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

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.

Serological diagnosis of human brucellosis: analysis of seven cases with neurological and cardiological manifestations.

Serum Tube Agglutination (STA) test was used as routine test to detect antibrucellar antibodies in diagnosis of brucella infection in sera (n = 75) and CSF (n = 14) from 78 patients with neurological (n = 60) and cardiological (n = 15) complaints in whom brucellosis was suspected, over a period of two and a half years from January, 1997 to July 1999. Seven (neurological-six and cardiac-one) serum samples (9.33%) were positive by STA, while none of the CSFs were positive. STA titres ranged from 1:10 to 1:1280. We report the findings of these seven cases with neurological and cardiac manifestations in whom STA were found positive. Treatment was accomplished in two cases (neurological-one and cardiac-one), while remaining five cases either were treated empirically with antitubercular treatment or lost for follow up. However these reported cases should alert clinicians to investigate for Brucella infection in cases of pyrexia of unknown origin and this condition in cases of chronic meningitis with unproven aetiology.

Immunodiagnosis of tuberculous meningitis: detection of antibody reactivity to antigens of Mycobacterium tuberculosis and Cysticercus cellulosae in cerebrospinal fluid tuberculous meningitis patients by ELISA.

Enzyme-linked immunosorbent assay (ELISA) was standardized and evaluated for detection of antibody response in cerebrospinal fluid (CSF) to antigens of Mycobacterium tuberculosis and Cysticercus cellulosae. Sonicated extracts of heat killed M. tuberculosis H37Rv and C. cellulosae were prepared and used in ELISA to detect respective antibody response in CSFs for a definitive diagnosis as to tuberculous meningitis (TBM)/neurocysticercosis (NCC). ELISA was performed in a total of 201 CSF samples, which include Group I: chronic infections of the central nervous system (CNS) with possible diagnosis of TBM, tuberculoma, or NCC (n = 70), and Group II: control group of patients with infectious neurological (n = 19), non-infectious neurological (n = 82), and non-infectious non-neurological conditions, i.e., spinal anaesthesia CSFs (n = 30). Specificity in this study was 99.9% and no true cross-reactivity between antimycobacterial antibodies and C. cellulosae antigens and vice-versa was observed. However, in 17.14% of CSFs (12/70), both antimycobacterial and anticysticercal antibodies were detected, 50% of these cases were diagnosed as TBM. But none of the proven NCC cases showed presence of antimycobacterial antibodies. Results of this study would indicate that it would be beneficial if both antibody and antigen responses are detected in CSFs to infectious aetiologies such as M. tuberculosis, C. cellulosae, and C. neoformans in order to enhance the diagnostic accuracy and proper management, as these diseases are highly endemic in underdeveloped and developing countries.

Comparative evaluation of cysticercal antigens and immunoassays in the diagnosis of neurocysticercosis.

Enzyme linked immunosorbent assay (ELISA), dot immunobinding assay (DIA) and passive haemagglutination assay (PHA) were evaluated for the detection of anticysticercal antibodies in cerebrospinal fluids (CSF) from neurocysticercosis (NCC) patients. Results from the three tests were similar. Higher titres of antibodies were observed to the antigens in porcine whole-cyst sonicate than to those in vesicular fluid or scolex or membrane sonicates. Affinity purified parasitic antigens showed a higher degree of specificity and sensitivity in PHA than in ELISA or DIA. Western blot analyses with cyst antigens showed that CSF antibodies from confirmed NCC patients consistently recognized a protein in the region of 64-68 kDa. Other proteins, of 110, 94-97, 80, 72-75, 52, 45, 26-28 and 16-18 kDa, showed heterogenous reactivity, whereas the partially purified antigen of 64-68 kDa showed a high degree of sensitivity and specificity.

Antigenic analysis of Cysticercus cellulosae by crossed immunoelectrophoresis & its role in immune diagnosis of neurocysticercosis.

The antigenic composition of Cysticercus cellulosae cysts excised from infected pig and autopsied human brain was analysed by crossed immunoelectrophoresis with an intermediate gel technique using rabbit hyperimmune serum. Normal pork muscle and human brain antigen were used to differentiate parasite derived components from that of host. Attempts were made to look for the rich source of parasitic immunodominant antigens by analysing preparations of different parts of cyst namely scolex and fluid using rabbit hyperimmune serum. Twenty three antigenic components were identified in sonicate extract of porcine cyst, of which 15 were parasite derived. On comparison with antigens of whole cyst sonicate, scolex showed 10, cyst fluid 9 and human cyst sonicate 11 parasite derived antigens. Serum and cerebrospinal fluid (CSF) of neurocysticercotic patients reacted with 12 parasite derived antigens of porcine cyst sonicate (PCS) in a heterogenous manner. It was also noticed that human cyst sonicate (HCS) lacked 4 of the parasite derived antigens present in the PCS.