Does hepatitis E viral load and genotypes influence the final outcome of acute liver failure during pregnancy?

BACKGROUND: Hepatitis E is a major health problem in developing countries including India. The incidence and mortality rate in pregnant women with fulminant hepatic failure (FHF) due to hepatitis E virus (HEV) has been reported to be significantly higher, specifically in Asian women. Pregnancy is usually associated with an altered status of sex steroid hormones and immunity. Steroid hormones directly influence the replication through their effects on viral regulatory elements. Moreover, pregnant women in Asia generally suffer from folate deficiency, which is known to cause reduced immunocompetence leading to greater risk of multiple viral infections and higher viral load. OBJECTIVES: To correlate and analyze the viral load and genotypes of HEV in acute liver failure with that of acute viral hepatitis among pregnant and nonpregnant women. MATERIALS AND METHODS: A total of 100 FHF and 150 acute viral hepatitis (AVH) patients (50, 75 pregnant and 50, 75 nonpregnant, respectively), were included in the study. These cases were evaluated on the basis of history, clinical examination, liver function profile, and serological test of hepatitis A, B, C, and E using commercially available ELISA kits. Quantification of HEV RNA-positive samples was carried out. RESULTS: Out of 100 FHF and 150 acute viral hepatitis (AVH) patients, 28 (56%) and 22 (29.3%) pregnant and 7 (14%) and 8 (16%) nonpregnant, respectively, were HEV RNA-positive. HEV viral load in FHF pregnant women was 5.87 x 10(4)+/- 1.5 x 10(5) microL/mL as compared to AVH pregnant women 343.29 +/- 216.44 microL/mL and FHF and AVH nonpregnant 199.2 +/- 225.5 microL/mL and 13.83 +/- 7.8 microL/mL, respectively. Sequencing data of all the positive samples of FHF and AVH pregnant and nonpregnant women showed genotype 1. CONCLUSION: HEV viral load was found to be significantly higher (P < 0.05) in pregnant patients compared to the nonpregnant. Pregnancy appears to be a risk factor for viral replication. The viral copies of HEV in FHF pregnant women were comparatively higher when compared to AVH pregnant women, which may be related to the severity of the disease in these patients. We could detect only one genotype (genotype 1) in our study population. Thus in the absence of other genotypes in this population, the impact of genotype could not be adequately assessed in this study.

Emergence of an independent lineage of dengue virus type 1 (DENV-1) and its co-circulation with predominant DENV-3 during the 2006 dengue fever outbreak in Delhi.

OBJECTIVES: The sudden emergence of dengue virus type 1 (DENV-1) and its co-circulation with predominant DENV-3 was the hallmark of the 2006 dengue fever outbreak in Delhi. Viruses that circulated between 1996 and 2005 in the City have been well characterized, but the genomic diversity in 2006 strains is not known. The present study was undertaken to reveal the emerging molecular genotype(s) and evolutionary trend of the viruses responsible for the dengue fever outbreak in Delhi during 2006. STUDY DESIGN: The CprM gene junction of the DENV isolates from the 2006 Delhi dengue fever outbreak were subjected to nucleotide sequencing. Comparative phylogenetic analysis was done using DENV-1 and DENV-3 sequences retrieved from the global database. RESULTS: Multiple sequence alignment revealed only substitutions, with no insertions or deletions. A dendrogram indicated emergence of a distinct lineage of DENV-1 (having similarity with the Comoros/Singapore 1993 and Delhi 1982 strains, but quite different from the Delhi 2005 lineage) and microevolution of the pre-circulating DENV-3. These findings point towards the circulation of two independent lineages of DENV-1 in Delhi during 2005 and 2006. CONCLUSIONS: It is feared that the introduction of an independent lineage of the outbreak-associated strain of DENV-1 and its co-circulation with the deeply-rooted strain of DENV-3 in Delhi may result in yet another, possibly more severe outbreak in the near future.

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.

Detection of M. tuberculosis in clinical samples of diversified nature by IS6110 based PCR.

Performance of the polymerase chain reaction technique based on IS6110 sequence was evaluated in clinical samples obtained from pulmonary and extrapulmonary cases of tuberculosis. One hundred and seventy two samples were processed for detection of M. tuberculosis by ZN stained smear examination, LJ medium culture, BACTEC radiometric culture and PCR tests amplifying 123bp region of IS6110 sequence. A significant difference was seen in the sensitivities of different tests, the figures being 83% for PCR test, 35.2% for smear examination, 47.16% for LJ culture and 53.45% for BACTEC culture (p < 0.05). However, no significant difference was found as far as specificity was concerned. PCR test sensitivity in. pulmonary and extrapulmonary clinical samples were 90.14% and 77.27% 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. PCR based on IS6100 sequence is highly sensitive method for the early diagnosis of pulmonary and extrapulmonary tuberculosis.

Does mutation of hepatitis A virus exist in North India?

BACKGROUND: Human hepatitis A, a widespread infectious disease that is hyperendemic in vast areas of the world, results in the infection of the liver. Different human HAV strains of diverse geographic origin are remarkably closely related. HAV exploits all known mechanisms of genetic variation to ensure survival, including mutation and genetic recombination. OBJECTIVES: The aim of the study was to undertake an in-depth analysis of the mutation in three groups: (i) mild acute hepatitis (m-AH), (ii) severe acute hepatitis (s-AH), and (iii) fulminant hepatitis (FHF) A patients, who were tested positive for HAV RNA. MATERIALS AND METHODS: A total of 500 patients of acute viral hepatitis (AVH) were screened for HAV-IgM positivity from January 2003 to December 2004. HAV RNA positivity was subject to reverse transcription of RNA followed by polymerase chain reaction (RT-PCR) for the detection of HAV RNA. The HAV RNA positive cases were subject to single-stranded conformational polymorphism (SSCP). RESULTS: Out of 500 acute cases of hepatitis, 80 (16%) were positive for HAV-IgM. HAV RNA was detected in 34 (42.5%) cases by RT-PCR. Twenty-four (70.5%) were m-AH, seven (20.5%) were s-AH, and three (8.8%) were FHF. All the positive samples were subject to SSCP. No mobility shift was observed with respect to any screened samples by PCR-SSCP. Four (m-AHI-54, m-AHI-80, s-AHI-341 and FHFI-195 suspected cases were directly sequenced to prove that there was no point mutation. CONCLUSION: SSCP demonstrates no mobility shift in the VP1/P2A region of the HAV genome. No point mutation was observed in the four suspected cases by sequencing. However a large study from different geographical locations is needed to achieve a logical conclusion about the existence of HAV mutation in the Indian population.

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.

Detection of M. tuberculosis in clinical samples of diversified nature by IS6110 based PCR.

Performance of the polymerase chain reaction technique based on IS6110 sequence was evaluated in clinical samples obtained from pulmonary and extrapulmonary cases of tuberculosis. One hundred and seventy two samples were processed for detection of M. tuberculosis by ZN stained smear examination, LJ medium culture, BACTEC radiometric culture and PCR tests amplifying 123bp region of IS6110 sequence. A significant difference was seen in the sensitivities of different tests, the figures being 83% for PCR test, 35.2% for smear examination, 47.16% for LJ culture and 53.45% for BACTEC culture (p < 0.05). However, no significant difference was found as far as specificity was concerned. PCR test sensitivity in. pulmonary and extrapulmonary clinical samples were 90.14% and 77.27% 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. PCR based on IS6100 sequence is highly sensitive method for the early diagnosis of pulmonary and extrapulmonary tuberculosis.