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The clinical and histological criteria used to diagnose lymphadenitis caused by Mycobacterium tuberculosis complex organisms have poor specificity. Acid-fast staining and culture has low sensitivity and specificity. We report a novel method for diagnosis of tuberculosis that uses immunohistochemistry to detect the secreted mycobacterial antigen MPT64 on formalin-fixed tissue biopsies. This antigen has not been detected in nontuberculous mycobacteria. Polymerase chain reaction (PCR) for amplification of IS6110 from DNA obtained from the biopsies was used as a gold standard. Fifty-five cases of granulomatous lymphadenitis with histologically suspected tuberculosis obtained from Norway and Tanzania were evaluated. Four known tuberculosis cases were used as positive controls, and 16 biopsies (12 foreign body granulomas and four other non-granulomatous cases) as negative controls. With immunohistochemistry, 64% (35/55) and with PCR, 60% (33/55) of granulomatous lymphadenitis cases were positive. Using PCR as the gold standard, the classical tuberculosis histology had sensitivity, specificity, positive and negative predictive values of 92, 37, 60, and 81%, respectively, and immunohistochemistry had sensitivity, specificity, positive and negative predictive values of 90, 83, 86, and 88%, respectively. The observed agreement between PCR and immunohistochemistry was 87% (j ¼ 0.73). Immunohistochemistry with anti-MPT64 antiserum is a rapid, sensitive, and specific method for establishing an etiological diagnosis of tuberculosis in histologic specimens. Immunohistochemistry has the advantages over PCR of being robust and cheap, and it can easily be used in a routine laboratory.

To study the location and mechanism of apoptosis within the human tuberculosis (TB) and leprosy lesions, parallel sections were analyzed for mycobacterial antigens (M.Ag), Fas ligand (FasL), Fas, CD68 and Mac387 by immunohistochemistry, and apoptotic cells by the terminal deoxynucleotidyl-transferasemediated dUTP-digoxigenin nick end labelling method. Cutaneous leishmaniasis and foreign body granulomas were analyzed for comparison. The heavily infected macrophages in multibacillary TB and leprosy granulomas very strongly expressed FasL, indicating that a mycobacterial infection can induce an increased expression of FasL in a population of infected macrophages, which may protect them from the attack of Fas-expressing lymphocytes. However, macrophages with high levels of leishmania amastigotes did not selectively express FasL, suggesting that this phenomenon is specific for the mycobacteria. Interestingly, in the well-formed TB granulomas, 84% of the multinucleated giant cells strongly expressed FasL. The expression of Fas was weak (34%) or absent. A higher number (33%) of epithelioid cells expressed FasL than Fas (23%). Lymphocytes were scanty among the epithelioid cells. The frequency of apoptotic cells was higher in the epithelioid cells (0.25%) than the mononuclear cells in the mantle zone (0.14%). Thus, the epithelioid cells and the multinucleated giant cells by virtue of the increased expression of FasL may make these granulomas an immune privileged site for mycobacteria.

Mustafa T, Phyu S, Nilsen R, Jonsson R, Bjune G. A Mouse Model for Slowly Progressive Primary Tuberculosis. Scand J Immunol 1999;50:127-136 The progression from primary Mycobacterium tuberculosis infection to disease is usually slow in humans. The aim of this study was to develop and characterize a mouse model for slowly progressive primary tuberculosis, using the intraperitoneal (i.p.) route of infection, and to compare it with our previously described model of latent M. tuberculosis infection. B6D2F1 hybrid mice inoculated with 1.5 × 10 6 colony-forming units (CFUs) of M. tuberculosis H37Rv were followed-up for 70 weeks. Lungs, livers and spleens were examined for bacillary growth, histopathological changes and mycobacterial antigens (MPT64, ManLAM and multiple antigens of M. tuberculosis), by immunohistochemical staining. The infection was found to pass through three distinctive phases. During phase 1, mice were healthy despite development of small granulomas and an increasing number of bacilli in the lungs. During phase 2, mice were unwell but mortality was low. The count of M. tuberculosis and the granuloma size stabilized. The granulomas contained an increasing population of large, vacuolated macrophages. During phase 3, mice became moribund and died rapidly, but the M. tuberculosis count remained relatively stable. The inflammatory infiltrates filled Ϸ 80% of the lung parenchyma and the lesions were not well demarcated. Rapidly progressing inflammation, rather than an increase in the M. tuberculosis count, seems to contribute more to mortality.

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.

Background Pakistan is fifth among high burden countries for tuberculosis. A steady increase is seen in extrapulmonary tuberculosis (EPTB), which now accounts for 20% of all notified TB cases. There is very limited information on the epidemiology of EPTB. This study was performed with the aim to describe the demographic characteristics, clinical manifestations and treatment outcomes of EPTB patients in Pakistan. Method We performed descriptive analysis on routinely collected data for cohorts of TB patients registered nationwide in 2016 at health facilities selected using stratified convenient sampling. Findings Altogether 54092 TB including 15790 (29.2%) EPTB cases were registered in 2016 at 50 study sites. The median age was 24 years for EPTB and 30 years for PTB patients. The crude prevalence of EPTB in females was 30.5% (95%CI; 29.9-31.0) compared to 27.9% (95%CI; 27.3-28.4) in males. The likelihood of having EPTB (OR), was 1.1 times greater for females, 2.0 times for children, and 3.3 times for residents of provinces in the NorthWest .

Background: The aim of this study was to evaluate the diagnostic potential of immunohistochemistry using an antibody to the secreted mycobacterial antigen MPT64, in abdominal and lymph node tuberculosis.

Mycobacterium tuberculosis (MTB) persists in host macrophages (Mfs) because it has developed mechanisms to escape Mf killing. In vitro studies have shown that MTB can induce and inhibit apoptosis by causing the expression of Bax and Bcl-2, respectively, suggesting that the infected cells' fate depends on pro-and antiapoptotic signals. In the present study, we investigated the role of Bcl-2 in MTB infection in situ. The aim was to study the pattern and distribution of Bcl-2 and Bax in cellular infiltrates of MTB-infected B6D2F1 hybrid mice and correlate the expression with the presence of MTB antigens (MAgs). Using formalin-fixed lung tissues (n 45), our results showed a significant difference in the percentage of Mfs stained for Bcl-2 or MAgs and Bax (P < 0.0001). Bcl-2 expression was increased in a population of Mfs and corresponded in intensity, colocalization and percentage with that of MAgs on the same cells, while Bax expression was reduced. In lymphocyte aggregates, Bcl-2 and Bax did not show any differences. We conclude that overexpression of Bcl-2 in Mfs containing MTB may be associated with intracellular survival of the bacilli, thus demonstrating one way by which MTB can escape the host's cellular response and killing.