Browsing by Author "Mathai, A"
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Item A comparative evaluation of Dot immunobinding assay (Dot-Iba) and polymerase chain reaction (PCR) for the laboratory diagnosis of tuberculous meningitis(DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE, 2002)The results of a Dot immunobinding assay (Dot Iba) for the detection of mycobacterial antigen in the cerebrospinal fluid (CSF) of 45 patients with tuberculous meningitis (TBM) were compared with the results of a polymerase chain reaction (PCR) for the detection of Mycobacterium tuberculosis. In eight patients with culture proven TBM, Dot-lba gave positive results, while PCR yielded positive results only in six patients. The overall sensitivities of Dot-lba and PCR in 37 patients with culture negative (probable) TBM were 75.67% and 40.5% respectively. Dot-lba, in contrast to PCR is a rapid and relatively easier method. More importantly, Dot-lba is suitable for the routine application for the laboratory diagnosis of TBM and therefore best suited to laboratories in the developing world. (C) 2002 Elsevier Science Inc. All rights reserved.Item A dot-immunobinding assay (dot-Iba) for rapid diagnosis of pulmonary tuberculosis.(Indian journal of experimental biology, 2001)IgG antibody to Mycobacterium tuberculosis from the sera of patients with 'definite' pulmonary tuberculosis (PT) was isolated and coupled with Cyanogen bromide-Sepharose 4B. Using an immunoabsorbent affinity chromatography, 14 kDa antigen was recovered from the culture filtrates of M. tuberculosis. With this mycobacterial antigen, a dot immunobinding assay (Dot-Iba) was developed for the detection of specific antibody to M. tuberculosis in the sera of patients with PT and controls. The assay gave positive results in all the 12 sputum-smear positive [acid fast bacilli (AFB)] patients with PT and gave negative results in the 50 sera from control groups. The Dot-Iba as described in this study, is simple, rapid and specific for laboratory diagnosis of PT.Item A newer approach for the laboratory diagnosis of tuberculous meningitis(DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE, 2001) Mathai, A; Radhakrishnan, VV; George, SM; Sarada, CIn this prospective study, a simple method was standardized for measuring circulating mycobacterial antigen in the cerebrospinal fluid (CSF) for the laboratory diagnosis of tuberculous meningitis (TBM). The heat-inactivated CSF specimens from tuberculous and nontuberculous patients were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) (SDS-PAGE) and they were subsequently transferred onto nitrocellulose membrane (NCM) Using a rabbit polyvalent antibody to M tuberculosis, a heat stable 82 kDa mycobacterial antigen was demonstrated in the CSFs of patients with TBM. This antigen was conspicuous by it!; absence in the CSFs of non-tuberculous subjects. Due to inactivation of CSF; specimens, there is a minimal risk of handling of infectious material in the laboratory. Besides, this newer approach is simple, inexpensive and can be readily applied in any routine clinical laboratory and it is particularly suited to developing countries. (C) 2001 Elsevier Science Inc. All rights reserved.Item Autoimmune encephalitis: Clinical diagnosis versus antibody confirmation(ANNALS OF INDIAN ACADEMY OF NEUROLOGY, 2015) Cyril, AC; Nair, SS; Mathai, A; Kannoth, S; Thomas, SVContext: Autoimmune encephalitis is a heterogeneous disorder which is being diagnosed with increasing frequency. The diagnosis of these disorders is based on the detection of autoantibodies and characteristic clinical profiles. Aims: We aimed to study the antibody profile in encephalitis patients with suspected autoimmune etiology presenting to a tertiary care center. Settings and Design: The subjects were selected by screening all patients with clinical profile suggesting autoimmune encephalitis admitted in the neuromedical intensive care unit (ICU) of a tertiary care center in South India. Materials and Methods: Patients who fulfilled modified Zuliani et al.'s, criteria for autoimmune encephalitis were identified during the period December 2009-June 2013. Blood samples from these subjects were screened for six neuronal antibodies. Statistical analysis used: Chi-square test was applied to compare the antibody positive and negative patients. Results: Out of 1,227 patients screened, 39 subjects (14 males: 25 females) were identified with a mean age of 15.95 years and 19 cases were assessed in the acute and 20 in the convalescent phase of the illness. Seizure (87.8 %) was the most common presenting symptom; status epilepticus occurred in 23 (60.5%) patients during the course of the illness. Fourteen (35.9%) patients were N-methyl-D-aspartate receptor (NMDAR) antibody-positive and all were negative for the other antibodies tested. Conclusions: One-third of patients presenting with acute noninfective encephalitis would be positive for NMDAR antibodies with the remaining two-thirds with clinically suspected autoimmune encephalitis being antibody-negative. There are few markers in the clinical and investigative profiles to distinguish antibody-positive and -negative patients.Item Characterization of mycobacterial antigens by Elisa and immunoblot methods.(Indian journal of pathology & microbiology, 1995)Antibodies to two mycobacterial antigens viz - culture filtrate antigen (CFA) and Mycobacterium tuberculosis antigen 5 were raised in rabbits. Enzyme-linked immuno sorbent assay (ELISA) and immunoblot methods were used for the evaluation of the specificity of the rabbit antibodies to M. tuberculosis. Immunoblot method is more sensitive than ELISA for the detection of antibodies to M. tuberculosis in the rabbit sera. It is being emphasised that characterisation of the mycobacterial antigens and evaluation of the specificity of the antimycobacterial antibodies are essential prior to their applications as an adjunct in the laboratory diagnosis of human mycobacterial disease.Item Circulating immune complexes in cerebrospinal fluid of patients with tuberculous meningitis.(Indian journal of experimental biology, 1991)Circulating immune complexes (ICs) were isolated from cerebrospinal fluids (CSFs) of patients with tuberculous meningitis (TBM), non-tuberculous neurological diseases by a polyethylene glycol (PEG) precipitation method. Mycobacterium tuberculosis antigen 5 was detected in CICs of 30% patients with TBM, by sandwich ELISA. CIC level decreases during antituberculosis chemotherapy and therefore its detection can provide a method to monitor the therapeutic schedule in patients with TBM.Item Circulating tumour necrosis factor alpha & soluble TNF receptors in patients with Guillain-Barre syndrome(INDIAN JOURNAL OF MEDICAL RESEARCH, 2003)Background & objectives: Tumour necrosis factor-alpha (TNF-alpha) is regarded as one of the immune factors that can induce demyelination of peripheral nerves in patients with Guillian-Barre syndrome (GBS). This present study was undertaken to find out the role of TNF-alpha and soluble TNF receptors in the pathogenesis of GBS; and to study the effect of intravenous immunoglobulin (ivlg) therapy on the serum TNF-a and soluble TNF receptors in patients with GBS.Methods: Thirty six patients with GBS in progressive stages of motor weakness were included in this study. The serum TNF-alpha and soluble TNF receptors (TNF-RI, TNF-RII) were measured in the serum samples of these patients before and after ivIg therapy by a sandwich ELISA.Results: Of the 36 patients with GBS, 26 (72.2%) showed elevated serum TNF-alpha levels prior to ivIg therapy. Following a complete course of ivIg therapy there was a progressive decrease in the serum TNF-alpha concentrations in these 26 patients. On the other hand, the soluble TNF receptors, particularly TNF-RII showed an increase in the serum of GBS patients following ivIg therapy.Interpretation & conclusion: The results indicate that ivIg reduces the serum TNF-alpha. concentrations in the GBS patients having elevated levels prior to ivlg therapy. Elevated serum levels of soluble TNF receptors following ivIg therapy may play a protective role by inhibiting the demyelinating effect of TNF-alpha in the peripheral nerves of patients with GBS.Item Correlation between the isolation of Mycobacterium tuberculosis and estimation of mycobacterial antigen in cisternal, ventricular and lumbar cerebrospinal fluids of patients with tuberculous meningitis.(Indian journal of pathology & microbiology, 1993)In the study Mycobacterium tuberculosis was isolated in the cerebrospinal fluid (CSF) specimens of patients with tuberculous meningitis (TBM) by the conventional bacteriological technique. The isolation rate of M. tuberculosis was found to be 11.5% in lumbar, 75% in ventricular and 87.5% in cisternal CSFs. Low isolation rate of M. tuberculosis in lumbar CSF is due the low density of tubercle bacilli in lumbar CSF than in cisternal CSF. However M. tuberculosis antigen 5 is present in significant concentration in CSFs. The antigen concentration in CSF was estimated by an inhibition enzyme-linked immunosorbent assay (ELISA). Since CSF specimens can not be collected from ventricular or cisternal routes for the routine bacteriological investigations in patients with TBM, estimation of M. tuberculosis antigen 5 concentration in lumbar CSF by an inhibition ELISA may be considered as an adjunct in the laboratory diagnosis of TBM. This is particularly relevant in those patients in whom bacteriological methods fail to demonstrate M. tuberculosis in CSF specimens.Item Detection of heat stable mycobacterial antigen in cerebrospinal fluid by dot-immunobinding assay(NEUROLOGY INDIA, 2003)Background: Isolation of Mycobacterium tuberculosis in cerebrospinal fluid (CSF) specimen in patients with tuberculous meningitis (TBM) is infrequent and carries low sensitivity. Thus development of an alternative laboratory diagnostic test is essential for the early diagnosis and treatment of TBM. Objective: A simple, rapid Dot immunobinding assay (Dot-Iba), for the laboratory diagnosis of TBM is devised. This method minimizes the risk of handling infectious material in the laboratory. Method: The Dot-Iba was standardized with heat-inactivated M tuberculosis antigen (PPD). The heat-inactivated CSF from TBM and non-TBM patients was similarly assayed and it can detect antigen upto 1ng/ml in CSF Result: A positive result was obtained in all the five culture positive patients with TBM and in 20/25 probable TBM. A negative result was obtained in 38/40 CSF from disease control group. The overall sensitivity and specificity of Dot-Iba was 83.3% and 95% respectively. Conclusion: Dot-Iba can be used as an adjunct for the laboratory diagnosis of TBM, particularly in culture negative TBM patients and also in those clinical situations where no laboratory tests are available to distinguish between TBM and partially treated pyogenic meningitis.Item Diagnosis of tuberculous meningitis by enzyme-linked immuno-sorbent assay (ELISA), using an affinity chromatography purified mycobacterial antigen.(The Journal of the Association of Physicians of India, 1994)Using an immunoabsorbent affinity chromatography, a mycobacterial antigen was isolated from culture filtrate of H37Ra Mycobacterium tuberculosis (MTB). The immunoabsorbents were prepared by coupling cynogen bromide-activated Sepharose-4B with human IgG antibody to MTB. Cerebrospinal fluids (CSF) from 10 culture positive, 30 culture negative patients with tuberculous meningitis (TBM) were assayed, for IgG antibody to this mycobacterial antigen by ELISA. CSFs from 50 patients with non-tuberculous neurological diseases were selected as control group. At a selected 'cut off' titre of 1:80, 21 out of 30 CSFs from culture negative patients gave positive results. No false negative result was observed in CSF from 10 culture positive patients with TBM. No false positive results were recorded in CSFs of 50 patients with non-tuberculous neurological diseases. Technical aspects involved in the isolation of this myobacterial antigen and its potential applications in the laboratory diagnosis of TBM have been emphasised in this study.Item Diagnostic utility of polymerase chain reaction and immunohistochemical techniques for the laboratory diagnosis of intracranial tuberculoma(CLINICAL NEUROPATHOLOGY, 2001)In an attempt to establish a tuberculous etiology, polymerase chain reaction (PCR) and immunohistochemical (IHC) methods were undertaken in formalin-fixed paraffin sections of ten surgical specimens of intracranial tuberculoma. The control group included an equal number of intracranial fungal granuloma. Both PCR and IHC methods did not yield false-positive results in fungal granuloma. PCR was found to be less sensitive (60%) than IHC method (80%) in this study. IHC method definitely possesses several operational advantages over PCR and is more suited to laboratories in developing countries for establishing a tuberculous etiology particularly in those patients in whom the conventional bacteriological methods did not confirm the diagnosis of tuberculoma.Item Dot-immunobinding assay.(Methods in molecular biology (Clifton, N.J.), 2009)Dot-immunobinding assay (Dot-Iba) is a simple and highly reproducible immunodiagnostic method. Antibody or antigen is dotted directly onto nitrocellulose membrane (NCM) discs. The diagnostic material to be checked can be incubated on this disc. Presence of antigen-antibody complex in NCM discs can be directly demonstrated with enzyme-conjugated antiglobulins and substrate. Development of a purple-pink colored, insoluble substrate product in the NCM will be considered a positive result in the assay. This assay allows the processing of multiple specimens at a time and the entire operational procedures require only 4-6 h. Dot-Iba is rapid, and the technical steps involved in the assay are much simpler than in the other immunoassays such as enzyme-linked immunosorbant assay in detecting circulating antigen and antibody in clinical samples. The Dot-Iba showed an overall sensitivity of 60% for tuberculous meningitis diagnosis and no false positive results were encountered. Hence, this assay is highly specific for the diagnosis of paucibacillary diseases such as extrapulmonary tuberculosis. Dot-Iba is best suited to laboratories in developing world where there are constraints in laboratory resources.Item Evaluation of purified protein derivative in the laboratory diagnosis of tuberculous meningitis.(The Journal of the Association of Physicians of India, 1992)An enzyme-linked immunosorbent assay (ELISA) was standardised for the quantitation of IgG antibody in cerebrospinal fluid (CSF) specimens of patients with tuberculous meningitis (TBM). Purified protein derivative (PPD1) from H37Ra M tuberculosis was used as the antigen in the assay. The sensitivity of the ELISA with this antigen was evaluated in the CSF of 10 culture positive and 40 culture negative patients with TBM. The specificity of the assay was evaluated in the CSF of 50 patients with non-tuberculous neurological diseases (control group). The results obtained with this antigen were compared with commercially available tuberculin purified protein derivative (PPD2) and BCG antigens. PPD2 gave false negative results (50%) in culture positive patients with TBM, and BCG antigen gave false positive results in 32% of non-tuberculous subjects. PPD1 gave a sensitivity of 60% in culture negative patients with TBM and no false positive reactions in the non-tuberculous group. PPD1 antigen, in contrast to other mycobacterial antigens, can be very easily prepared in any routine laboratory, and this antigen is recommended for use as an aid in the laboratory diagnosis of TBM, particularly in culture negative patients with TBM.Item Humoral immune reactions in tuberculous meningitis.(Indian journal of medical sciences, 1991)Humoral immune reactions as reflected in sera and cerebrospinal fluid (CSF) of 50 patients with Tuberculous meningitis (TBM) were studied. CSFs and sera from 50 patients with nontuberculous neurological diseases were selected as controls. CSFs of patients with TBM showed high titres of circulating antimycobacterial antibodies than in nontuberculous subjects. The CSF-IgG index is significantly higher in patients with TBM. Humoral immune reaction could be applied in the laboratory diagnosis of TBM, particularly when repeated bacteriological methods are negative for M. tuberculosis in CSFs.Item Immune complex levels and plasmapheresis in Guillain-Barre syndrome.(Acta neurologica, 1993)Circulating IC levels were assayed serially in 12 G.B.S. patients treated with PE and an attempt was made to predict the outcome of treatment, from the levels of circulating IC. It was found that there was no significant correlation between the levels of circulating IC and the outcome of treatment with PE, in G.B.S. patients.Item Immunocytochemical method for early laboratory diagnosis of tuberculous meningitis(CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, 2002)A simple immunocytochemical method was standardized for the direct demonstration of mycobacterial antigen in cerebrospinal fluid (CSF) specimens of patients with tuberculous meningitis (TBM). CSF-cytospin smears were prepared from 22 patients with a clinical diagnosis of TBM and also from an equal number of patients with nontuberculous neurological diseases (disease control). Immunocytological demonstration of mycobacterial antigens in the cytoplasm of monocytoid cells was attempted, by using rabbit immunoglobulin G to Mycobacterium tuberculosis as the primary antibody. Of the 22 CSF-cytospin smears from TBM patients, 16 showed positive immunostaining, while all of the CSF-cytospin smears from the disease control showed negative immunostaining for mycobacterial antigen. The technical aspects of this immunocytological method for the demonstration of mycobacterial antigens are simple, rapid, and reproducible, as well as specific, and therefore can be applied for the early diagnosis of TBM, particularly in patients in whom bacteriological methods did not demonstrate the presence of M. tuberculosis in the CSF.Item Immunohistochemical demonstration of mycobacterial antigens in intracranial tuberculoma.(Indian journal of experimental biology, 1991)Mycobacterial antigens have been demonstrated immunohistochemically in the paraffin sections of 10 intracranial tuberculous granulomas and the results were compared with the detection of acid fast bacilli by conventional Ziehl-Neelsen method. In none of the 10 specimens, acid fast bacilli were demonstrated while mycobacterial antigens were characterised as diffusely staining granular brownish-pink material within the cytoplasm of giant cells and macrophages. In 14 specimens of granulomatous lesions due to non-tuberculous aetiology, immunohistochemical stains were negative for mycobacterial antigen. Thus demonstration of mycobacterial antigen will be not only useful in establishing mycobacterial aetiology of a caseating intracranial granuloma but also can be used as an alternative method to the conventional Ziehl-Neelsen method.Item Intravenous immunoglobulin reduces serum tumor necrosis factor alpha in patients with Guillain-Barre Syndrome(NEUROLOGY INDIA, 2003)Background: Tumor necrosis factor a TNF-alpha has a possible role in the pathogenesis of the Guillain-Barre'syndrome (GBS). Alms: To study the effect of intravenous immunoglobulin (IVIg) on serum TNF-alpha concentrations in patients with GBS. Material and Methods: The effect of IVIg on TNF-alpha was evaluated in 36 patients with GBS. Serum TNF-alpha concentration was measured by enzyme-linked immunosorbent assay (ELISA). The sera of 22 (61%) patients with GBS showed elevated concentrations of TNFalpha (35-182 pg/ml) and these sera were individually incubated in vitro with IVIg (0.25mg/ml) at 370 degreesC for 24 hours. Results: The serum TNF-alpha concentrations in the 22 GBS patients with elevated levels showed a steady decline (60.34-19.78 pg/ml) following incubation with IVIg. These 22 patients also received IVIg therapy, and serum TNF-alpha concentrations showed a significant decline (65.5-9.75 pg/ml) at the end of the therapy. At the time of discharge from the hospital, there was a positive correlation between neurological recovery and decline in TNF-alpha concentrations in these 22 GBS patients. Conclusions: The results of this study indicate that elevated levels of TNF-alpha occur in a proportion of patients with GBS and in these patients elevated serum TNF-alpha levels decline with IVIg therapy.Item Rapid diagnosis of tuberculous meningitis by a dot immunobinding assay to detect mycobacterial antigen in cerebrospinal fluid specimens(JOURNAL OF CLINICAL MICROBIOLOGY, 1999)In the present prospective study, a dot immunobinding assay (Dot-Iba) was standardized to measure the circulating mycobacterial antigen in cerebrospinal fluid (CSF) specimens for the laboratory diagnosis of tuberculous meningitis (TBM), Immunoglobulin G antibody specific for Mycobacterium tuberculosis in a CSF specimen from a patient with culture-proven TBM was isolated and was coupled with activated cyanogen bromide-Sepharose 4B. By immunosorbent affinity chromatography, a 14-kDa antigen was isolated from the culture filtrate of M, tuberculosis. Antibody to the 14-kDa mycobacterial antigen was raised in rabbits. The Dot-Iba in this study gave no false-positive results with CSF specimens from patients with nontuberculous neurological diseases. The assay gave positive results for all five patients with culture-proven TBM, The Dot-Iba described in the present report is simple, rapid, sensitive, specific, and, more importantly, suitable for routine application in laboratories in developing countries.Item Serum tumor necrosis factor-alpha in Guillain-Barre syndrome and its relation to plasma exchange(NEUROLOGIST, 2002)BACKGROUND- To correlate the serum tumor necrosis factor-alpha (TNFalpha) concentrations before, during and following plasma exchange in patients with Guillain-Barre syndrome (GBS). In this prospective study, 21 GBS patients were selected. Patients in clinical stages III to V were subjected to plasma exchange. The control group included equal numbers of age-matched patients with other neurological diseases and healthy voluntary blood donors. A sandwich ELISA method was applied to estimate serum TNFalpha concentrations in test and control groups.REVIEW SUMMARY-Twelve GBS patients had elevated serum TNFalpha levels that ranged between 74 and 182 pg/mL. All 12 GBS patients showed a steady decrease in the TNFalpha concentration following plasma exchange and also showed a positive correlation with neurological recovery.CONCLUSIONS- We conclude that serum TNFalpha concentrations are elevated in 57.1 % of GBS patients and TNFalpha level decreases following plasma exchange.