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  Table of Contents    
ORIGINAL ARTICLE  
Year : 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


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

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Date of Web Publication11-Oct-2014
 

   Abstract 

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.

Keywords: Granulomatous inflammation, immunohistochemistry, Mycobacterium tuberculosis, polyclonal antibody, tuberculous granuloma

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;57:574-8

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 2019 Dec 10];57:574-8. Available from: http://www.ijpmonline.org/text.asp?2014/57/4/574/142667



   Introduction Top


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.


   Materials and methods Top


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.


   Results Top


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: Photomicrograph showing epithelioid cell granulomas in a section from lymph node (H and E, ×200). Inset shows acid-fast bacilli in foci of caseation necrosis (Ziehl– Neelsen, ×1000)

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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: Photomicrograph showing brown diffuse antigenic dust in giant cells (anti Bacillus Calmette-Guérin [BCG] antibody, ×100). Inset shows brown rods in a giant cell (anti-BCG antibody, ×200)

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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: Results on ZN staining and IHC (n = 100)

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   Discussion Top


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: Comparison of AFB studies done on lymph nodes

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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: Comparative review of the literature on IHC with polyclonal and monoclonal antibodies for Mycobacterium tuberculosis

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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.


   Conclusion Top


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.

 
   References Top

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Sharma SK. Introduction. In: Tuberculosis. 2 nd ed. New Delhi: Jaypee; 2009. p. 1-6.  Back to cited text no. 1
    
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Prapanna P, Srivastava R, Arora VK, Singh N, Bhatia A, Kaur IR. Immunocytochemical detection of mycobacterial antigen in extrapulmonary tuberculosis. Diagn Cytopathol 2014;42:391-5.  Back to cited text no. 2
    
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Mukherjee A, Kalra N, Beena KR. Immuno-histochemical detection of mycobacterial antigen in tuberculous lymphadenitis. Indian J Tuberc 2002;49:213-6.  Back to cited text no. 3
    
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Park K. Epidemiology of communicable diseases. In: Park's Textbook of Preventive and Social Medicine. 22 nd ed. Jabalpur: Bhanot; 2013. p. 132-333.  Back to cited text no. 4
    
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Mustafa T, Wiker HG, Mfinanga SG, Mørkve O, Sviland L. Immunohistochemistry using a Mycobacterium tuberculosis complex specific antibody for improved diagnosis of tuberculous lymphadenitis. Mod Pathol 2006;19:1606-14.  Back to cited text no. 5
    
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Raviglione MC, O'Brien RJ. Tuberculosis. In: Fauci AS, Kasper DL, Longo DL, Braunwald E, Hauser SL, Jameson JL, editors. Harrison's Principles of Internal Medicine. 17 th ed. New York: McGraw-Hill; 2008. p. 1006-20.  Back to cited text no. 6
    
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Silva BB, Santos LG, Costa PV, Pires GC, Borges SA. Clinical case report: Primary tuberculosis of the breast mimicking carcinoma. Am J Trop Med Hyg 2005;73:975-6.  Back to cited text no. 7
    
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McAdam AJ, Sharpe AH. Infectious diseases. In: Kumar V, Abbas AK, Fausto N, Aster JC, editors. Robbins and Cotran Pathologic Basis of Disease. 8 th ed. Pennsylvania: Saunders; 2010. p. 331-98.  Back to cited text no. 8
    
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Wiley EL, Mulhollan TJ, Beck B, Tyndall JA, Freeman RG. Polyclonal antibodies raised against Bacillus Calmette-Guerin, Mycobacterium duvalii, and Mycobacterium paratuberculosis used to detect mycobacteria in tissue with the use of immunohistochemical techniques. Am J Clin Pathol 1990;94:307-12.  Back to cited text no. 9
    
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Foulds J, O'Brien R. New tools for the diagnosis of tuberculosis: The perspective of developing countries. Int J Tuberc Lung Dis 1998;2:778-83.  Back to cited text no. 10
    
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Nahid P, Pai M, Hopewell PC. Advances in the diagnosis and treatment of tuberculosis. Proc Am Thorac Soc 2006;3:103-10.  Back to cited text no. 11
    
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Bancroft JD. Immunohistochemical techniques. In: Theory and Practice of Histological Techniques. 6 th ed. China: Churchill Livingstone; 2008. p. 433-72.  Back to cited text no. 12
    
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Radhakrishnan VV, Mathai A, Radhakrishnan NS, Rout D, Sehgal S. Immunohistochemical demonstration of mycobacterial antigens in intracranial tuberculoma. Indian J Exp Biol 1991;29:641-4.  Back to cited text no. 13
    
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Purohit MR, Mustafa T, Wiker HG, Mørkve O, Sviland L. Immunohistochemical diagnosis of abdominal and lymph node tuberculosis by detecting Mycobacterium tuberculosis complex specific antigen MPT64. Diagn Pathol 2007;2:36.  Back to cited text no. 14
    
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Sumi S, Radhakrishnan VV. Evaluation of immunohistochemistry with a panel of antibodies against recombinant mycobacterial antigens for the diagnosis of tuberculous lymphadenitis. Int J Med Sci 2009;1:215-9.  Back to cited text no. 15
    
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Padmavathy L, Rao LL, Ramanadhan S. Mycobacterial antigen in tissues in diagnosis of cutaneous tuberculosis. Indian J Tuberc 2005;52:31-5.  Back to cited text no. 16
    
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[PUBMED]    
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Goel MM, Budhwar P. Immunohistochemical localization of mycobacterium tuberculosis complex antigen with antibody to 38 kDa antigen versus Ziehl Neelsen staining in tissue granulomas of extrapulmonary tuberculosis. Indian J Tuberc 2007;54:24-9.  Back to cited text no. 18
    
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Barbolini G, Bisetti A, Colizzi V, Damiani G, Migaldi M, Vismara D. Immunohistologic analysis of mycobacterial antigens by monoclonal antibodies in tuberculosis and mycobacteriosis. Hum Pathol 1989;20:1078-83.  Back to cited text no. 19
    
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Baba K, Dyrhol-Riise AM, Sviland L, Langeland N, Hoosen AA, Wiker HG, et al. Rapid and specific diagnosis of tuberculous pleuritis with immunohistochemistry by detecting Mycobacterium tuberculosis complex specific antigen MPT64 in patients from a HIV endemic area. Appl Immunohistochem Mol Morphol 2008;16:554-61.  Back to cited text no. 20
    
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Oliveira BF, Takay FC, Shida TM, Santo RM, Souza AC Jr, Matayoshi S. Orbital tuberculosis diagnosed by immunohistochemistry: Case reports. Rev Inst Med Trop Sao Paulo 2004;46:291-4.  Back to cited text no. 21
    

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Correspondence Address:
Reetu Kundu
Department of Pathology, Government Medical College and Hospital, Sector 32-A, Chandigarh - 160 030, Punjab
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.142667

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