Acta Oto-Laryngologica. 2013; Early Online, 1–4

ORIGINAL ARTICLE

Interferon-g release assay in the diagnosis of laryngeal tuberculosis

BING FEI*1,2, ZHENG WU*3, KEHUA MIN2, JINGBO ZHANG2, CHUANLIAN DING2 &HAO WU1

1Department of Otolaryngology-Head and Neck Surgery, Affiliated Hospital of Nantong University, Nantong, Jiangsu,PR China, 2Department of Otolaryngology, Jin Hu People’s Hospital, Huai An, Jiangsu, PR China and 3Department ofOtolaryngology, Chang Shu Second People’s Hospital, Su Zhou, Jiangsu, PR China

AbstractConclusion: The T-SPOT test for Mycobacterium tuberculosis infection (T-SPOT.TB) can be used for early diagnosis oflaryngeal tuberculosis (TB). Objective: The incidence of TB is increasing on a global scale. Laryngeal TB is the most commonextrapulmonary form of TB and its early diagnosis is still difficult. This study investigated the performance of the interferon-grelease assay in the diagnosis of laryngeal TB.Methods: A total of 83 patients with laryngeal neoplasms were confirmed to havelaryngeal TB by pathology, acid-fast staining, and/or fluorescence quantitative PCR. In addition, 52 patients with vocal cordpolyps were enrolled as the control group. Two groups underwent both T-SPOT.TB and tuberculin skin test (TST). Results:T-SPOT.TB was positive in 75 cases in the laryngeal TB group and 4 cases in the control group, showing a sensitivity of 90.3%(75/83) and a specificity of 92.3% (48/52). The TST was positive in 42 cases and 20 cases, respectively, in these two groups.Obviously, TST and T-SPOT.TB were significantly different in terms of sensitivity when applied for detection of laryngeal TB(p < 0.05).

Keywords: T-SPOT.TB, tuberculin skin test, diagnosis, specificity

Introduction

Worldwide, tuberculosis (TB) remains one of themost important infectious causes of mortality. In2010, there were an estimated 8.8 million incidentcases and approximately 1.4 million people died fromthis disease [1]. A vast pool of individuals with latentTB infection (LTBI) persists in developing countries,posing a major barrier to global TB control [2]. Chinais among the 22 countries with the heaviest TBburdens and has the second largest TB populationin the world. There are about 0.55 billion peopleinfected, which is a much higher infection rate thanthe global rate of 1/3 of the population all over theworld have infected TB [3]. The prevalence of TB hasincreased for a variety of reasons including theoveruse of antibiotics, mutation of Mycobacteriumtuberculosis, antibiotic resistance, diabetes mellitus,

and AIDS and other immunodeficiency disorders [4].Laryngeal TB is the most common type of extrapul-monary TB and varies greatly in disease onset andsigns and symptoms, leading to a high rate of misdi-agnosis [5]. The T-SPOT test for Mycobacteriumtuberculosis infection (T-SPOT.TB) has been widelyused in the diagnosis of pulmonary TB, but its appli-cation in the diagnosis of laryngeal TB has not beenreported. In this study, we investigated the perfor-mance of T-SPOT.TB in the diagnosis of laryngealTB.

Materials and methods

Patients

A total of 83 patients (46 men and 37 women; age,30–65 years; mean, 47.1 years) were found to have

Correspondence: Professor Hao Wu, Department of Otolaryngology-Head and Neck Surgery, Affiliated Hospital of Nantong University, Nantong, JiangsuProvince, 226001, People’s Republic of China. Tel: +86 13706294560. E-mail: entwuhao@163.com *These authors contributed equally to this work.

(Received 22 July 2013; accepted 21 September 2013)

ISSN 0001-6489 print/ISSN 1651-2251 online � 2013 Informa HealthcareDOI: 10.3109/00016489.2013.850174

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laryngeal TB between August 2007 and December2012. Among them, 45 patients had hoarseness, and38 suffered from sore throat and foreign body sensa-tion in the throat. Video laryngoscopy showed that thelesions were located at the anterior and middle partsof the vocal cords in 44 patients and in the posteriorpart in 39 patients. The morphological findings wereas follows: pale and edematous mucous membranesnear the lesions along with the development of localspotty shallow ulcers (n = 20); hyperplasia of granu-lation tissue, with its surface covered with pseudo-membrane (n = 23); granulation-like neoplasms(n = 22); and nodular uplift (n = 18). In seven patientswith a previous history of pulmonary TB, chest X-rayshowed old changes; nevertheless, chest X-rayshowed no abnormality in the remaining patients.All the patients had good nutritional status andHIV tests were negative.Any one of the following criteria satisfies a diagnosis

of laryngeal TB: (a) biopsy of the lesion shows gran-uloma with caseous necrosis; (b) Mycobacteriumtuberculosis is found by pathology; or (c) laryngealTB is confirmed by fluorescence quantitative PCR forpathologically suspected TB.

Control group

A total of 52 patients (28 men and 24 women; age,31–66 years; mean, 48.3 years) with vocal cord polypsduring the same period were enrolled as the controlgroup. All presented with hoarseness. Video laryn-goscopy showed polypoid changes. The lesions weresurgically removed and pathologically confirmed.None of these 52 patients had a history of TB orany TB-related symptoms, and chest X-ray showedno abnormalities.This study was approved by the Ethics Committee

of Jin Hu County Hospital and all the patients gavetheir written informed consent. T-SPOT.TB andtuberculin skin test (TST) were performed by thesame senior physician.

Fluorescence PCR

The DNA from the paraffin-embedded tissues wasextracted and amplified using the Real Art� M.tuberculosis RG PCR Kit (QIAGEN, Hilden,Germany) following the manufacturer’s protocol.Real-time PCR was performed on a Rotor-Gene2000 (Corbett Research, Sydney, Australia). TheLAN real-time PCR detection system (LG LifeScience, Seoul, Korea) was used to measure the fluo-rescence during PCR. A positive result was indicatedwhen the signal was observed in each channel and thecycle threshold (CT) value was less than 35 cycles.

The selected cut-off value for the differential diag-nosis was 1.14 � 103 copies per ml for quantitativeanalysis [6].

T-SPOT.TB test

A commercial T-SPOT.TB kit (Oxford ImmunoteLtd, Oxford, UK) was used. Mononuclear cells wereisolated from 5 ml of peripheral blood by densitygradient centrifugation. The cells were washed withphosphate-buffered saline (PBS) and adjusted to adensity of 2.5 � 106/ml. In a 96-well plate, thefollowing were added to the respective wells: cellculture medium (blank control); phytohemagglutinin(positive control); ESAT-6 (early secreted antigenictarget, 6 kDa); and M. tuberculosis secretory proteinCFP-10. An additional 100 ml cell suspension wasadded to each well. The cells were incubated at 37�Cfor 16–20 h [7]. The culture medium was discardedand the wells were washed with PBS four times. Thecells were incubated with alkaline phosphatase-labeled mouse anti-human interferon (IFN)-g mono-clonal antibody at 4�C for 1 h. The wells were washedwith PBS four times. Nitro-blue tetrazolium/5-bromo-4-chloro-3’-indolylphosphate (BCIP/NBT)color development substrate was added for 7 minat room temperature. Deionized water was addedto stop the reaction. The criteria for T-SPOT.TBpositivity were: (1) when the blank control well had0–5 blots, the number of blots in the antigen A/Bwell – number of blots in the blank control well ‡6; or(2) when the blank control well had ‡6 blots, thenumber of blots in the antigen A/B well ‡ 2 � numberof blots in the blank control well.

Tuberculin skin test (TST)

TSTs were performed using theMantouxmethod [8].To perform this test, 0.1 ml of purified protein deriv-ative (PPD) tuberculin containing 5 tuberculin units(TU) was intradermally injected into the ulnar side ofthe forearm. The results were observed 48–72 h later.The interpretation criteria were as follows: a reactionof ‡5 mm of induration (swelling) with or withoutblisters or rupture is considered to be TST-positive;absence of induration (swelling) is regarded asnegative.

Statistical analysis

Data were analyzed using SPSS 19.0 software. TheT-SPOT.TB and TST results were comparedbetween the laryngeal TB group and control groupusing a paired chi-squared test, with p < 0.05considered to be statistically significant.

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Results

T-SPOT.TB

In the laryngeal TB group, the T-SPOT.TB waspositive in 75 patients and negative in 8 patients. Inthe control group, the T-SPOT.TB was positive in4 patients and negative in 48 patients. Hence, theT-SPOT.TB had a sensitivity of 93.3% (75/83) andspecificity of 92.3% (48/52) in detecting laryngeal TB.

TST

In the laryngeal TB group, the TST was positive in42 patients and negative in 41 patients. In the controlgroup, the TST was positive in 20 patients andnegative in 32 patients. Hence, the TST had asensitivity of 50.6% (42/83) and specificity of61.5% (32/52) in detecting laryngeal TB.

Comparison of T-SPOT.TB and TST for the detectionof laryngeal TB

The comparison of the two tests is summarizedin Table I.

Discussion

The fourth national epidemiological survey of TB inChina showed that the infection rate and prevalenceof TB were still high and showed no sign of decreasing[9], which might have resulted from long-termimmunization, widespread use of anti-TB drugs,and mutation to the L-type of M. tuberculosis [10].Laryngeal TB is a chronic infectious disease caused bylaryngeal infection with M. tuberculosis, and is themost common form of extrapulmonary TB inthe respiratory system. Previously, laryngeal TB wasusually secondary to advanced pulmonary TB, withsevere systemic and mild laryngeal symptoms. How-ever, the current form of laryngeal TB is characterizedby mild systemic and severe local symptoms, such ashoarseness and sore throat [11].

Traditionally, laryngeal TB has certain clinicalfeatures. In particular, it is often located in the pos-terior larynx (e.g. arytenoid cartilage). However, it isnow more often seen in the anterior part of the larynxincluding the vocal cords [12], ventricular band,and epiglottis. This makes the diagnosis somewhatdifficult.Pathological examination is the gold standard for

diagnosis of laryngeal TB. The typical pathology oflaryngeal TB is epithelioid granuloma and cheesynecrosis, with lymphocytic infiltration and scatteredLangerhans giant cells. Possible reasons for this lowrate of pathologically confirmed laryngeal TB include:(1) inadequate sampling of the laryngeal tissue due tothe risk of compromised vocal quality; and (2) sam-ples were compressed during the sampling procedure,which led to atypical pathological results.TST is the traditionally used immunoassay for

cellular immunity against M. tuberculosis, but itsspecificity is low due to cross-reaction with BCGand other Mycobacterium strains. Our study alsoshowed that TST is low in diagnostic performancefor laryngeal TB. The specificity of TST could beincreased by setting the mean diameter of indurationat ‡10 mm as the threshold for positivity [13], whichmight lead to increased false negativity.Activated T lymphocytes can release IFN-g upon

stimulation by specific antigens of M. tuberculosis[14], which is also the mechanism of the T-SPOT.TBtest. This novel test can determine the infection stateby detecting the amount of IFN-g released by T cells[14]. In addition, the T-SPOT.TB test is ableto differentiate TB from granulomatous lesionssuch as sarcoidosis and Wegener’s granulomatosis.T-SPOT.TB provides definite results in a manner of‘all or none,’ and requires less than 24 h to perform.According to Lalvani [15], the T-SPOT.TB test

had a sensitivity of 83.0–97.0% (pooled sensitivity,90%) for active TB in adults with normal immunefunction. In their meta-analysis, Pai et al. [16] foundthat the specificity of the T-SPOT.TB in diagnosingTB was 93.0%.In the present study, the T-SPOT.TB test was

positive in 75 patients with confirmed laryngealTB, yielding a sensitivity of 90.3%; in the controlgroup, the sensitivity reached 92.3%. These findingsare consistent with the literature.In summary, the sensitivity of the T-SPOT.TB is

remarkably higher than that of the conventional testsin terms of detecting M. tuberculosis; this test willtherefore play a role in the early diagnosis of laryngealTB. However, our subjects did not include youngchildren, and the role of the T-SPOT.TB in thediagnosis of TB in children requires furtherinvestigation.

Table I. Comparison of T-SPOT.TB and TST findings.

T-SPOT.TB

Test + – Total

TST (+) 36 6 42

TST (–) 39 2 41

Total 75 8 83

p Value 0.000

Diagnosis of laryngeal tuberculosis 3

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Declaration of interest: The authors report noconflicts of interest. The authors alone are responsiblefor the content and writing of the paper.

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