Modified immunohistological staining allows detection of Ziehl–Neelsen‐negative Mycobacterium tuberculosis organisms and their precise localization in human tissue

Abstract

The diagnosis of mycobacterial infection depends on the Ziehl-Neelsen (ZN) stain, which detects mycobacteria because of their characteristic acid-fast cell wall composition and structure. The histological diagnosis of tuberculosis (TB) comprises various aspects: (1) sensitive detection of mycobacteria; (2) precise localization of mycobacteria in the context of granulomatous lesions; (3) 'staging' of disease according to mycobacterial spread and granulomatous tissue integrity. Thus, detection of minute numbers of acid-fast bacteria in tissue specimens is critical. The conventional ZN stain fails to identify mycobacteria in numbers less than 10(4) per ml. Hence many infections evade diagnosis. PCR is highly sensitive, but allows neither localization within tissues nor staging of mycobacterial disease, and positive findings frequently do not correlate with disease. In this study, an anti-Mycobacterium bovis bacille Calmette-Guérin polyclonal antiserum (pAbBCG) was used to improve immunostaining, which was compared to the ZN stain in histological samples. Screening of tissue samples including lungs, pleural lesions, lymph nodes, bone marrow, and skin for mycobacterial infection revealed that pAbBCG staining detects infected macrophages harbouring intracellular mycobacteria or mycobacterial material as well as free mycobacteria that are present at low abundance and not detected by the ZN stain. The positive pAbBCG staining results were confirmed either by PCR analysis of microdissected stained tissue or by culture from tissue. This immunostaining approach allows precise localization of the pathogen in infected tissue.

New insights into the function of granulomas in human tuberculosis

“…Staining with a polyclonal serum specific for mycobacterial antigens detected APCs containing abundant mycobacterial material in the periphery. In contrast, the necrotic core of numerous granulomas did not reveal any positive staining, suggesting that these necrotic centres were sterile [21,53]. We conclude that the preferred localization of dormant M. tuberculosis during latency is the peripheral rim of the leukocyte infiltrate in the tuberculous lesion.…”

Immunohistochemistry using a Mycobacterium tuberculosis complex specific antibody for improved diagnosis of tuberculous lymphadenitis

“…This has also been shown by others. 11,20,21 Among the acid-fast bacilli-negative lymphadenitis cases, immunohistochemistry with Dako anti-BCG was negative and not more sensitive than acid-fast staining. In contrast, MPT64 was demonstrated in the majority of cases.…”

“…9 A diagnosis is confirmed by the presence of acid-fast bacilli and/ or isolation of M. tuberculosis in culture from biopsy specimens or fine-needle aspirates. Staining for acid-fast bacilli has low sensitivity, as its detection limit is 410 4 bacilli per slide, or 10 4 bacilli per ml of specimen, 10,11 and most cases of tuberculous lymphadenitis are paucibacillary. It does not differentiate mycobacterial species either.…”

“…Thus, immunohistochemistry is generally considered to be more sensitive than acid-fast staining and has been investigated in order to improve the diagnosis of tuberculosis, mainly by the use of a commercially available antiBacillus Calmette Guérin (BCG) antiserum (Dako), 11,[18][19][20] or in-house anti-mycobacterial antisera. 21 However, immunohistochemistry has so far not been shown to be more sensitive in extrapulmonary paucibacillary cases, which are difficult to diagnose.…”

Dissemination of Mycobacteria to the Thymus Renders Newly Generated T Cells Tolerant to the Invading Pathogen

“…1B, 1C). This has been attributed to mycobacterial envelope material present in the phagosomes of infected cells (21). Epithelial cells do not seem to be a target for M. avium infection, because no infected cells expressed K8 (Fig.…”