Immunodiagnosis of tuberculous meningitis: rapid detection of mycobacterial antigens in cerebrospinal fluid by reverse passive hemagglutination assay and their characterization by Western blotting

Abstract

Tuberculous meningitis (TBM) is one of the commonest chronic infections of the central nervous system (CNS). Diagnosis of TBM has been a problem as it causes various clinical manifestations which can be confused with those of other chronic infections of the CNS such as neurocysticercosis (NCC), neurobrucellosis and cryptococcal meningitis, that are prevalent in many underdeveloped and developing countries. Differential diagnosis of TBM can be made by detecting circulating mycobacterial antigens in CSF by immunoassays. In this study, a reverse passive hemagglutination (RPHA) has been developed using rabbit antimycobacterial IgG for detection of circulating mycobacterial antigens in CSFs from chronic infections of the CNS in order to develop a rapid, simple, sensitive and cost-effective method. Circulating mycobacterial antigens were characterized by immunoblot assay. The sensitivity limit of RPHA was 400 ng ml(-1). RPHA was specific as antimycobacterial IgG did not show any reaction with porcine Cysticercus cellulosae which was used as a control antigen. RPHA could detect mycobacterial antigens in CSF at a sensitivity level of 94.11% with a specificity of 99.0%. Immunoblot analysis of RPHA positive CSFs revealed predominantly 30-32 kDa and 71 kDa antigens whilst 6, 86, 120, 96 and 110 kDa showed varied degree of reactivity. Antigens of masses 30-32 and 71 kDa were absent in culture filtrate of Mycobacterium tuberculosis H37Rv grown in Proskeur-Beck liquid medium. RPHA is a rapid, simple and sensitive immunological method with a long shelf life of 6-8 weeks if stabilized coated erythrocytes are stored at +4 degrees C. RPHA could be used as an additional immunodiagnostic tool in both differential diagnosis and prognosis of TBM. Immunoblot results indicate that 30-32 kDa and 71 kDa antigens are cell wall derived.

Demonstration of Components of Antigen 85 Complex in Cerebrospinal Fluid of Tuberculous Meningitis Patients

“…The CSF of 30-kDa protein-positive CSF patients was subjected to SDS-PAGE and electroblotted to nitrocellulose membrane as per standard protocols (22). The membrane was probed with a 1:1,000 dilution of polyvalent antibodies (04.Ag85.1.11.32.rp) to Ag85 complex for 2 h at room temperature, followed by the secondary antibody, goat anti-rabbit IgG conjugated to horseradish peroxidase (HRP).…”

“…In most of the cases, diagnosis relies on clinical observations, imaging of the infected area, and detection of Mycobacterium tuberculosis in CSF by acid-fast bacillus (AFB) staining and culturing. A number of immunological and molecular methods have been reported, but these cannot be employed in standard pathology laboratories and are often not useful for early confirmative diagnosis of TBM (3,7,11,15,22,23,25,26,31,35).…”

Immunologic Diagnosis of Neurotuberculosis

“…Patil also identified 30-40kDa antigens in the CSF but lower amounts of 14kDa and 18-25kDa antigens (Patil et al, 1996). Katti identified mostly 30-32kDa and 71kDa antigens in the CSF of patients with TB meningitis (Katti, 2001). Literature data also reveals the predominance of 30kDa antigen in the CSF.…”

“…Moreover, quantitative determination of antigens could be useful for treatment monitoring. Attempts to detect mycobacterial antigens in neurotuberculosis have been made since 1984 employing mostly agglutination tests, ELISA with different variants or other methods (Venkatesh et al, 2007;Sumi et al, 2002;Radhacrishnan et al, 1990;Katti, 2001;Kashyap et al, 2009 a. Method efficiency.…”

“…The use of a polyclonal serum generally proved more efficient. The main limitation of this method was connected to the variable sensitivity (50-94%) and to the short shelf-life of red-cell labelled antibodies; both listed drawbacks could be amended (Katti, 2001). The method is interesting from the perspective of using a single sample for the detection of both antibodies and antigens in biologic products and for measuring antigens and antibody dilutions needed in treatment monitoring.…”