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

Abstract

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.

Laboratory Diagnosis of Extra-pulmonary Tuberculosis (EPTB) in Resource- constrained Setting: State of the Art, Challenges and the Need

“…Ethanol-fixed aspirate smears can be stored at room temperature, and can easily be transported if molecular facilities are not available at the local health centre. However, the sensitivity of conventional PCR is low in pauci-bacillary EPTB and nested PCR or real time PCR is required to achieve a better sensitivity than AFB staining or culture [4,5,16].…”

“…insertion element IS-6110 carried in multiple copies by most strains belonging to M. tuberculosis) are in routine use [5]. It is a suitable method for sensitive and fast detection of M. tuberculosis complex DNA in histological material including formalin-fixed tissues and dried scraped material [4,5,16]. This has major implications both in TB endemic and non-endemic areas especially when culture is negative or a fresh specimen is not available for further investigation and thus avoids over treatment and more invasive procedures.…”

“…In recent years, several reports have described immunostaining techniques as an alternative to conventional acid-fast staining [4,16]. The improvement in sensitivity may partly be attributed to the additional detection of degraded bacilli, unlike the ZN staining that requires intact bacilli.…”

“…Various assays for example immunochemistry assay, [4,5,16] have been validated in the research settings with reproducible results and warrants its operationalization as a routine diagnostic test. The availability of new guideline and tools such as Standards for Reporting of Diagnostics Accuracy (STARD) initiative and the Quality Assessment of Diagnostic Accuracy Studies (QUADAS) can facilitate the implementation of such tests.…”

Nested PCR in lung tissue for diagnosis of pulmonary tuberculosis

“…Histological AFB staining can be used to improve the diagnostic yield in suspected cases of TB with granulomatous inflammation; however, AFB staining of histological specimens has a low sensitivity, its detection limit is . 10 4 bacilli?slide -1 [7]. Furthermore, the sensitivity of AFB staining in formalin-fixed, paraffin-embedded tissues can be further reduced by the fixative fluids and organic solvents [8].…”