Low prevalence of hepatitis C viral infection in patients with non-alcoholic chronic liver disease in India.

To investigate the prevalence and profile of chronic liver disease due to hepatitis B (HBV) and C (HCV) infection in patients with non-alcoholic chronic liver disease in North India, 148 biopsy proven patients (73 with a history of transfusion and 75 non-transfused) were studied. Detection of hepatitis B included HBsAg, AntiHBc, and HBV DNA testing. Presence of HCV infection was investigated by EIA using second generation tests and confirmed by RIBA III and HCV RNA testing. Eighty three (56.1%) patients had cirrhosis related to hepatitis B, 13 (15.7%) of them had precore (HBeAg -ve, HBVDNA +ve) and 11 (13%) had surface (HBsAg-ve, IgM antiHBc-ve, HBVDNA +ve) mutation. Antibodies to HCV were found in 16 (10.8%) patients. Dual infection with HBV and HCV was seen in 20 (13.5%) patients. Twenty nine (19.5%) patients, had cryptogenic cirrhosis as none of the markers for HBV or HCV infection was positive. In conclusion, our results demonstrate that HBV was the most prevalent viral infection associated with chronic liver disease patients in North India. Prevalence of HCV infection was low. Studies to detect HBV mutants and other viruses should be done in patients with suspected cryptogenic cirrhosis of the liver.

Evaluation of PCR based test for the detection of Mycobacterium tuberculosis in coded sputum specimens.

A test based on amplification of 169 bp DNA specific to Mycobacterium tuberculosis was evaluated in a trial, on 50 coded samples which included 25 sputum specimens from radiologically, clinically and bacteriologically proven patients of pulmonary tuberculosis and 25 control specimens. At the end of the trial the code was broken and the results of PCR test were compared with those obtained with Ziehl Neelsen staining and culture test. The test appeared highly sensitive reacting 100 per cent in either of the hands with concordance rate of 76 per cent; 3 of 25 control samples gave false positive results.

Identification and characterization of mutations in 'X' region of a hepatitis B virus carrier.

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.

Polymerase chain reaction based diagnostic assay for identification of hepatitis B virus.

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.

Cloned single copy DNA sequences of Mycobacterium tuberculosis as DNA probes.

A recombinant genomic clone was isolated from a lambda gt 11 library of M. tuberculosis on the basis of lack of hybridization with M. avium and M. kansasi. The specificity and sensitivity of M. tb DNA probes, 2.5 and 2.3 kb in size, were assessed by Southern blot and dot blot hybridization. These did not cross hybridize to DNA of mycobacteria other than members of M. tb complex, nor with DNA of non mycobacterial origin. Sensitivity was determined to be 200 pg which is equivalent to 10(4) bacilli. Genomic Southern hybridization indicated single copy nature of the probes.

A bacteriophage M13 based sandwich hybridization assay for the detection of hepatitis B virus in human blood.

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.

Detection of hepatitis Β virus in blood samples negative for surface antigen, by DNA probe hybridization assay.

A DNA hybridization assay was developed using a cloned hepatitis Β viral genome to detect the presence of infectious virions in human serum. The merit of this assay was to put in evidence virus particles in 7 out of 133 sera that were negative for surface antigen (HBsAg) using routine serological methods. The usefulness of this assay was confirmed by actual visualization of the virus under electron microscope. Some serum samples although positive for surface antigen, did not give a hybridization signal by dot blot assay and might indicate cases of acute hepatitis.

Dipicolinic acid as a secondary metabolite in Penicillium citreoviride.

Synthesis of dipicolinic acid in Penicillium citreoviride showed typical kinetics of a secondary metabolite. Its synthesis resumed during idiophase and continued through stationary phase of growth. Total duration of synthesis was 100 h at the end of which its synthesis was arrested. Production of dipicolinic acid by the cells was subject to catabolite repression by glucose and was not subject to end product inhibition by exogenously added dipicolinic acid. Unlike the bacteria, dipicolinic acid synthesis in this mold was highly sensitive to inhibition by calcium ions in the growth medium. Calcium promoted sporulation but dipicolinic acid was not found to be present in detectable amounts in mold spores. Addition of dipicolinic acid and Ca2+ completely inhibited its de novo synthesis, an effect not observed when calcium was replaced by Mg2+ When the mold was grown in the presence of calcium alone, its inhibitory effects on de novo synthesis of dipicolinic acid were expressed only after some of this metabolite was first synthesised by the producer cells suggesting that the active feedback inhibitor is probably a Ca: dipicolinic acid complex. It is suggested that over-production of this metabolite is very important to the mold in increasing its survival potential in nature by retrieving the essential minerals from the environment through ligand: metal complex at a time when cells are in the process of dying, so that a proper mineral balance is maintained within the cells.