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.
Tanned sheep erythrocytes stabilized with pyruvic aldehyde and glutaraldehyde, called double-aldehyde-stabilized cells, were used to standardize passive hemagglutination assay (PHA) for detection of antibody responses to sonicate extract of Mycobacterium tuberculosis and Cysticercus cellulosae soluble antigens. PHA was performed in the following groups of cerebrospinal fluid (CSF) samples: group I - chronic infections of the central nervous system with the possible diagnosis of tuberculous meningitis (TBM), tuberculoma and neurocysticercosis (NCC) (n=88), and group II - controls which included (a) non-infectious non-neurological conditions (n=30), (b) infectious neurological conditions (n=21) and (c) non-infectious neurological conditions (n=133). PHA could detect anti-mycobacterial antibodies at the sensitivity level of 80.76% with a specificity of 92.4% and anti-cysticercal antibodies with a sensitivity of 100% and specificity of 92.94%. However, in 6.33% (i.e. 14/221) of group I and group II (c) CSFs both anti-mycobacterial and anti-cysticercal antibodies were detected. Immunoblot analysis of CSFs derived from TBM patients reacted predominantly to 120-kDa, 96-kDa, 65-kDa, 38-kDa, 26-kDa, 23-kDa, 19-kDa and 12-14-kDa and 4-6-kDa antigens of M. tuberculosis sonicate extract (MTSE), whilst CSFs of proven NCC reacted to >110-kDa, 96-kDa, 80-kDa, 66-68-kDa, 52-kDa and 26-28-kDa antigens of porcine whole cyst sonicate extract (PCSE). On immunoblot analysis, some of the CSFs of TBM patients were PHA positive for both MTSE and PCSE showed antibody reactivity to 70-kDa and 10-kDa antigens of C. cellulosae. Similarly CSF antibody of some Guillain Barre syndrome and myeloradiculopathy patients reacted with cysticercal antigens. But per se no cross-reactivity between MTSE and anti-cysticercal antibodies and vice-versa were observed. However, findings of this study should alert laboratory personnel especially in endemic areas to be extra careful in interpretation of antibody detection results.
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