Diagnosis of viral central nervous system infections using antipeptide antibody against viral antigen by ELISA.

Viral infections of the central nervous system (CNS) occur sporadically and have been extensively studied because of the potential for permanent neurological damage or death. The neurotropic viruses have been reported to lead to various CNS infections. The objective of the present study is to develop an antigen detection ELISA protocol for detection and quantification of viral antigen in CNS infections by assessing the usefulness of antipeptide antibodies against potential peptides of cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella zoster virus (VZV), Japanese encephalitis virus (JEV), dengue (DENV), West Nile virus (WNV) and Chandipura virus (CHPV). A total of 182 cerebrospinal fluid (CSF) samples from confirmed, suspected and non-viral infections of the CNS were evaluated using panels of antipeptide antibodies against synthetic peptides of viral proteins. The cases of confirmed and suspected viral infections of the CNS showed 77% and 11% positivity, respectively, for the detection of viral antigen using antipeptide against synthetic peptides of CMV, EBV, VZV and JEV. The concentration of viral antigen was also obtained by using antipeptide of respective viruses in CSF from both the groups. The viral antigen concentration was also correlated with viral load in confirmed cases of viral infection of the CNS. This study demonstrates the use of antipeptide against synthetic peptide derived from CMV, EBV, VZV and JEV in diagnostics of viral infections of the CNS using patients' CSF samples. Keywords: viral infection of the CNS; synthetic peptide; antipeptide antibody; viral load; antigen concentration.

Identification of an immunodominant epitope in glycoproteins B and G of herpes simplex viruses (HSVs) using synthetic peptides as antigens in assay of antibodies to HSV in herpes simplex encephalitis patients.

Herpes simplex encephalitis (HSE) is a severe viral infection of the central nervous system (CNS). Assay of antibody response is widely used in diagnostics of HSE. The aim of this study was to identify an immunodominant epitope determining the antibody response to herpes simplex viruses (HSVs) in cerebrospinal fluid (CSF) of HSE patients. The synthetic peptides that resembled type-common as well as type-specific domains of glycoproteins B (gB) and G (gG) of these viruses were evaluated for binding with IgM and IgG antibodies in CSF samples from HSE and non-HSE patients in ELISA. The QLHDLRF peptide, derived from gB of HSV was found to be an immunodominant epitope in the IgM and IgG antibody response. The patients with confirmed and suspected HSE showed in ELISA against this peptide 26% and 23% positivities for IgM, 43% and 37% positivities for IgG and 17% and 15% for both IgM and IgG antibodies, respectively. The total positivities of 86% and 75% for both IgM and IgG antibodies were obtained in the patients with confirmed and suspected HSE, respectively. These results demonstrate that a synthetic peptide-based diagnostics of HSE can be an efficient and easily accessible alternative. This is the first report describing the use of synthetic peptides derived from HSVs in diagnostics of HSE using patientsʹ CSF samples.

Evaluation of BioFM liquid medium for culture of cerebrospinal fluid in tuberculous meningitis to identify Mycobacterium tuberculosis.

The present study was designed to evaluate the sensitivity and specificity of liquid culture medium (BioFM broth) for the diagnosis of tuberculous meningitis (TBM) in cerebrospinal fluid (CSF). CSF samples from 200 patients (TBM group = 150 and non-TBM group = 50) were tested for culture of Mycobacterium tuberculosis in BioFM liquid culture medium. Out of 150 TBM cases, 120 were found to be culture positive, indicating a sensitivity of 80% in BioFM broth within 2-3 weeks of inoculation. Positive cultures were also observed for CSF from 32 (64%) out of 50 non-TBM patients in BioFM liquid culture medium within 4 days of sample inoculation. Therefore, according to our study, BioFM broth system yielded 80% sensitivity [95% confidence interval (CI): 67-93%] and 36% specificity (95% CI: 57-98%) for TBM diagnosis. Our results indicate that although BioFM broth allows the detection of positive cultures within a shorter time, it has a high potential for contamination or for the coexistence of M. tuberculosis and non-tuberculous meningitis (NTM). This coexistence may go undetected or potentially lead to erroneous reporting of results.

Determination of polymerase chain reaction efficiency for diagnosis of tuberculous meningitis in Chelex-100 extracted DNA samples.

SETTING: Polymerase chain reaction (PCR) offers great promise for the rapid, sensitive and specific diagnosis of tuberculous meningitis (TBM). However, the isolation of DNA of high quantity and quality from cerebrospinal fluid (CSF) samples is critical for successful PCR assays. OBJECTIVE: To develop and use a single-tube method for the isolation of PCR-compatible DNA from Mycobacterium tuberculosis using Chelex-100 chelating resin, which does not require organic solvents or detergents. DESIGN: The study focused on the standardisation of a suitable Chelex protocol and its evaluation in 32 CSF samples from TBM and non-TBM subjects. A simultaneous comparison was made with the conventional phenol/chloroform extraction method. RESULT: PCR was found to be more sensitive, more rapid and less technically demanding with the Chelex protocol than the conventional phenol/chloroform extraction method (sensitivity 84.2% vs. 73.6%). CONCLUSION: The single-tube method and the simplicity of the procedure permits early and reliable diagnosis of TBM and makes it an attractive method for routine laboratory assays.

Diagnostic value of early secreted antigenic target-6 for the diagnosis of tuberculous meningitis patients.

BACKGROUND: The early diagnosis of tuberculous meningitis (TBM) is very crucial, since delayed diagnosis can lead to various neurological manifestations. We have previously developed an in-house indirect enzyme-linked immunosorbent assay (ELISA) for TBM diagnosis using the Antigen 85 (Ag 85) complex. It has been suggested that the Ag 85 complex might give false-positive reactions for individuals vaccinated with Bacillus Calmette-Guérin (BCG). OBJECTIVES: In the present study, we describe a prospective evaluation demonstrating that early secreted antigenic target- 6 (ESAT-6), which is absent in Mycobacterium bovis BCG strains, is in the cerebrospinal fluid (CSF) of TBM patients. METHODS: We used an indirect ELISA to detect ESAT-6 antigens in the CSF of TBM patients using polyclonal antibodies against ESAT-6. RESULTS: Using the indirect ELISA method, we demonstrated a sensitivity and specificity of 80% and 94%, respectively, for the diagnosis of TBM. CONCLUSION: The detection of ESAT-6 in the CSF of TBM patients by indirect ELISA is a promising method and can be used to develop an immunodiagnostic assay with increased sensitivity and specificity.

Serodiagnosis of tuberculosis based on the analysis of the 65 kD heat shock protein of Mycobacterium tuberculosis.

OBJECTIVE: To evaluate the 65 kD heat shock protein (hsp) antigen in serum samples of tuberculosis (TB) patients by enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies (mAbs) specific to the 65 kD hsp antigen for the diagnosis of TB. DESIGN: Serum samples were obtained from 188 different groups of patients (confirmed TB [n = 24], clinically suspected TB [n = 48], non-TB disease controls [n = 74] and healthy individuals [n = 42]) and analysed by ELISA using mAbs to the 65 kD hsp antigen. The Kruskal Wallis test (non-parametric analysis of variance) with the Dunnett post test was used for statistical analysis. RESULTS: The method yielded 82% sensitivity and 89% specificity for the diagnosis of TB. The mean (+ or -SD) absorbance value of the 65 kD hsp antigen in TB patients (1.73 + or - 0.27) was significantly higher than in the non-TB disease control group (1.12 + or - 0.42, P < 0.001) and was also higher than among healthy individuals (1.06 + or - 0.42, P < 0.001). CONCLUSION: The detection of the 65 kD hsp antigen in serum samples from confirmed and suspected TB patients by ELISA using mAb against purified 65 kD antigen gives a reliable diagnosis and could be considered as a diagnostic marker for TB. The absence of the 65 kD hsp antigen in healthy control BCG-vaccinated subjects indicates the diagnostic value of this assay in regions of endemicity.

Demonstration of IgG antibodies to 30 Kd protein antigen in CSF for diagnosis of tuberculous meningitis by antibody-capturing ELISA.

OBJECTIVE: A simple and rapid immunological assay method has been developed to demonstrate the presence of IgG antibodies to 30Kd protein antigen (30Kdpa) and culture filtrate protein (CFP) in the CSF of patients with Tuberculous meningitis (TBM). METHOD: Antibody capturing Enzyme Linked Immunosorbent Assay (ELISA) was standardized with CFP antigen of MTB. The IgG antibodies were assayed in CSF sample from TBM and non-TBM patients against 30 Kdpa. RESULTS: The sensitivity and specificity of IgG antibodies for the diagnosis of suspected patients of TBM using 30 Kdpa was 80% and 91% respectively and the corresponding figures for CFP were 85% and 94% respectively. The sensitivity and specificity in two confirmed cases of TBM was 100%. CONCLUSION: The presence of this 30Kdpa in the CSF of suspected cases of TBM consistently would indicate that the selected protein band carries the candidate protein marker antigen, which is specific to M. tuberculosis and could be considered as a diagnostic marker for TBM.