Diagnostics for the tropical countries.
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Biomedical subjects
Publications and source records attributed to K Banerjee.
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A defective form of Hepatitis B virus (HBV) was identified in an apparently healthy voluntary blood donor, who was positive for the presence of HBV by dot blot hybridization, but did not have any serological markers of HBV infection. Two regions, part of X and part of surface antigen genes, were amplified by polymerase chain reaction, cloned and sequenced by Sanger's dideoxy chain termination method. The base sequence analysis revealed that the HBV mutant belonged to ayw serotype and showed three point mutations, in the form of deletions at nucleotides number 1402, 1438 and 1450. Such mutations in the 'X' region, and their likely presence elsewhere, could explain altered antigenic expression.
The immunogenicity of three different commercially available, well established Hepatitis B vaccines was determined. Three groups of high risk individuals were administered the vaccine, viz, Albugam (20 micrograms), Engerix-B (20 micrograms) and vaccine produced by Cheil Sugar & Co. (3 micrograms) respectively in a schedule of 3 doses as recommended by the manufacturers. A fourth group was administered a combination of the Albugam (2 doses) and Engerix-B (1 dose) vaccine. The sera collected 2 months after the 3rd dose showed seroconversion rates to be 100 percent and 95.34 percent respectively in the group which received the Albugam and Engerix-B vaccines respectively. The group which received a combination of both, the seropositivity rate was also 100 percent. The Chiel Sugar vaccine gave a seroresponse of only 52.38 percent. Geometric mean titres among the groups which received Albugam vaccine (501.30) were comparable to those receiving the combination of Engerix-B and Albugam (442.28). Those who received all 3 doses of Engerix-B vaccine showed a significantly lower GMT as compared to the above tow groups (GMT--43.14, p < .001, P < .001 respectively). The GMT in the group that received Cheil Sugar Vaccine was 3.81. Seroresponse was found to be inversely proportionate to age.
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An immunoglobulin G (IgG2b) class of monoclonal antibody (MoAb, NHA-1) raised against Japanese encephalitis virus (JEV) E glycoprotein, reacted with the viral antigen expressed in cytoplasm of the infected cells and also with the cell nuclei, by an indirect fluorescent antibody technique (FA). The NHA-1 reactivity to nuclei was found to be due to its recognizing a JEV cross-reactive epitope present on the nuclear histones. Adsorption with calf thymus histones (type II-AS) showed a drop in NHA-1 reactivity to both JEV and histones by an enzyme-linked immunosorbent assay (ELISA) and indirect FA; the drop was higher against the histones. The MoAb recognized specifically the viral antigens expressed on the infected porcine kidney cell surface by a modified indirect FA. ELISA carried out with glutaraldehyde-fixed antigens showed an almost 2-fold increase in the reactivity over unfixed JEV antigen but none for the histones. Thus, the results indicate that histones share a sequential homology with E glycoprotein of JEV, which might lead to an autoimmune disorder induced due to the molecular mimicry between these two antigens.
As beta 2 microglobulin (B2M) has been found to be elevated in immunological disorders and also in HIV infection, its levels were studied in 475 HIV seropositive, asymptomatic persons; 101 HIV seronegative persons from high risk groups for HIV and 99 healthy controls. The B2M levels in asymptomatic HIV seropositives are found to be significantly higher than healthy controls (1.0 mg/1 to 2.7 mg/1, P less than 0.001) and HIV seronegatives from high risk groups (1.1 mg/1 to 2.7 mg/1, P less than 0.001). Two hundred and thirty four (49.26%) seropositives showed increased levels of serum B2M. Thus, quantitative analysis of B2M may be useful as an early nonspecific marker of HIV infection and immune dysfunction. The prognostic value of B2M was assessed in a follow up study of 54 HIV seropositives in a 2 yr period. Within this period, B2M levels were found to be significantly increased in these subjects (1.2 mg/1 to 4.6 mg/1, P less than 0.001). Three of the subjects who showed high increase in the B2M levels, progressed to AIDS-related condition, whereas one progressed to persistent generalised lymphadenopathy. Thus, the rising levels of B2M appears to correlate well with disease progression.
A highly sensitive polymerase chain reaction (PCR)-based diagnostic assay was developed to detect the presence of hepatitis B virus (HBV) in human serum. The assay involved amplification of HBV DNA sequences using primers specific to HBV. This in vitro enzymatic amplification technique, when used in combination with molecular hybridization assay can detect HBV genome in the patient's serum, with a higher degree of sensitivity than achieved with dot blot assay. The assay can identify samples containing 3-10 virus particles.
Five hundred and twenty seven children between 7 months and 2 years of age were vaccinated with measles vaccine manufactured by the Serum Institute of India. The sero-conversion rate in children who had no antibodies previous to vaccination was 98.4% as tested in HI. Ninety per cent of children who had pre-vaccination measles antibodies showed a two-fold or more rise in HI antibodies. The side reactions of the vaccine were negligible.
An epidemic of viral hepatitis occurred at Rewa district of Madhya Pradesh during December 1989 through April 1990. A total of 302 cases were admitted to Gandhi Memorial Hospital. Few cases were reported from the adjacent rural areas. Twenty six of the 40 wards of the city were affected and 7 wards were most affected with attack rates ranging from 3-7 per cent. Seventy one per cent of the hospitalised cases were 15 to 35 years of age. Males constituted 72.2% of the cases. In 37 patients (12.2%) the illness had a fatal outcome. The cases fatality rate was 7.9% in males and 20.6% in females. There were leakages in water supply pipe lines at many places which ran parallel to or were laid across open gutters. The source of infection appeared to be water contaminated by sewage. Results of serological tests indicated a non-A, non-B hepatitis viral aetiology of the epidemic.
Administrations of hepatotoxicants namely carbon tetrachloride (CCl4:0.4 ml in 1.2 ml of liquid paraffin) and ANIT (1 ml of 1.5% solution in liquid paraffin) in Charles foster rats (force fed) and D-galactosamine (8 mg in water per swiss albino mouse, ip) induce the release of TNF-alpha in case of CCl4 and D-galactosamine. High TNF-alpha level was observed up to 48 hr in CCl4 and up to 24 hr in D-galactosamine treated animals. Elevated levels of biochemical like ALP and SGPT are also recorded. TNF-alpha level can be measured of tissue damage and prognosis in case of hepatitis.
Two hundred serum samples were tested to detect the presence of HIV-2 infection in Maharashtra state. The serum samples were derived from various groups including those with high risk behaviour for HIV infection. All samples were tested by three combined HIV-1 and HIV-2 ELISA kits. The reactivity was confirmed by LiaTek HIV 1+2 immunoblot. The study confirmed that HIV-2 infection exists in Maharashtra, as 14 samples showed antibodies to HIV-2 and 14 showed antibodies to both HIV-1 and HIV-2. Antibodies to HIV-2 or HIV-1 and HIV-2 were detected mainly in persons with high risk behaviour.
A two step hybridization procedure was developed to detect the presence of hepatitis B virus in blood samples using bacteriophage M13 radiolabelled DNA as probe. During the first step of hybridization, single-stranded bacteriophage M13 tg 130 DNA, with 3.2 kb HBV DNA cloned into it, was hybridized to target HBV DNA immobilized on nitrocellulose membrane filter. In the second step of hybridization, M13 DNA annealed to HBV target is detected with the help of double stranded form of M13 DNA. The assay offers minimum 4- to 6-fold higher sensitivity in comparison to single-step conventional hybridization assays. Additionally M13 DNA offers itself as universal probe.
During the Japanese encephalitis (JE) epidemic in 1988 at Gorakhpur, Uttar Pradesh, 34 cerebrospinal fluid (CSF) samples with 16 matching sera from 34 anti JEV IgM positive (confirmed JE) and 24 CSF samples with 4 matching sera from 24 anti JEV IgM negative (clinical encephalitis) patients were collected and tested for presence of JEV specific IgG by ELISA. Eighteen CSF samples and 8 matching sera from confirmed JE and 5 CSF samples and one matching serum from clinical encephalitis patients positive for JEV specific IgG were further assayed for subclass specificity using specific murine monoclonal antibodies. Almost all the samples exhibited IgG1 as the virus specific subclass. In addition to IgG1, one serum and one CSF sample each from two different confirmed JE patients showed the presence of virus specific IgG4 and IgG3 respectively. Half of the confirmed JE and clinical encephalitis patients exhibited intrathecal synthesis as evident from either elevated IgG index or CSF IgG/CSF albumin ratio. Most of the patients who recovered had predominantly virus specific IgG1 in CSF. It seems likely that IgG1 might have a protective role in clearance of virus from the central nervous system.
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Sera from healthy donors and four groups of subjects with acute viral hepatitis were tested for anti-HAV IgM by ELISA with hepatitis A virus antigen grown in tissue culture. The results were compared with those obtained by formalin inactivated HAV from 'HAVAB-M' test kit (Abbott Laboratories, USA). With both preparations of antigens, sera from healthy donors and patients suffering from acute hepatitis B or non-A, non-B did not show anti-HAV IgM antibodies whereas acute phase sera from hepatitis A patients showed strong reactions indicating the presence of anti-HAV IgM antibodies. The results indicate similarity in the specificity of both preparations of HAV against anti-HAV antibodies and encourage the use of tissue culture derived HAV for serological diagnosis of hepatitis A.
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The components of the circulating immune complexes (CICs) were characterised in asymptomatic HIV seropositive individuals. Forty four of 214 individuals (20.56%) showed the presence of CICs. Specific HIV anti-HIV CICs were detected in 33 of them (75%). The isotypic specificity of antibodies found to these CICs was measured. IgG and IgA immunoglobulin classes were detected in CICs.