Indian Journal of Pathology and Microbiology

: 2014  |  Volume : 57  |  Issue : 4  |  Page : 574--578

Relative value of immunohistochemistry in detection of mycobacterial antigen in suspected cases of tuberculosis in tissue sections

Rachneet Kohli1, Rajpal Singh Punia1, Robin Kaushik2, Reetu Kundu1, Harsh Mohan1,  
1 Department of Pathology, Government Medical College and Hospital, Chandigarh, Punjab, India
2 Department of General Surgery, Government Medical College and Hospital, Chandigarh, Punjab, India

Correspondence Address:
Reetu Kundu
Department of Pathology, Government Medical College and Hospital, Sector 32-A, Chandigarh - 160 030, Punjab


Background: The diagnosis of tuberculosis (TB) depends on identification of the infecting organism. The diagnosis presents as a challenge due to its diverse clinical presentation and low yield of acid-fast bacilli (AFB) in tissue sections. Aim: The aim of the present study is immunohistochemical localization of tubercle bacilli or their components that persist in the granulomas, but have lost the property of staining with acid-fast stain, assess the advantage of immunostaining over conventional Ziehl-Neelsen (ZN) staining and further to study the staining pattern on immunohistochemistry (IHC). Materials and Methods: The study population comprised 100 suspected cases of TB. Tissue sections from these were subjected to hematoxylin and eosin, ZN and IHC staining using polyclonal antibody to Mycobacterium tuberculosis followed by a comparative analysis of the results. Cases of lepromatous leprosy were used as a positive control. Results: Acid-fast bacilli were identified by ZN stain in 23% of cases. IHC identified 72% cases. In the present study, IHC had higher sensitivity (95.56%) and negative predictive value (96.43%), but lower specificity (35.06%) and positive predictive value (30.56%) than ZN stain which had the sensitivity, specificity, positive predictive value and negative predictive values of 30.56%, 96.43%, 95.65% and 41.56% respectively. Conclusion: Immunohistochemistry is a simple and sensitive technique for localization of tubercle bacilli and their components on tissue sections. It can be easily incorporated in routine histopathology laboratory and serve as an efficient diagnostic adjunct to conventional ZN staining. This will help reduce the practice of prescribing empirical antitubercular treatment based on clinical suspicion alone.

How to cite this article:
Kohli R, Punia RS, Kaushik R, Kundu R, Mohan H. Relative value of immunohistochemistry in detection of mycobacterial antigen in suspected cases of tuberculosis in tissue sections .Indian J Pathol Microbiol 2014;57:574-578

How to cite this URL:
Kohli R, Punia RS, Kaushik R, Kundu R, Mohan H. Relative value of immunohistochemistry in detection of mycobacterial antigen in suspected cases of tuberculosis in tissue sections . Indian J Pathol Microbiol [serial online] 2014 [cited 2021 Nov 30 ];57:574-578
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Tuberculosis (TB) continues to intimidate the human race since time immemorial not only due to its effects as a medical malady, but also as a social and economic tragedy. [1] With an estimated world prevalence of 40 million cases and 10 million new cases added annually, TB is one of the most important infectious diseases. [2] The situation in India is grimmer where nearly one-fourth of the global burden exists with approximately 1.8 million persons developing TB every year. [3],[4]

Primarily considered a pulmonary disease, TB can affect almost any organ, with lymph node involvement being the commonest form of extra-pulmonary TB. [5],[6] The gold standard for diagnosis is detection of the etiologic agent, Mycobacterium tuberculosis, by using Ziehl-Neelsen (ZN) staining or culture. [7],[8] However, fresh, unfixed tissue with live bacilli is usually not available for culture. [9] Moreover, it has a low sensitivity and may take weeks to months before organisms can be identified. [2]

Ziehl-Neelsen staining has low sensitivity on tissue sections although is rapid and inexpensive. Immunohistochemistry (IHC) can be used easily on tissues while awaiting culture results and has great utility in cases with low bacterial load, or those partially treated. [3] In recent times, the use of polymerase chain reaction has been reported which in developing countries is limited by the expense, easy contamination and technical expertise. [10],[11]

The aim of the present study is immunohistochemical localization of tubercle bacilli or their components that persist in the granulomas having lost the property of staining with acid-fast stain, assess the advantage of immunostaining over conventional ZN staining and further to study IHC staining pattern.


The study protocol was approved by the Institutional Ethics Committee and informed consent was obtained from all the patients. Tissue specimens from 100 suspected cases of TB were processed for routine histological examination with hematoxylin and eosin (H and E) staining. ZN staining for acid-fast bacilli (AFB) was done. Polyclonal anti-Bacillus Calmette-Guérin antibody (Genxbio, India) in a dilution of 1:50 was used as primary antibody for IHC. For visualizing system, one step Envision method (HRP-streptavidin-biotin method; Dako, Germany) using diaminobenzidine as chromogen was used. [12] Tissue sections of diagnosed cases of lepromatous leprosy with high bacillary index were used as positive controls. This is because Mycobacterium leprae shows cross-reactivity with M. tuberculosis giving positive immunostaining when polyclonal antibody is used. Other nontubercular granulomatous lesions show no immunoreactivity. [2]

Clinical suspicion of TB was based on clinical examination and investigations like routine hematological investigations, chest X-ray, ultrasonography, computed tomography/magnetic resonance imaging and fine needle aspiration. Diagnosis of granulomatous inflammation was made on routine sections. A comparison was then made between ZN staining and IHC and respective sensitivity, and specificity values were determined.


The age of 100 patients ranged from 02 to 75 years (median 28 years) with a mean of 31.5 ± 16.15 years. The maximum numbers of cases included were in the third decade of life. There were 52 male and 48 female patients. The most common presentation was low-grade pyrexia followed by cervical lymphadenopathy. The duration of symptoms at the time of biopsy/excision varied from few days to years.

Routine histopathology sections in all patients showed granulomatous lesions [Figure 1]. The morphological features observed were the type of cells comprising the granuloma (epithelioid cells and lymphocytes), presence/absence of giant cells, type of giant cells (Langhans' and foreign body) and presence/absence of caseation necrosis. Epithelioid cells, lymphocytes and giant cells (Langhans' and foreign body) were consistently seen in all the cases. Foci of caseation necrosis were seen in 82/100 cases. Maximum number of cases comprised of lymph nodes 21/100 with 19/21 (90.5%) showing caseation necrosis.{Figure 1}

Ziehl-Neelsen staining showed AFB in 23/100 cases. The AFB were seen as beaded rods in the granuloma in association with the epithelioid cells as well as in areas of caseation [Figure 1]. The number of AFB was found to be sparse in distribution in all the AFB positive cases 23/23 (100%). Caseation was present in 22 of 23 (95.7%) AFB positive cases. Of 100 cases, maximum ZN positivity was seen in lymph nodes. Nine of 21 cases (42.9%) of lymph nodes showed AFB. Caseation was present in all the AFB positive lymph nodes. The number of AFB was found to be sparse. Of the remaining 79% cases, 16% specimens comprised of tissue from spine and 15% were from resected specimens from the gastrointestinal tract; showing AFB in 1/16 (6.3%) and 4/15 (26.7%) cases respectively. The rest 48% cases formed the miscellaneous category.

Immunohistochemistry was positive in 72/100 cases. Caseation necrosis was present in 61/72 (84.7%) cases showing IHC positivity. IHC positivity ranged from 0% to 100%; depending on the type of tissue. Skin biopsies showed a very low positive rate of 14.3%. Lymph nodes, resected specimens of intestine and tissue from spine showed IHC positivity in 85.7%, 66.7% and 56.3% cases respectively. Furthermore, ZN detected AFB in 1/18 (5.6%) noncaseating granulomas while IHC detected mycobacterial antigen in 11/18 (61.1%) noncaseating granulomas. Pattern of mycobacterial antigen distribution in IHC positive cases was studied by the type of rods (solid, beaded and fragmented) and presence/absence of antigenic dust in caseous area and in giant cell/epithelioid cell cytoplasm [Figure 2]. Antigenic dust in giant cell/epithelioid cell cytoplasm was seen as the predominant pattern followed by antigenic dust in caseous area. Other patterns included fragmented, beaded and solid rods in descending order in both caseating and giant cell/epithelioid cell cytoplasm. More than one pattern was often present in different or same area in a given section.{Figure 2}

In the present study, the diagnostic yield improved by 50% with IHC as 50/100 AFB negative cases were positive with IHC [Table 1]. IHC had higher sensitivity (95.56%), but lower specificity (35.06%) than ZN staining, which had sensitivity and specificity of 30.56% and 96.43% respectively.{Table 1}


Tuberculosis remains the number one killer infectious disease affecting adults in developing countries. The situation is more complicated when one considers countries such as India where TB disproportionately affects the young. In the present study, maximum numbers of cases included were in the third decade of life with male to female ratio of 1.1:1. The laboratory diagnosis of TB is usually established by detection of AFB and later confirmed by isolation of an organism. However, ZN staining has a very low positivity rate on paraffin sections and culture requires several weeks and may be negative because of previous therapy.

A diagnosis is usually made on the basis of classical histomorphology of chronic granulomatous inflammation suggestive of TB. These histological features can be found in various conditions and diseases other than TB and moreover in immunocompromised TB patients these features can be atypical, leading to considerable difficulty and delay in diagnosis. [5],[8] The 100 cases included in the current study were subjected to routine H and E staining and all showed granulomatous lesions. Epithelioid cells, lymphocytes and giant cells were consistently seen in all the cases.

Ziehl-Neelsen staining is a technique which is positive only when there are more than 10,000 organisms/ml of specimen. [3],[5] Furthermore, antimycobacterial therapy can alter capsule integrity to render organisms nonacid-fast. [9] As the AFB get engulfed and phagocytosed by the macrophages, only fragments of bacilli are left in the lesion which are not identified by ZN stain. [13] ZN staining was done in all the 100 cases included in the present study. Range of ZN stain positivity reported in literature varies between 0% and 44%. [3],[5],[13] Due to intensive phagocytic activity by macrophages in tuberculous granulomas, the morphological characteristics of AFB often get distorted. This may account for the low detectability of ZN staining in the above studies. In the present study, it was 23%. A comparison with studies done on lymph nodes is drawn in [Table 2]. [3],[14],[15]{Table 2}

Immunohistochemical staining procedure is a simple and sensitive technique which has been used to identify mycobacteria in cultures, sputum as well as other smears and tissue sections. Polyclonal and monoclonal antibodies have been raised against various components of mycobacteria, and these antibodies are available commercially. [3] The result of IHC staining (72% positivity) in the current study is similar to some other studies. [16] Higher positivity on IHC using a polyclonal antibody has been reported by some studies to the tune of 87%. [3],[17] The positivity in the current study is slightly better than some studies using a polyclonal antibody. [5] The difference could be due to smaller sample sizes used in these studies as compared to the present study comprising of a bigger sample size of 100 cases. The results of studies using monoclonal antibodies were found to be superior to the present study with 100% IHC positivity. [18],[19] A comparison with various studies on IHC for Mycobacterium tuberculosis is shown in [Table 3]. [3],[5],[9],[13],[14],[16],[17],[18],[19],[20],[21]{Table 3}

The sensitivity of IHC depends on various factors like distribution of mycobacterial antigen within the granuloma, the clinical stage of disease, duration of antitubercular treatment received prior to biopsy and specificity of the primary antibody. In a study by Goel and Budhwar, [18] all cases of lymph nodes (16/16), specimens from the gastrointestinal tract (2/2), female genital tract (7/7) and bone and joints (6/6) were positive for IHC. In the present study, 18/21 (85.7%) lymph nodes, 15/23 (65.2%) gastrointestinal specimens, 17/24 (80.9%) tissues from bone and joints and 2/3 (66.7%) of specimens from female genital tract were positive for IHC. The reason for 100% positivity in the previous study could be due to the use of monoclonal antibody.

The classical histomorphology of tuberculous granulomatous inflammation is not a diagnostic problem in a tissue biopsy. However, when the sections show noncaseous epithelioid granulomas mimicking TB, which is a common occurrence in biopsies, it poses a diagnostic dilemma. The positive immunostaining with antibodies in these cases rules out the differential diagnosis of sarcoidosis and other nonspecific granulomas.

There are only sporadic reports comparing ZN staining with IHC in experimental and clinical granulomata with ZN positivity ranging from 0% to 44% [3],[5],[13] and IHC positivity ranging from 48% to 100%. [13],[17],[20] Low AFB positivity in the present study could be because only the intact bacilli took up the stain. The positive immunostaining in cases where AFB were absent or scarce indicated that the concentrated debris derived from mycobacteria apparently retained its antigenic property although it had lost its AFB staining property.


Immunohistochemistry can be applied in routine laboratory for diagnosis of granulomas due to mycobacterial etiology as it is more sensitive than ZN method. The commercially available antibodies to attenuated strains of mycobacteria do detect mycobacterial antigens in tissue sections by staining whole organisms, fragments and debris. The technical aspects of IHC are simple. The immunologic reagents are stable at 4 °C and can be used for at least 6 months. The method is quicker than culture and can be useful to provide a diagnosis in high endemic areas. Immunostaining may be a useful adjunct to conventional methods to reach an unequivocal diagnosis of mycobacterial infection.


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