5],[6] In these methods, TB-specific antigens are used to stimulate the peripheral blood T lymphocytes, then the increased IFN-γ levels are detected by enzyme-linked immunosorbent assay (ELISA) and the TB infection is confirmed indirectly. At present, two products are applied with this theory, one is the QuantiFERON-TB Gold In-Tube (QFT-GIT; Cellestis, Carnegie, VIC, Australia) and the other is T-SPOT.TB (Oxford, Immunotec, Abingdon, UK). They have much higher sensitivity and specificity than TST, [7] so they are recommended as a replacement for TST. But the T-SPOT.TB is expensive and complex‚ and requires the high technology of technicians. Because the QFT-GIT needs relative less laboratory conditions, facilities, requirements, and the results can be observed within 2 days, it is suitable for developing countries.

Recently, in China, a new TB-related interferon gamma release assay (TB-IGRA) was developed for TB infection diagnosis (Beijing Wantai Biological Pharmacy Enterprise Co., Ltd., Beijing, China). The aim of this study was to compare the TB-IGRA with QFT-GIT assay for TB infection diagnosis in China.

 Materials and Methods



Study population

From May 2009 to December 2011, 458 TB patients recruited from Center of TB Diagnosis and Therapy in Huzhou Central Hospital (case group) were enrolled in this study. All these patients were diagnosed according to the diagnosis standards and classification of TB in adults and children. [8] The patients in our study were not on anti-TB drugs treatment. Meanwhile, samples from 378 healthy individuals were collected from the Center of Physical Examination in Huzhou Central Hospital. Active TB was excluded on the basis of symptom review and chest radiographs (control group). All the subjects were adults, aged from 18 to 65 years. The subjects who had immunosupression, including primary immunodeficiency disorders, were on immunosuppressive drug therapy, or had HIV infection, diabetes, or cancer were excluded.

This study was approved by the Faculty of Health Sciences Human Research Ethics Committee of the Huzhou Central Hospital. All subjects provided written informed consent before participating in this study.

QFT-GIT assay

The QFT-GIT assay was performed according to the manufacturer's instructions. [9]

A positive test was defined as a value of TB antigen minus nil value ≥0.35 IU/ml and TB antigen minus nil ≥25% of nil value. A nil value of >8.0 IU/ml or a mitogen minus nil of <0.5 IU/ml was classified as indeterminate.

TB-IGRA

This assay included two procedures, culture in vitro and qualitative analysis of IFN-γ. Briefly, 1 ml of peripheral venous blood samples collected from each participant were injected in three special culture tubes for the TB-IGRA: One test tube coated with M. tuberculosis-specific antigens (ESAT-6, CFP-10), one positive control tube containing Phytohemagglutinin (PHA), and one negative control tube. Then the three tubes were incubated at 37°C for 22 ± 2 h. Following this, the tubes were centrifuged at 3000 g for 10 min and the serum was stored at -20°C until detection. The IFN-γ levels were detected by sandwich-ELISA. The TB-IGRA has been established by the manufacturer based on receiver operator characteristic (ROC) curve analysis to achieve optimum sensitivity and specificity.

A positive result was defined as the concentration of IFN-γ ≥14 pg/ml (test tube minus negative tube) and ≥25% of control.

A concentration of IFN-γ of negative control >400 pg/ml or a positive control minus negative of <20 pg/ml was defined as indeterminate.

Statistical analysis

Patients with indeterminate TB-IGRA or QFT-GIT results were excluded for analyses. Statistical analyses were performed by the χ2 analysis test or Fisher's exact test for categorical variables. Data were analyzed with SPSS 12.0 statistical software package.

 Results



Characteristics of study populations

Four hundred and fifty-eight active TB patients and 378 healthy individuals were enrolled in this study. Patients included 395 pulmonary TB cases and 63 extra-pulmonary TB cases. Extra-pulmonary TB patients included tuberculous meningitis (n = 32), tuberculous pleurisy (n = 13), urinary TB (n = 5), skeletal TB (n = 5), fallopian tube TB (n = 4), tuberculous peritonitis (n = 2), and intestinal TB (n = 2).

The demographic and clinical characteristics of these subjects are shown in [Table 1]. There were no significant differences in age, gender, and BCG vaccination rates between the TB group and control group (P > 0.05, respectively). Patients with TB had a higher history of previous TB rate (P < 0.05) than those in the control group.{Table 1}

Comparison of TB-IGRA and QFT-GIT in TB patients

The test results of TB-IGRA and QFT-GIT are shown in [Table 2]. Among the TB patients, 382 cases gave positive TB-IGRA results. In the control group, 22 cases gave positive TB-IGRA results. The TB-IGRA assay had a sensitivity of 83.4%, specificity of 94.2%, positive predictive value of 94.5%, and negative predictive value of 82.4%. With the QFT-GIT assay, 373 cases gave positive results in the TB patients group and 11 cases gave positive results in the control group. The sensitivity, specificity, positive predictive value, and negative predictive value of QFT-GIT test were 81.4%, 97.1%, 97.1%, and 81.2%, respectively [Table 4]. There were no performance differences between these two assays in TB infection diagnosis for active TB patients.{Table 2}

Comparison of TB-IGRA and QFT-GIT in pulmonary TB and extra-pulmonary TB

The results of TB-IGRA and QFT-GIT in pulmonary TB and extra-pulmonary TB are shown in [Table 3]. There were no performance differences between these two assays in sensitivity, specificity, positive predictive value, and negative predictive value in TB infection diagnosis for pulmonary TB and extra-pulmonary TB [Table 4]. The performance of the two assays in pulmonary TB and extra-pulmonary TB was comparable.{Table 3}{Table 4}

 Discussion



Early diagnosis of TB infection is the crucial process for the prevention and treatment of TB. At present, the sputum culture with microbiological confirmation is the gold standard for clinical diagnosis of active TB, but this method takes a long time that cannot meet the clinical demand.

TST, a traditional test for TB infection screening, was limited because it was affected by NTM and BCG vaccination, especially in China, with 98% inoculation rate of BCG.

In recent years‚ IGRAs based on T-cell immune response have been developed. IGRA is a new immunological diagnosis technology for MTB infection, having high sensitivity and specificity. [10] At present, QFT-GIT and TSPOT are the main assays based on this theory. Among these assays, QFT-GIT uses ELISA; because of less requirements and conditions, it has been approved by the Food and Drug Administration and recommended by the Center for Disease Control and Prevention for diagnosis of TB infection. [11]

The new TB-IGRA is also based on the T-cell immune response, like the QFT-GIT. The immune system is activated with MTB infection, which leads to generation of specific effector T cells and memory T cells for MTB, and T-cell activation and cytokine secretion (such as IFN-γ) happens after specific lymphocyte cells encounter MTB. The difference between the two assays is that the specific antigens of QFT-GIT are synthesis polypeptides, whereas the specific antigens of TB-IGRA are recombinant proteins, expressed by genetically engineered bacterium; so, cost of this assay is low.

Our study showed that the sensitivity of QFT-GIT for TB infection diagnosis in patients with active TB was 81.4%, for patients with smear-positive pulmonary TB was 82.2%, and for patients with smear-negative pulmonary TB was 81.1%. The results were in accordance with previous reports. [12],[13],[14] Additionally, the sensitivity of QFT-GIT for TB infection diagnosis in patients with extra-pulmonary TB was also high (80.9%). The results were consistent with those reported in previous studies. [15],[16]

In this study, we found that the sensitivity of TB-IGRA for TB infection diagnosis in active TB patients was 83.4%, for smear-positive pulmonary TB was 86.6%, and for smear-negative pulmonary TB was 82.3%. The results also showed that the TB-IGRA was a powerful auxiliary fool for TB infection diagnosis in extra-pulmonary TB, and its sensitivity for extra-pulmonary TB was 80.9%. There were no significant differences found in TB infection diagnosis for pulmonary TB and extra-pulmonary TB by using this assay, and the previous study also supported this view. [15] The results indicated that there were no performance differences of sensitivity between TB-IGRA and QFT-GIT.

The specificity of TB-IGRA and QFT-GIT assays were 94.2% and 97.1%, respectively; the results were consistent with many previous reports. [12],[13] But some studies indicated that the specificity was lower than that obtained in our study. [17],[18],[19],[20] It might be related to ethnic and geographic diversion, the different status of study objects, and the latent infection rate of healthy population.

Our results were also consistent with a recent meta-analysis, [21] which showed that the pool sensitivity of QFT-GIT was 81% (95%CI 0.78-0.83) and the pool specificity of QFT-GIT was 99% (95%CI 0.98-1.00).

The results also indicated that there were no performance differences of sensitivity and specificity between TB-IGRA and QFT-GIT, the TB infection diagnostic values of the TB-IGRA and QFT-GIT were comparable, and the results can be observed within 2 days. So, a high sensitivity and specificity assay for TB infection diagnosis is achievable. Furthermore, the advantage of the TB-IGRA is its low cost and suitability for Chinese conditions.

In summary, such convenient, rapid, and low-cost diagnostic technique might be used as an important assisting tool for TB infection diagnosis in the Chinese clinical setting.

 Acknowledgment



This work was supported by a grant from the Major National Science and Technology Projects for infectious diseases (2008ZX10003-004) and the Foundation Project for Science and Technology of Huzhou City (No. 2010YS15).

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