Glycoprotein B Genotyping in Congenital/perinatal Cytomegalovirus Infection in Symptomatic Infants.

Background: Molecular epidemiological studies on circulating strains of CMV in cogenital/perinatal infections have not been done earlier in this region. Objective: To study the glycoprotein B genotypes in babies with symptomatic congenital/perinatal CMV infection and to assess the possible influence of genotype on the outcome of the infection. Methods: Clinical samples (blood and urine) of symptomatic babies are sent to the Virology Department of NCDC, Delhi for the diagnosis of congenital infections. 375 clinical samples of infants (newborn - 6 months old) were included for the study. Serum samples were subjected to ELISA for detection of IgM antibodies against CMV. DNA isolation and amplification of CMV genomic DNA targeting gB gene fragment by nested PCR, was carried out in the samples. The amplified fragment including the cleavage site was subjected to RFLP using restriction enzymes Rsal and Hinf1. They were also verified by sequencing using Big Dye Terminator chemistry. Results: 75 samples out of 375 tested were confirmed positive for CMV infection by serology and PCR. Both RFLP and sequencing of gB gene fragment showed that gB 1, 2 and 3 genotypes were in circulation. gB 3 was the most prevalent genotype in symptomatic infants. Hepatosplenomegaly was the most common feature in gB-3 genotype of CMV. gB2 congenital CMV infection was more commonly associated with long term sequelae.

Congenital CMV infection; diagnosis in symptomatic infants.

Background: Samples from babies exhibiting clinical symptoms suggestive of congenital infection are referred regularly to NICD, New Delhi,, from Government Hospitals located in Delhi and a home for abandoned children (Palna), for the diagnosis of etiological agents like toxoplasma, rubella, CMV and herpes. Blood samples of mothers of most of the affected babies are also received. Objective: Evaluation of rapid and accurate technique for the diagnosis of congenital CMV infection. Materials and Methods: One hundred and twenty five blood samples suggestive of symptomatic congenital CMV infection were selected from samples received at NICD during the period June 2005-March 2007. A request to collect and send the urine samples of the selected babies was sent to the respective hospitals. Serum samples of the babies were tested for CMV-IgM antibodies using µ-capture ELISA. Mothers' serum samples were subjected to CMV-IgM and IgG class antibodies assay by commercial ELISA kits. DNA isolation and amplification was performed in urine samples and some of the serum samples using a commercial PCR kit for detection of HCMV. Blood and urine samples from 20 normal babies were included in the study. Results: Twenty Seven serum samples (21.6%) of infants, of the 125 tested, were positive for CMV-IgM antibodies. Twenty five samples (20%) showed amplification of CMV -DNA. All 25 samples positive for PCR were positive for CMV IgM antibodies. Sera of 73 mothers, out of 75 tested (97.3%), were positive for CMV IgG antibodies. However, none of them was positive for CMV IgM antibodies. Mothers of all 27 positive babies were positive for CMV-IgG antibodies. Serum and urine samples from 20 normal babies were negative for ELISA and PCR. Conclusion: µ-capture ELISA technique was found to be more sensitive than PCR (92.6%) for detection of congenital CMV infection. ELISA is also rapid, less cumbersome and cost effective for diagnosis of CMV infection.

Histone as future drug target for malaria.

Malaria continues to be a major cause of mortality and morbidity in tropical countries and affecting around 100 countries of the world. As per WHO estimates, 300-500 million are being infected and 1-3 million deaths annually due to malaria. With the emerging knowledge about genome sequence of all the three counterparts involved in the disease of malaria, the parasite Plasmodium, vector Anopheles and host Homo sapien have helped the scientists to understand interactions between them. Simultaneous advancement in technology further improves the prospects to discover new targets for vaccines and drugs. Though the malaria vaccine is still far away in this situation there is need to develop a potent and affordable drug(s). Histones are the key protein of chromatin and play an important role in DNA packaging, replication and gene expression. They also show frequent post-translation modifications. The specific combinations of these posttranslational modifications are thought to alter chromatin structure by forming epigenetic bar codes that specify either transient or heritable patterns of genome function. Chromatin regulators and upstream pathways are therefore seen as promising targets for development of therapeutic drugs.

Occurrence & nucleotide sequence analysis of hepatitis G virus in patients with acute viral hepatitis & fulminant hepatitis.

BACKGROUND & OBJECTIVE: Association of hepatitis G virus (HGV) with acute viral hepatitis (AVH) and fulminant hepatitis (FH) is not clearly understood.This study was designed to asses the occurrence of HGV infection and its relationship with other hepatotropic viruses in patients with FH and AVH and also to determine the nucleotide sequence of HGV isolates. METHODS: The study included 100 patients of FH and 125 of AVH on the basis of clinical examination, liver function test and serology for hepatitis A, B, C and E virus. HGV RNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and direct sequencing for 4 randomly selected samples followed by phylogenetic analysis. RESULTS: Of the 100 patients with FH, 30 were negative for hepatitis viruses A, B, C and E by serology (non A-non E) while 60 were negative in the AVH group. In the non A-non-E hepatitis group, HGV was positive in 16.66 per cent (5/30) cases of FH, 10 per cent (6/60) cases of AVH and 6 per cent (6/100) of healthy controls. The difference in HGV seropositivity between FH and AVH patients was statistically not significant compared to healthy controls, while HBV and HCV infections were significant. The four isolates sequenced seemed to be of same type and close to Chinese strain of HGV (Y13755.1 Y13756.1 Y15407, and U67782) on phylogeny. INTERPRETATION & CONCLUSION: In HGV infection was not found to be clinically significant as well as nonpathogenic in the patients of FH and AVH and appeared to be an innocent bystander in the course of the disease. The four sequenced HGV isolates showed close pairing with Chinese strains.

Diagnostic potential of IS6110, 38kDa, 65kDa and 85B sequence-based polymerase chain reaction in the diagnosis of Mycobacterium tuberculosis in clinical samples.

PURPOSE: The correlation between the presence of specific gene sequence of M. tuberculosis and specific diagnosis of clinical tuberculosis is not known. This study compared the results of polymerase chain reaction (PCR) amplification of M. tuberculosis specific DNA sequences (IS6110, 65kDa, 38kDa and mRNA coding for 85B protein) from different clinical samples of pulmonary and extrapulmonary tuberculosis. METHODS: One hundred and seventy-two clinical samples from suspected tuberculosis patients were tested for smear examination, culture (LJ and rapid BACTEC 460 TB system) and PCR. PCR was performed with specific primers for the targets: IS6110, 65 kDa, 38 kDa and 85 B. RESULTS: Each PCR test was found to have a much higher positivity than conventional test and BACTEC culture (P < 0.05). Smear positive samples (56) and the samples (36) showing positive results by conventional methods (smear and LJ medium culture) and BACTEC were found to be positive by all PCR protocols. No significant difference was found between the four PCR protocols (P> 0.05). The primer specific for amplifying the 123bp IS6110 fragment gave the highest positivity (83%), followed by 65kDa, 38kDa and 85B RT-PCR in descending order. CONCLUSIONS: These data suggest that the presence of IS6110 correlates more closely with the diagnosis of clinical tuberculosis than that of 65kDa, 38kDa and 85B proteins.

Prompt laboratory diagnosis in timely containment of a plague outbreak in India.

A focal outbreak of pneumonic plague occurred in a hamlet of village Hatkoti, district Shimla, Himachal Pradesh in the first fortnight of February, 2002. A total of 16 cases with 4 deaths were reported. Diagnosis of plague was confirmed by the laboratory in 10 (63%) cases. Y. pestis was isolated from clinical samples of 3 cases and confirmed by bacteriophage lysis. Molecular tests confirmed the presence of Y. pestis specific pla and F1 genes in 4 cases; DNA fingerprinting had identity with the known sequence of plague bacilli. Paired samples from 5 cases showed more than 4 fold rise and 1 case showed more than 4 fold fall in antibodies against F1 antigen of Y. pestis. The present communication emphasises that timely and systematic laboratory investigations give confirmatory diagnosis in shortest possible time which forms the backbone of the outbreak control in a timely fashion and prevents confusion and controversy.

Protein antigen b (Pab) based PCR test in diagnosis of pulmonary and extra-pulmonary tuberculosis.

BACKGROUND AND OBJECTIVES: Diagnosis of tuberculosis (TB) is largely based on microscopy and culture examination which are either less sensitive, or time consuming. In the present study a PCR (polymerase chain reaction) test based on DNA sequence coding for a 38-kilodalton protein antigen b (Pab) ,specific for Mycobacterium tuberculosis was compared with Ziehl-Neelsen (ZN) stained AFB (acid fast bacilli) smear examination, culture based on conventional Lowenstein-Jensen (LJ) medium and radiometric BACTEC 460 system for the diagnosis of TB using clinical samples obtained from pulmonary and extra-pulmonary cases of TB. METHODS: Clinical samples obtained from 168 patients of suspected TB (pulmonary and extrapulmonary) were subjected to ZN smear examination, LJ culture, radiometric BACTEC culture and a PCR test by amplifying 419 bp sequence coding for Pab, a glycoprotein of molecular weight 38 kDa. RESULTS: A significant difference was seen in the sensitivity of different tests, the figures being 74.2 per cent for PCR test, 53.4 per cent for BACTEC culture, 47.1 per cent for LJ medium based culture and 35.2 per cent for ZN smear examination (P<0.05). However, there was no significant difference between different tests as far as specificity was concerned. PCR test sensitivity in pulmonary and extra-pulmonary clinical samples were 74.3 and 71.5 per cent respectively, being significantly higher (P<0.05) when compared with sensitivity of other tests. The mean detection time for M. tuberculosis was 24.0 days by LJ media culture, 12.8 days by BACTEC culture and less than 1 day by smear examination and PCR test. INTERPRETATION AND CONCLUSION: PCR test is more sensitive than ZN smear examination, LJ medium culture and BACTEC culture for diagnosing TB in pulmonary and extra-pulmonary clinical samples.

Molecular characterization of mutation associated with rifampicin and isoniazid resistance in Mycobacterium tuberculosis isolates.

Nucleotide changes in catalase peroxidase (Kat G) gene and gene encoding the beta subunit of RNA polymerase (rpo B), responsible for isoniazid and rifampicin drug resistance were determined in the clinical isolates of Mycobacterium tuberculosis by PCR-RFLP, Line probe assay and DNA sequencing. PCR-RFLP test was performed by HapII cleavage of an amplified fragment of Kat G gene to detect the transversion 315AGC-->ACC(Ser-->Thr) which is associated with INH drug resistance. The Line probe assay kit was evaluated to detect the mutation in 81bp RMP resistance determining region of rpo B gene associated with RMP drug resistance. These results were validated by DNA sequencing and drug susceptibility test. Kat G S 315 T mutation was found in 74.19% strains of M. tuberculosis from Delhi. This mutation was not found in any of the susceptible strains tested. The line probe assay kit and DNA sequencing identified 18 isolates as RMP resistant with specific mutation, while one of the RMP resistant strain was identified as RMP susceptible, with a concordance of 94.73% with the phenotypic drug susceptibility result. Majority (8 of 19, 42.1%) of resistant isolates involved base changes at codon 531 of rpo B gene. Both PCR-RFLP and Line probe assay test can be used in many of the clinical microbiology laboratories for early detection of isoniazid and rifampicin drug resistance in clinical isolates of M. tuberculosis.

Comparative study of PCR, smear examination and culture for diagnosis of cutaneous tuberculosis.

Diagnosis of tuberculosis (TB), especially cutaneous TB by conventional laboratory method is unreliable and time consuming. We assessed the utility of Polymerase Chain Reaction (PCR) test vis a vis other laboratory tests in 37 clinical samples of skin biopsy from equal number of patients with different variants of cutaneous TB. The PCR test amplifying 165bp region of 65kDa antigen coding gene specific for M. tuberculosis was performed on skin biopsy samples obtained from cases with a strong clinical evidence of cutaneous TB. The samples were also subjected to other laboratory tests e.g. smear examination, conventional (LJ based culture) and rapid BACTEC culture and histopathological examination for mycobacteria. Significant difference (p<0.05) was observed in the sensitivity of PCR test vis-a-vis other tests e.g. smear examination, LJ and BACTEC culture. PCR test showed a higher sensitivity than histopathological examination but the difference was not found to be statistically significant (p>0.05). PCR test showed the maximum positivity of 79.4% followed by histopathology (73.5%), BACTEC culture (47.5%), LJ media culture (29.4%) and smear examination (5.8%). The sensitivity and specificity of PCR test employing culture as the "gold standard" were 95.2% and 100%. The mean time taken for a positive result in different tests were less than 24 hours for smear examination, 1 day for PCR test, 23.42 days for BACTEC culture and 38.02 days for LJ culture. These results show that PCR amplification of 165bp region of 65kDa antigen coding gene of M. tuberculosis is a rapid and sensitive test for diagnosis of cutaneous TB using skin biopsy samples.

Comparison of the conventional diagnostic modalities, bactec culture and polymerase chain reaction test for diagnosis of tuberculosis.

PURPOSE: To evaluate the performance of 65 kDa antigen based PCR assay in clinical samples obtained from pulmonary and extrapulmonary cases of tuberculosis. METHODS: One hundred and fifty six samples were processed for detection of Mycobacterium tuberculosis by ZN smear examination, LJ medium culture, BACTEC radiometric culture and PCR tests. RESULTS: A significant difference was seen in the sensitivities of different tests, the figures being 74.4% for PCR test, 33.79% for ZN smear examination, 48.9% for LJ culture and 55.8% for BACTEC culture (P< 0.05). However, there was no significant difference (P>0.05) as far as specificity of different tests was concerned. PCR test sensitivity in pulmonary and extrapulmonary clinical samples were 72.7% and 75.9% respectively and found to be significantly higher (P< 0.05) when compared with those of other tests. The mean detection time for M.tuberculosis was 24.03 days by LJ medium culture, 12.89 days by BACTEC culture and less than one day by PCR test. CONCLUSIONS: PCR is a rapid and sensitive method for the early diagnosis of pulmonary and extrapulmonary tuberculosis.

An outbreak of human anthrax in Mysore (India).

Anthrax is a zoonotic disease caused by Bacillus anthracis. Intestinal anthrax though a rare entity mostly ends with fatal outcome. Very few cases of intestinal anthrax are reported. Present outbreak of intestinal anthrax is unique in itself that four cases succumbed to the illness within a span of 48-72 hours in a small hamlet of Mysore district of Karnataka, after consuming diseased deer meat. Confirmation of the diagnosis was carried out at NICD, Delhi by bacteriological culture isolation, biochemical tests, animal pathogenicity and polymerase chain reaction (PCR). This outbreak clearly indicates surveillance of anthrax in animals in endemic areas is an essential part in the control of the disease with intersectoral coordination between the departments of health, animal husbandary, agriculture and forest.

Antimicrobial susceptibility pattern and prevalence of extended spectrum beta-lactamase (ESBLs) producing strains of Klebsiella pneumoniae from a major hospital in New Delhi.

Extended spectrum beta-lactamases (ESBLs) are plasmid mediated enzymes capable of hydrolyzing penicillins, broad spectrum-cephalosporins and monobactams. The ESBL producing K. pneumoniae strains are being reported from around the world including India. The present study was taken up to evaluate the ESBL production and in-vitro susceptibility of K. pneumoniae isolates from a hospital. The bacterial isolates collected during 2003 included 51 K. pneumoniae biochemically confirmed isolates from 395 patients admitted in various wards of a major hospital in New Delhi. The isolates were from pus, wound, pleural fluid, urine and tracheal aspirate of patients attending respiratory, urology and burns wards. Antimicrobial susceptibility was carried out by Kirby Bauer's disc diffusion technique using NCCLS criteria. A screening of ESBL production was done by Double-disc synergy test (DDST) and using E-test ESBL strips. The frequency of resistance among K. pneumoniae for the cephalosporins (cefoxitin, cefuroxime, cefotaxime, ceftazidime, and cefepime) and non-cephalosporins (aztreonam, piperacillin, chloramphenicol and trimethoprim-sulfamethoxazole) were in the range of 39.2-88.0% and 51.0-90.2% respectively. 14 different antimicrobial resistance profiles were recognized ranging from resistance to only four (n=6, 11.7%) to as many as ten (n=9, 17.7%). Among the 51 isolates of K. pneumoniae strains, a total of 36 (70.6%) could be identified as ESBL producers, that correlates with the high frequency of multi-drug resistant K. pneumoniae The study shows alarming rise in ESBL production among K. pneumoniae strains and high rate of resistance to a wide range of cephalosporin and non-cephalosporin group of antimicrobials.

Relationship of Xba1 and EcoR1 polymorphisms of apolipoprotein-B gene to dyslipidemia and obesity in Asian Indians in North India.

BACKGROUND: Genetic investigation of dyslipidemia and obesity prevalent in the Indian population form the basis of this study. METHODS AND RESULTS: The frequency of restriction fragment length polymorphisms (Xba1 and EcoR1) of the apolipoprotein-B gene was investigated in a case-control study of 30 hyperlipidemic and 40 normolipidemic subjects. By univariate analysis, old age, higher body mass index, waist-hip ratio and sum of four skinfolds were found to be significantly associated with hyperlipidemia. The frequencies of X- and E+ alleles of the apolipoprotein-B gene were significantly higher in North Indians in the state of New Delhi (0.83 and 0.91, respectively) as compared to the observations made in Caucasians in previous studies, but was similar to the frequency reported in Indians settled in Singapore and the UK. There were no significant differences in the allele or genotype frequencies of either Xba1 or EcoR1 polymorphisms between the hyperlipidemic and normolipidemic groups. On multiple logistic regression analysis considering body mass index, waist-hip ratio, percentage body fat and genotypes as independent variables, no association was observed between the apolipoprotein-B genotypes and serum lipid components. Further, there were no associations between apolipoprotein-B polymorphisms and generalized obesity (as assessed by body mass index, sum of four skinfolds, and percentage total body fat) and abdominal obesity (as measured by waist circumference and waist-hip ratio). CONCLUSIONS: We conclude that apolipoprotein-B (Xba1 and EcoR1) polymorphisms do not appear to influence serum lipid levels and parameters of generalized andregional obesity in the study sample.

A comparative evaluation of immunotoxicity of malathion after subchronic exposure in experimental animals.

The effects of sub-chronic doses of malathion exposure on humoral and cell-mediated immune (CMI) responses were studied in male albino mice, rats and rabbits using sheep red blood cells (SRBC), tetanus toxoid and ovalbumin as antigens. The humoral immune response was assessed by estimating serum immunoglobulin (IgM and IgG) concentrations, antibody titre against antigens and splenic-plaque forming cells (PFC). The CMI response was studied by using the leucocyte migration inhibition (LMI) and macrophage migration inhibition (MMI) tests. In general there were (a) attenuation in antigen induced antibody response, (b) suppression of PFC, and (c) marked inhibition of LMI and MMI factors. Sub-chronic malathion exposure induced differential degrees of humoral and CMI suppression in these experimental animals. However, both cellular and humoral immune responses were decreased in a dose-time dependent pattern and a consistent trend was observed. The threshold level of the malathion for inducing immune suppression depends on the animal species, type of antigen used, and the method of immunological assay. In view of the widespread use of malathion a comparative assessment of immune responses using different experimental animals and antigens is an important aspect of its safety evaluation.

Influence of subchronic exposure to lindane on humoral immunity in mice.

Lindane suppressed both primary and secondary antibody responses to sheep red blood cell (SRBC) in albino mice, the effects being more pronounced on the secondary than the primary response. However, a longer duration of pesticide exposure induced similar degrees of immunosuppression on both responses. The sequential study of plaque forming cells (PFC) kinetics revealed that suppression of plaque formation not only occurred at peak days but also on pre and post peak days, and there was no delay in peak antibody formation. Moreover, reduction in the primary PFC was not associated with decrease in the antibody response to SRBC. The results indicate that lindane suppresses both primary and secondary humoral immune responses in a time and dose dependent manner, and suggest a threshold susceptibility to exposure.

A comparative evaluation of immunotoxicity of DDT and its metabolites in rats.

Effects of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) and its metabolites, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethene (DDE), 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (DDD) and 2,2-bis(p-chlorophenyl) acetic acid (DDA) were comparatively evaluated on humoral and cell mediated immune (CMI) responses in rats. Rats were given a diet containing 200 ppm of the various test compounds for 6 weeks and were subsequently immunized with ovalbumin. DDT, DDE and DDD, all induced differential degrees of humoral and cellular immune suppression. There were (a) increases in albumin/globulin ratios, (b) suppression of IgM and IgG levels, and (c) attenuations in ovalbumin induced antibody responses. In CMI studies, there were marked inhibitions of (a) leucocyte and macrophage migration factors, and (b) delayed type hypersensitivity (DTH) reaction. Whereas, these effects were most marked with DDE and DDD, DDA did not elicit such immunomodulatory effects. It is inferred that suppression of immune responses by immediate DDT metabolites, DDE (and DDD and not DDA) is an important determinant of the toxicity of DDT (DDE > DDD > DDT) and the influence of this environmental pollutant in health and disease.

Resolution of enzyme bands of Plasmodium knowlesi as a function of substrate concentration.

An extra band of isoenzyme lactate dehydrogenase (LDH) was obtained in case of Plasmodium knowlesi free parasite as compared to normal monkey blood. This extra band could be resolved due to the decreasing amount of substrate concentration. Agarose electrophoresis technique was used to separate the isoenzyme bands.