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The T-SPOT.TB Test for Diagnosis of Breast Tuberculosis Lei Zhong, PhD,1 Xing-Lu Zhou, MS,1 Juan Li, MS,2 Yan-Mei Zhang, PhD,2 Yu-Fei Jiao, PhD,2 Bao-Liang Guo, PhD,1 Zhao-Qi Yan, PhD,1 and Jian-Guo Zhang, PhD1* Lab Med Winter 2015;46:5-19 DOI: 10.1309/LMFBRT05MWV3AYIM

ABSTRACT Objective: To assess the diagnostic value of the T-SPOT.TB test in cases of breast turberculosis (BTB) in China.

Results: The sensitivity of the T-SPOT.TB test for detection of BTB (84.6%) was significantly greater than that of TST (53.8%) (P .05).

Methods: We enrolled 13 female patients with primary BTB as the BTB test group and 10 healthy volunteers as the control group. The 2 groups underwent T-SPOT.TB tests and tuberculin skin tests (TSTs) before receiving a core-needle biopsy or excision biopsy. We then collected and analyzed T-SPOT.TB and TST data.

Conclusion: The T-SPOT.TB test could be a useful adjunct to current tests for diagnosis of BTB and could be used for early diagnosis of this condition.

Globally, tuberculosis (TB) is an important infectious cause of mortality. In 2011 an estimated 8.7 million new cases of TB and 1.4 million deaths from TB were expected worldwide.1 China is among the 22 countries with the heaviest TB burden and has the second largest TB population in the world.2 Breast TB (BTB) is a rare form of extrapulmonary TB that was first documented by Sir Astley Cooper in 1829.3 The incidence of BTB varies from 0.1% to 3.0% of all breast diseases4 and approximately 0.1% to 0.5% of all cases of tuberculosis.5 BTB is sometimes difficult to diagnose due to its nonspecific

clinical appearance imaging results. This results in delays in diagnosis and initiation of treatment, which is a serious issue because early diagnosis and treatment are key for successful cure of this disease. Mycobacterial culture, although it is the current gold standard, produces high false-negative rates. Therefore, a faster, more sensitive, and more specific test for the diagnosis of BTB is needed. The T-SPOT.TB test (Oxford Immunotec Global PLC, Abingdon, England) has been widely used in the diagnosis of pulmonary TB and has been demonstrated colloquially to be useful for the diagnosis of some types of extrapulmonary TB. However, its application in the diagnosis of BTB has not been reported, to our knowledge. In this study, we investigated the sensitivity and specificity of the T-SPOT.TB test for diagnosing BTB in clinically suspected cases of the disease.

Abbreviations TB, tuberculosis; BTB, breast tuberculosis; HIV, human immunodeficiency virus; AFB, acid-fast bacilli; PHA, phytohemagglutinin; ESAT-6, Mycobacterium tuberculosis earlysecreted antigen of 6 kDa; CFP10, ESAT-6–like protein esxB; SFCs, spot-forming cells; PPD, purified protein derivative; PCR, polymerase chain reaction; AFB, acid-fast bacilli; ZN, Ziehl-Neelsen; BCG, Bacillus Calmette-Guérin; IFN, interferon; LTBI, latent tuberculosis infection; H, isoniazid; R, rifampin; Z, pyrazinamide; E, ethambutol; WHO, World Health Organization; H&E, hematoxylin-eosin Departments of 1Breast Surgery and 2Pathology, Second Affiliated Hospital of Harbin Medical University, Harbin, China *To whom correspondence should be addressed. [email protected]

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Keywords: breast tuberculosis, T-SPOT.TB, TST, diagnosis, BTB, sensitivity

Materials and Methods Study Population and Specimens Between January 2010 and September 2013, a total of 13 patients who had been diagnosed with primary BTB were enrolled in this study; we designated them collectively as the BTB group. All patients in our study were women, although

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BTB is also reported in males. The mean age of the patients was 35.4 years (range, 18 to 49 years), and the majority (76.9%) were 20 to 40 years old. None of the patients had a history of pulmonary or extrapulmonary TB revealed via systemic radiologic examination and ultrasonography. Further, none of the patients exhibited any constitutional symptoms of TB, such as fever, weight loss, night sweats, or anorexia. The patients were well nourished and their human immunodeficiency virus (HIV) test results were negative. On examination, each patient had unilateral symptoms; contralateral breast examination results were normal. The clinical presentations included a painless lump in the breast in all 13 patients, ulceration of the skin/sinuses in 4 patients, and formation of more than 1 abscess in 2 patients. BTB was diagnosed by examination of acid-fast bacilli (AFB)– stained smears from core-needle-biopsy specimens in patients and confirmed via histological examination.

Control Group Ten healthy female volunteers (age, 21 to 45 years; mean, 30.1 years) were enrolled as the control group. None of these participants had a history of TB or any TB-related symptoms; their systemic examination results showed no abnormalities.

Ethics Committee Approval This study was approved by the Medical Ethics Committee of the Second Affiliated Hospital of Harbin Medical University, Harbin, China. All patients and volunteers were informed verbally and in writing of the potential benefits and risks of the study; all patients signed informed consent paperwork.

T-SPOT.TB Test We performed the T-SPOT.TB test according to the recommendations of the manufacturer. Mononuclear cells were isolated from 5 mL of peripheral blood from the study participants via density gradient centrifugation, washed twice, and the cell concentration was adjusted such that each of the 4 wells of the assay plate contained approximately 250,000 cells. The cells were left unstimulated (negative control) or were stimulated with 50 μl of phytohemagglutinin (PHA; positive control), with 50 μl of the Mycobacterium tuberculosis early-secreted antigen of 6 kDa (ESAT-6) and 50 μl of ESAT-6–like protein esxB (CFP-10) peptides in separate wells. Last, we incubated the cells at 37ºC for 16 to 20 hours.6

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Image 1 Representative results of the T-SPOT.TB test (Oxford Immunotec Global PLC, Abingdon, England; original magnification ×2) in 2 patients. A, Negative result. B, Positive result.

The interpretation criteria for a positive T-SPOT.TB test result were as follows: 5 or less spots in the blank control well and a difference of 6 or more spots in the antigen A/B well relative to the blank control well or 6 or more spots in the blank control well and a product of 2 or greater for spots in the antigen A/B well multiplied by the number of spots in the blank control well. We counted the number of spot-forming cells (SFCs) in each well using a CTLImmunoSpot S5 Versa Analyzer (Cellular Technology Ltd., Shaker Heights, OH (Image 1).

TS We performed TSTs using the Mantoux method.7,8 Each study participant was injected intradermally in the left forearm with 0.1 mL of 5 IU purified protein derivative (PPD) after extraction of blood for the T-SPOT.TB test. We observed the results after 48 to 72 hours. A physician (LZ) measured and recorded the diameters of both axes of skin induration. The interpretation criteria were as follows: a reaction of 5 mm or greater of induration (swelling) with or without blisters or rupture was regarded as indicating TST positivity; the absence of induration (swelling) was regarded as indicating TST negativity (Image 2). The T-SPOT.TB and TST were performed by the same physician before biopsy.

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group, these results were positive in 2 patients and negative in 8 patients. Thus, the T-SPOT.TB test had a sensitivity of 84.6% (11/13) and specificity of 80.0% (8/10) in detecting BTB.

TST In the BTB group, TST results were positive in 7 patients and negative in 6 patients. In the control group, TST results were positive in 4 patients and negative in 6 patients. Thus, the TST had a sensitivity of 53.8% (7/13) and specificity of 60.0% (6/10) in detecting BTB. A comparison of results from the 2 tests is summarized in Table 1. The overall sensitivity of the T-SPOT.TB test was significantly higher than that of TST (P

The T-SPOT.TB Test for Diagnosis of Breast Tuberculosis.

To assess the diagnostic value of the T-SPOT.TB test in cases of breast turberculosis (BTB) in China...
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