Rapid diagnosis of tuberculosis in aspirate, effusions, and cerebrospinal fluid by immunocytochemical detection of Mycobacterium tuberculosis complex specific antigen MPT64.

Diagn Cytopathol. 2012 Sep;40(9):782-91.

Purohit MR, Mustafa T, Wiker HG, Sviland L.

Centre for International Health, University of Bergen, Bergen, Norway.

 

Abstract

The aim of the study was to evaluate the diagnostic potential of immunocytochemical staining for the detection of Mycobacterium tuberculosis complex-specific antigen MPT64, in tuberculous lymph node aspirates, cerebrospinal fluid, and effusions from pleura and abdomen. One hundred ninety patients with a diagnosis of tuberculosis (cases) and 80 patients with nontuberculous lesions (controls) were enrolled and differentiated on the basis of clinical features, histology, cytology, clinical biochemistry, Ziehl-Neelsen staining, Lowenstein-Jensen culture, and response to antituberculous therapy. Cervical lymph nodes fine-needle aspirate (n = 150), cerebrospinal fluid (n = 27), pleural fluid (n = 41), and peritoneal fluid (n = 52) were collected and stained with anti-MPT64 and anti-BCG antibodies using immunocytochemistry. Nested-PCR for IS6110 was done for comparison and to calculate the diagnostic indices of the ICC staining. ICC staining with anti-MPT64 was positive in 128/190 (67.4%) tuberculous smears and in 4/80 (5%) control smears thus giving sensitivity of 67.4% and the specificity of 95%, while anti-BCG was positive in 112 (58.9%) tuberculous smears and in 16/80 (20%) control smears with sensitivity of 58.9% and specificity of 80%. When diagnostic validation of ICC was done using PCR as the gold standard, the overall sensitivity, specificity, positive- and negative-predictive values for ICC staining in smears with anti-MPT64 was 96, 96, 95, and 97%, respectively, while the corresponding values for anti-BCG were 87, 88, 86, and 88%. ICC staining using anti-MPT64 represents a robust and simple method for establishing an early etiological diagnosis of M. tuberculosis complex infection in extrapulmonary tuberculosis.

PMID: 21416644

 

Supplement:

Tuberculosis (TB) is among the leading causes of morbidity and mortality worldwide. Primarily considered a pulmonary disease, TB can affect almost any organ system. Extrapulmonary TB accounts for approximately 14-40 % of all TB infections. (1,2). There is more extrapulmonary disease in young children (3), and in human immunodeficiency virus (HIV)-TB co-infection (4-6). The diagnosis of TB in general and extrapulmonary TB in particular has always been challenging. The historic methods of detection of Mycobacterium tuberculosis , the causative agent of TB, from pulmonary and extrapulmonary samples by acid-fast bacilli (AFB) staining and microscopy and/or isolation of M. tuberculosis by culture are still the mainstay of diagnosis. The detection limit of the AFB microscopy is around > 104 bacilli per ml of specimen, while most cases of extrapulmonary TB often have lower bacterial load which is often below the detection limit of AFB staining with a result that the test is negative in about 50% of pulmonary TB cases (7), and about 90% of extrapulmonary TB cases (8-11). Furthermore AFB microscopy does not differentiate between mycobacterial species. Mycobacterial culture takes several weeks and its sensitivity is low (up to 22%) in paucibacillary TB (8-11). Constitutional symptoms associated with TB (such as fever, weakness, and weight loss) may be infrequent and the clinical presentation is often varied and non-specific. Tuberculin skin testing and interferon-gamma release assays for the specific detection of TB (QuantiFERON® and T-SPOT®) are available but they cannot discriminate between latent and active infection. Molecular methods have better sensitivity and specificity, but these assays have not been optimally validated for extrapulmonary TB. Conventional M. tuberculosis polymerase chain reaction (PCR) is shown to have the same sensitivity as culture, and nested-PCR has better sensitivity (8-11). Furthermore they are expensive, have high technology requirements, and are susceptible to contamination, restricting their use in routine diagnostic settings in high TB burden countries with limited resources. Histological features of TB overlap with other granulomatous conditions, and may be atypical at various extrapulmonary sites and with concomitant immunosuppression (12), posing confirmatory challenge. These diagnostic confirmatory challenges lead to diagnostic delay, under-reporting, and either under- or over- treatment which causes development of drug resistance and increase in morbidity and mortality. There is, therefore, a great need for improvement in the diagnosis of TB.

There has been considerable focus on the development of better diagnostic methods for pulmonary TB for the past years (13), but the diagnostic challenges of paucibacillary extrapulmonary TB remain to be addressed. Immunohistochemistry and immunocytochemistry (referred as immunochemistry) are standard diagnostic procedures in clinical pathology laboratories. However their use in diagnosis of TB has been limited to only a few studies where the assay could not distinguish between pathogenic and atypical mycobacteria (14-16). One reason for this might be the lack of a specific anti-mycobacterial antibody suitable for all types of tissues and fluids. We have studied the in situ expression of nine different secreted and three somatic mycobacterial proteins, including the M. tuberculosis complex specific secretory protein MPT64 by using immunohistochemistry with in-house polyclonal antibodies in various types of extrapulmonary paucibacillary TB lesions. These results showed that MPT64 was consistently expressed intra-cellularly in all the TB granulomas, while other proteins were not detectable in the lesions where mycobacteria were below the detection limit of AFB microscopy and culture (submitted for publication). This led to the hypothesis that MPT64 has a special ability of accumulation in the infected host cells which makes it possible to detect it in lesions with only a small amount of mycobacteria. Further work confirmed the hypothesis and led to the development of a diagnostic method based on the detection of this protein by using immunochemistry on various extrapulmonary samples including fluids, lymph node aspirates and biopsies (8-11). These studies have shown that the assay can be applied to a wide range of clinical specimens from different geographical locations including Tanzania, India, South Africa, and Norway with a significantly higher sensitivity and specificity as compared to the conventional methods of AFB microscopy and solid culture (Table 1-2). Due to the low sensitivity of culture and conventional PCR in paucibacillary extrapulmonary TB, we used M. tuberculosis specific nested-PCR for detection of bacillary DNA. Clinical diagnosis and good response to treatment was used to define TB cases and used as gold standard for validation of the assay (Table 1-2). The sensitivity and specificity of the assay was similar to M. tuberculosis specific nested-PCR. All the culture positive cases were positive with this assay. The assay performed equally well in HIV co-infected TB cases with atypical histological features (9) (fig.1). MPT64 antigen is present only in M. tuberculosis complex and this test can differentiate between pathological and atypical mycobacteria. The test is significantly faster as compared to the culture as the results are available within one working day. These findings clearly show that detection of the MPT64 antigen can improve the diagnosis of paucibacillary extrapulmonary TB cases where microscopy, culture and conventional PCR are not sensitive due to low bacterial load.

 

Table 1: Positive results of various diagnostic procedure in TB* and non-TB biopsies from various extrapulmonary sites (modified from ref. (9-11)

Diagnostic Procedure

Cervical lymph node biopsies n(%)

Abdominal biopsies n(%)

Pleural biopsies

(HIV coinfection) n(%)

TB*

(n=120)

Non-TB (n=32)

TB*

(n=33)

Non-TB (n=18)

TB*

(n=25)

Non-TB (n=11)

AFB stain

14(12)

0

0

0

2 (8)

0

LJ Culture**

27(22)

0

4 (12)

0

3(12)

0

Immunochemistry-MPT64

96(80)

4(12)

25(76)

0

20 (80)

0

Nested-PCR

104(87)

3(9)

28(85)

0

16 (64)

2 (18)

*A TB case was defined based on the following criteria: fluid /biopsy showing AFB on stain and/or M. tuberculosis on culture and/or presence of typical TB epithelioid granulomas on histology and good response to anti-tuberculous treatment. **LJ: Löwenstein-Jensen culture medium

 


Table 2: Positive results of various diagnostic procedure in TB and non-TB effusions and aspirates from various extrapulmonary sites (modified from ref.(8))

Diagnostic Procedure

Lymph node aspirate n (%)

Cerebrospinal fluid n (%)

Pleural fluid n (%)

Ascitic fluid n(%)

TB (n=120)

Non-TB (n=30)

TB (n=16)

Non-TB (n=11)

TB (n=21)

Non-TB (n=20)

TB (n=33)

Non-TB (n=19)

AFB stain

15(12)

0

0

0

0

0

0

0

LJ** Culture

24(20)

0

1 (6)

0

4(19)

0

3(9)

0

Immunochemistry -MPT64

84(70)

2(7)

10 (62)

0

16(76)

1(5)

18(54)

1(5)

Nested-PCR

88(74)

1(3)

10 (62)

0

15(74)

0

16(48)

0

 

Tehmina Mustafa

Figure1: Immunohistochemical staining in HIV- TB coinfected pleural biopsies with typical and atypical histology (top panel), and HIV-negative Lymph node biopsies (bottom panel) showing strong, granular reddish brown intra-cellular staining with anti-MPT64. The areas in squares are further magnified in subsequent sections or insets.

 

Reference:

1.   WHO. 2012. Global tuberculosis report 2012. Geneva, Switzerland: World Health Organization, 2012. World Health Organization, 2012..

2.   Folkehelseinstituttet. 2013. Tuberkuloseveilederen. http://www.fhi.no/artikler/?id=83300.

3.   Safdar, N., S. G. Hinderaker, N. A. Baloch, D. Enarson, M. A. Khan, and O. Mørkve. 2010. Diagnosis and outcome of childhood tuberculosis: implementing public health policy in three districts of Pakistan. Int. J. Tuberc. Lung Dis. 14: 872-877.

4.   Small, P. M., G. F. Schecter, P. C. Goodman, M. A. Sande, R. E. Chaisson, and P. C. Hopewell. 1991. Treatment of tuberculosis in patients with advanced human immunodeficiency virus infection. N Engl J Med 324: 289-94.

5.   Clark, R. A., S. L. Blakley, D. Greer, M. H. Smith, W. Brandon, and T. L. Wisniewski. 1991. Hematogenous dissemination of Mycobacterium tuberculosis in patients with AIDS. Rev. Infect. Dis. 13: 1089-1092.

6.   Seibert, A. F., J. Haynes, Jr., R. Middleton, and J. B. Bass, Jr. 1991. Tuberculous pleural effusion. Twenty-year experience. Chest 99: 883-886.

7.   Qureshi, S. A., O. Mørkve, and T. Mustafa. 2008. Patient and health system delays: health-care seeking behaviour among pulmonary tuberculosis patients in Pakistan. J. Pak. Med. Assoc. 58: 318-321.

8.   Purohit, M. R., T. Mustafa, H. G. Wiker, and L. Sviland. 2012. Rapid diagnosis of tuberculosis in aspirate, effusions, and cerebrospinal fluid by immunocytochemical detection of Mycobacterium tuberculosis complex specific antigen MPT64. Diagn. Cytopathol. 40: 782-791.

9.   Baba, K., A. M. Dyrhol-Riise, L. Sviland, N. Langeland, A. A. Hoosen, H. G. Wiker, and T. Mustafa. 2008. 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. 16: 554-561.

10.   Purohit, M. R., T. Mustafa, H. G. Wiker, O. Mørkve, and L. Sviland. 2007. Immunohistochemical diagnosis of abdominal and lymph node tuberculosis by detecting Mycobacterium tuberculosis complex specific antigen MPT64. Diagn. Pathol. 2: 36.

11.   Mustafa, T., H. G. Wiker, S. G. Mfinanga, O. Mørkve, and L. Sviland. 2006. Immunohistochemistry using a Mycobacterium tuberculosis complex specific antibody for improved diagnosis of tuberculous lymphadenitis. Mod. Pathol. 19: 1606-1614.

12.   Lucas SB. 2003. Histopathology. In Clinical Tuberculosis, 3rd ed. Davies PDO, ed. Arnold, London. 74-87.

13.   UNITAID. 2012. Tuberculosis diagnostics technology landscape report, 2012. http://www.unitaid.eu/images/marketdynamics/publications/UNITAID-Tuberculosis-Landscape_2012.pdf (accessed Dec 23, 2012).

14.   Sumi, M. G., A. Mathai, S. Reuben, C. Sarada, and V. V. Radhakrishnan. 2002. Immunocytochemical method for early laboratory diagnosis of tuberculous meningitis. Clin. Diagn. Lab Immunol. 9: 344-347.

15.   Barbolini, G., A. Bisetti, V. Colizzi, G. Damiani, M. Migaldi, and D. Vismara. 1989. Immunohistologic analysis of mycobacterial antigens by monoclonal antibodies in tuberculosis and mycobacteriosis. Hum. Pathol. 20: 1078-1083.

16.   Ulrichs, T., K. Lefmann, M. Reich, L. Morawietz, A. Roth, V. Brinkmann, G. A. Kosmiadi, P. Seiler, P. Aichele, H. Hahn, V. Krenn, U. B. Gobel, and S. H. Kaufmann. 2005. Modified immunohistological staining allows detection of Ziehl-Neelsen-negative Mycobacterium tuberculosis organisms and their precise localization in human tissue. Journal of Pathology 205: 633-640.

 

 

Multiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier SchönmannMultiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier Schönmann