Multi-channel programmable power supply with temperature compensation for silicon sensors.

Silicon Photo-Multipliers (SiPMs) are increasingly becoming popular for discrete photon counting applications due to the wealth of advantages they offer over conventional photo-detectors such as photo-multiplier tubes and hybrid photo-diodes. SiPMs are used in variety of applications ranging from high energy physics and nuclear physics experiments to medical diagnostics. The gain of a SiPM is directly proportional to the difference between applied and breakdown voltage of the device. However, the breakdown voltage depends critically on the ambient temperature and has a large temperature co-efficient in the range of 40-60 mV/°C resulting in a typical gain variation of 3%-5%/°C [Dinu et al., in IEEE Nuclear Science Symposium, Medical Imaging Conference and 17th Room Temperature Semiconductor Detector Workshop (IEEE, 2010), p. 215]. We plan to use the SiPM as a replacement for PMT in the cosmic ray experiment (GRAPES-3) at Ooty [Gupta et al., Nucl. Instrum. Methods Phys. Res., Sect. A 540, 311 (2005)]. There the SiPMs will be operated in an outdoor environment subjected to temperature variation of about 15 °C over a day. A gain variation of more than 50% was observed for such large variations in the temperature. To stabilize the gain of the SiPM under such operating conditions, a low-cost, multi-channel programmable power supply (0-90 V) was designed that simultaneously provides the bias voltage to 16 SiPMs. The programmable power supply (PPS) was designed to automatically adjust the operating voltage for each channel with a built-in closed loop temperature feedback mechanism. The PPS provides bias voltage with a precision of 6 mV and measures the load current with a precision of 1 nA. Using this PPS, a gain stability of 0.5% for SiPM (Hamamatsu, S10931-050P) has been demonstrated over a wide temperature range of 15 °C. The design methodology of the PPS system, its validation, and the results of the tests carried out on the SiPM is presented in this article. The proposed design also has the capability of gain stabilization of devices with non-linear thermal response.

Identification of a novel surface mutant of hepatitis B virus in a seronegative chronic liver disease patient.

Hepatitis B virus (HBV) with mutations in the envelope proteins can emerge during natural infections, vaccinations or interferon therapy and appears occasionally to escape virus elimination or detection. The implications of such mutations at the molecular level are often obscure. We report the identification of a new surface mutant of HBV. This mutant was identified, and isolated from a chronic liver disease patient, negative for HBsAg as well as other serological markers but positive for HBV DNA. Several mutations were observed in the surface antigen gene out of which a Thr118-Ala118 change was predicted to have a destabilizing effect on the structural integrity of the 'a' determinant and also alter the antigenicity profile of the mutant HBsAg. Besides a RNA hairpin loop was predicted for the transcript generated by the small surface protein of this mutant, which could have an inhibitory effect at the translational level. These observations thus indicate that mutations in the surface gene could lead to a considerable decrease or complete absence of properly folded surface antigen which in turn could explain the absence of reactive HBsAg in the serum of the patient.

Post-transfusion hepatitis type B following multiple transfusions of HBsAg-negative blood.

BACKGROUND/AIMS: Post-transfusion hepatitis continues to occur, though with decreasing frequency, even after screening donor blood for HBsAg, anti-HBc, anti-HCV and alanine aminotransferase activity. Data from developing countries on the frequency and type of post-transfusion hepatitis are scarce. We undertook this prospective study to determine the incidence and type of post-transfusion hepatitis at our center after transfusion of blood negative for HBsAg by ELISA. METHODS: Forty-one patients undergoing open-heart surgery, who had received 3 or more units of HBsAg-negative blood, were followed up. Serum samples of donor units transfused to recipients who developed post-transfusion hepatitis-B were subjected to HBV DNA amplification by the polymerase chain reaction, using two sets of X-gene specific primers which amplified a 250-bp fragment of the HBV DNA. RESULTS: We found that six of the 41 patients (14.6%) developed post-transfusion hepatitis; four of them (66.6%) developed icteric post-transfusion hepatitis B, whereas two (33.3%) developed anicteric post-transfusion hepatitis C. These six recipients received a total of 48 units of blood and 30 of these 48 units could be subjected to HBV DNA amplification by polymerase chain reaction. Eleven donor samples were polymerase chain reaction positive and had been transfused to three of the four patients who had developed post-transfusion hepatitis B. CONCLUSIONS: We conclude that post-transfusion hepatitis B continues to be the most common cause of post-transfusion hepatitis in India. Screening of donor units for HBsAg by ELISA does not exclude all blood units infectious for hepatitis B virus. Additional measures to ensure safety of blood supply should be sought.

Frequency and clinical profile of precore and surface hepatitis B mutants in Asian-Indian patients with chronic liver disease.

BACKGROUND: Infection due to hepatitis B virus (HBV) could be due to wild or mutant (precore or surface) viruses. The prevalence and clinical profile of different viral forms in patients with chronic liver disease has not been established. METHODS: One hundred and twenty patients with histologically proven HBV-related chronic liver disease were studied. Patients with dual infection with HCV/HDV/HIV, past history of interferon therapy, or autoimmune hepatitis were excluded. Eighteen (15.5%) patients had the precore mutation (HBsAg +ve, HBeAg -ve/anti-HBe +ve, HBV DNA +ve), and 13 (10.8%) had the surface gene mutations (HBsAg -ve, HBeAg -ve, IgG anti-HBc, and HBV DNA +ve). The remaining 89 (74.2%) patients were infected with wild type HBV. The course of all patients with mutant forms and 41 of those with the wild type form was followed for a mean (+/- SD) of 4.4 +/- 2.4 yr. RESULTS: Compared with wild-type-infected patients, those with surface mutation were younger (39.9 +/- 14 vs. 30.1 +/- 12.4 yr, p < 0.05). Patients with precore mutations had a shorter illness than those with surface mutant (p < 0.01) and wild forms (p < 0.05). Histologically, patients with precore type had more active liver disease than wild type (39% vs. 15%, p < 0.05). Patients with precore mutations were always symptomatic, often presenting with ascites (67%) and jaundice (55%). Patients with surface mutant forms often presented with quiescent cirrhosis (77%) or cirrhosis with hepatoma (15%). CONCLUSIONS: One-fourth of HBV-related chronic liver disease in Asian Indians is attributable to mutant HBV forms. The presence of variant viruses alters the natural history of the disease, with the precore variance having a more aggressive course and the surface mutant, a more quiescent but unfavorable course, compared with the wild type.

Efficacy of low-dose alpha interferon therapy in HBV-related chronic liver disease in Asian Indians: a randomized controlled trial.

BACKGROUND/AIMS: Interferon therapy has been shown to be effective in Western patients with chronic hepatitis due to hepatitis B viral infection, but not in Asian Chinese. Its efficacy in Asian Indian subjects with chronic HBV infection is not known. METHODS: Forty-one patients with HBV-related chronic liver disease received randomly either: (a) recombinant alpha 2b interferon (n = 20) 3 MIU, subcutaneously, three times a week for 4 months, or (b) no treatment (n = 21). Patients were followed up for 12 months after completion of therapy. RESULTS: In the interferon-treated group, complete response (loss of HBV-DNA and HBeAg) was significantly higher than spontaneous clearance in the control group (50% vs. 4.8% p < 0.05). Seroconversion to anti-HBe was seen in 35% of the treated and 4.8% of the control group (p < 0.05) at 4 months; it was noticeably higher in patients with chronic hepatitis than in those with cirrhosis. In the responders, alanine aminotransferase levels nearly normalized. One year after interferon therapy, HBeAg and HBV-DNA clearance was observed in 65% of patients, with HBsAg clearance in 15%. Reactivation was not seen in any patient. Side-effects were transient and minimal. CONCLUSION: Low-dose recombinant alpha interferon therapy is quite effective and safe in Asian Indians with chronic liver disease due to hepatitis B infection.

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.

Hind III genomic polymorphism of the C3b receptor (CR1) in patients with SLE: low erythrocyte CR1 expression is an acquired phenomenon.

Expression of the human erythrocyte C3b receptor (CR1-CD35) and its Hind III RFLP was studied in a group of 37 patients with SLE, 15 consanguineous relatives of the patients and 48 healthy normal subjects. The CR1 number on erythrocytes was quantitated by ELISA using a mAb to CR1. Serum levels of complement proteins (C3, C4, C3d) and circulating immune complexes (CIC) were estimated simultaneously in controls and relatives. The patients were followed up during the course of the treatment. The CR1/erythrocyte (CR1/E) in patients were found to be significantly low in comparison to controls. The gene frequencies for the alleles H and L (7.4 and 6.9 kb Hind III restriction fragments) in the patients were 0.75 and 0.25, respectively, which did not differ significantly from the controls (0.77 and 0.23 in normal subjects and 0.79 and 0.21 in consanguineous relatives of the patients). However, patients expressed fewer CR1/E within each genotype than their relatives and healthy subjects. CR/E was found to be stable in consecutive samples in controls. In patients, the numbers varied between low and high during the course of the treatment. The variation in the numbers was significantly correlated with C3d and CIC as well as with the severity of the disease. Our results suggest that low levels of CR1 on erythrocytes in SLE patients are required during the course of the disease and that the 6.9 kb restriction fragment does not play a role in causing susceptibility to the disease.

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.

Molecular definition of unique species status of Mycobacterium w; a candidate leprosy vaccine strain.

Mycobacterium w, a candidate leprosy vaccine strain, is an atypical cultivable mycobacterium. Based on its growth and metabolic properties, M. w was listed in Runyon Group IV, along with other rapid growers such as M. fortuitum, M. smegmatis, M. chelonae and M. vaccae. However, M. w was not fully identical to any one of these. In the present study, a molecular biology approach was used to define the species identity of M. w in a manner that allows reliable comparison to be made with over 30 known mycobacterial species. A 383-bp region, present at the amino terminus of the conserved mycobacterial 65-kDa gene, has been polymerase chain reaction (PCR) amplified in M. w and DNA sequence was determined. A comparison of the M. w DNA sequence with those of M. tuberculosis, M. avium, M. paratuberculosis and M. fortuitum revealed a species-specific polymorphism, i.e., the presence of nucleotide substitutions unique to M. w. In an alternate approach, a 441-bp region, also a part of the 65-kDa gene, has been PCR amplified in M. w and a Hae III restriction pattern was generated.(ABSTRACT TRUNCATED AT 250 WORDS)

C3b receptor (CR1) genomic polymorphism in rheumatoid arthritis. Low receptor levels on erythrocytes are an acquired phenomenon.

The number of complement receptor 1 (CR1, CD35) molecules on erythrocytes is genetically determined by two codominant alleles. The numerical expression of CR1 on erythrocytes correlates with a HindIII-RFLP or CR1 gene using CR1-1, a complementary DNA probe. We have found low CR1 on erythrocytes in patients with rheumatoid arthritis (RA) in an Indian population. Low levels in RA patients may be acquired or genetically determined. Fifty-two patients with RA, 48 nonrelated healthy subjects and 19 consanguineous relatives of patients were genotyped. CR1 numbers on erythrocytes were quantitated by the enzyme-linked immunosorbent assay using monoclonal anti-CR1 antibody. Normal subjects and patients were followed up for a period of 6 months to evaluate the stability of their CR1 expression. The gene frequency for allele H and L (7.4- and 6.9-kb HindIII restriction fragment, respectively), which correlated with high and low expression of CR1 on erythrocytes was 0.77 and 0.23 in the normal controls. Gene frequency in RA patients was 0.78 and 0.22 for H and L allele, which did not differ significantly from either controls or relatives (0.80 and 0.20 for H and L allele, respectively). However, RA patients expressed fewer CR1 on erythrocytes within each genotype than their relatives and controls. CR1 on erythrocytes were found to be stable in consecutive samples in controls. In RA patients, the number varied between low and high during the course of the disease. The variation in number was significantly correlated (p < 0.05, r = -0.85 to -0.98) with disease activity as monitored by erythrocyte sedimentation rate. Our results suggest that low levels of CR1 on erythrocytes in patients with RA are not inherited, rather they are acquired during the course of the disease.

Molecular cloning and characterization of contiguously located repetitive and single copy DNA sequences of Mycobacterium tuberculosis: development of PCR-based diagnostic assay.

Screening of a lambda gt11 genomic library has been used as an approach for molecular cloning of the Mycobacterium tuberculosis repetitive DNA shown to be present on a previously described 5.6-kb Alu I genomic fragment. The recombinant clone R18.8.2, which strongly hybridized with the radiolabeled 5.7-kb Alu fragment, carried two Eco RI inserts of 2 kb and 1.4 kb in size. Southern hybridization of each of these fragments to restriction endonuclease-cleaved M. tuberculosis DNA clearly demonstrated the 2 kb to contain the repetitive DNA sequence, while the 1.4 kb is represented in a single copy. When replica plaque lifts from the genomic library were probed, the 5.6-kb genomic fragment and the cloned 2-kb repetitive insert hybridized to an identical number of plaques, indicating the similarity and the high copy number of the repeating unit shared by the two fragments. Restriction mapping and Southern hybridization patterns indicated that the 2-kb repetitive and the 1.4-kb single-copy DNA sequences originated from a contiguous piece of genomic DNA. Both fragments were found to be unique to members of the M. tuberculosis complex, except that the 2-kb insert exhibited a weak hybridization with M. kansasii DNA. Finally, a 169-bp region from one end of the single-copy sequence has been amplified by polymerase chain reaction (PCR) in a manner specific to members of the M. tuberculosis complex. The sensitivity of the PCR was such that as few as 9-10 bacilli could be detected.

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.

Potentials of Shigella flexneri Y strain TSF21 as a candidate vaccine against shigellosis: safety, immunogenicity and protective efficacy in Bonnet monkeys.

A thymine-requiring and temperature-sensitive mutant of Shigella flexneri Y was tested in Bonnet monkeys for safety, immunogenicity and protective efficacy. A dose of 10(11) cells when fed orally mimicked natural infection in having invaded epithelial cells, but was otherwise clinically non-reactogenic. Animals immunized with two oral doses, each dose consisting of 1 x 10(11) mutant bacteria, were fully protected when challenged, with respect to the lack of any clinical symptoms or detectable histological abnormalities in the intestinal mucosa. Unimmunized animals when similarly challenged developed frank dysentery and the intestinal mucosa showed severe histological abnormalities. Titres of serum antibodies increased by about 11-fold of the base level in animals immunized with a dose of 10(11) cells, but not with lower doses. The challenge bacteria appeared to be phagocytised by macrophages. In some monkeys of a particular group, congestive patches were seen in the stomach, but not in any other part of the gut, after the animals were fed with the virulent parent strain. The lesions were relatively severe in the immunized groups of animals.

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.

Repetitive DNA sequence of Mycobacterium tuberculosis: analysis of differential hybridization pattern with other mycobacteria.

In order to generate specific DNA probes for Mycobacterium tuberculosis, restriction fragment length analysis was carried out with some of the mycobacterial species that fall within the tuberculosis complex. The presence of specific bands of 5.6 kb and 4.8 kb was revealed in the AluI DNA digest of M. tuberculosis. The hybridization profile of the 5.6-kb AluI DNA sequence, as judged by the Southern blot and dot blot hybridization experiments, revealed the presence of this sequence in M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. bovis BCG as multiple copy and M. kansasii as single copy but this sequence was not present in M. avium, M. smegmatis, or M. vaccae genomes. Genomic clones corresponding to the 5.6-kb AluI fragment from M. tuberculosis H37Rv library made in the lambda gt11 expression vector were isolated.