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Genome Equivalents (genome + equivalent)
Selected AbstractsFulminant hepatitis after allogenic bone marrow transplantation caused by reactivation of hepatitis B virus with gene mutations in the core promotor regionEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 3 2006Kiyoshi Kitano Abstract:, Under immunosuppressive conditions after hematopoietic stem cell transplantation (HSCT), even if hepatitis B virus (HBV) antigen is negative but hepatitis B surface antibody (HBsAb) or hepatitis B core antibody (HBcAb) is presented, HBV reactivates and sometimes causes fulminant hepatitis. However, it remains unclear which patients will develop fulminant hepatitis, or whether fulminant hepatitis is caused by host-related factors or by virus-related factors. A 30-yr-old man with a history of aplastic anemia since 3 yr of age underwent allogenic BMT, when HBsAb and HBcAb were positive but HBs antigen (HBsAg) was negative. The donor was negative for HBsAg, HBsAb and HBcAb. After transplantation, the patient was complicated by acute graft-vs.-host disease (GVHD), cytomegalovirus infection, intestinal thrombotic microangiopathy and aspergillus colitis. Chronic GVHD was well controlled by FK506 and prednisolone. Twenty months after transplantation, the patient was admitted with general fatigue and liver dysfunction and was found to be positive for HBsAg and HBeAg. His serum HBV-DNA level was >8.8 log of the genome equivalent (LGE)/mL. Therefore, he was diagnosed as having hepatitis B caused by HBV reactivation and 100 mg/d lamivudine treatment was started. However, jaundice and hepatic failure deteriorated and became fatal. On analysis of the HBV-DNA, two adjacent gene mutations in the core promoter region (T1762/A1764) were detected. Increased replication of the mutated HBV might have caused HBV reactivation which progressed to fulminant hepatitis. [source] smcL as a novel diagnostic marker for quantitative detection of Listeria ivanovii in biological samplesJOURNAL OF APPLIED MICROBIOLOGY, Issue 3 2010D. Rodríguez-Lázaro Abstract Aims:, To develop a novel molecular tool for the quantitative detection of the ruminant pathogen Listeria ivanovii in different biological matrices. Methods and Results:, A real-time PCR (RTi-PCR) for the quantitative and species-specific identification of L. ivanovii was designed to target the region of the smcL gene. The assay includes an internal amplification control (IAC) to avoid false-negative results. The smcL -IAC RTi-PCR assay was 100% selective and allowed the detection of as little as one genome equivalent in 45% of reactions. The quantification accuracy was excellent, as demonstrated by its high linearity (R2 > 0·9989) and PCR efficiency (E > 0·984) over a 6-log dynamic range, down to 10 genome equivalents. Finally, the applicability of this assay was evaluated with artificially contaminated biological matrices implicated in the transmission of this bacterium such as sheep raw milk, blood and amniotic fluid. The smcL -IAC RTi-PCR assay allowed the detection of as few as 50 colony forming unit numbers (CFUs) per 25 ml of raw milk, 43 CFUs per 1 ml of blood or 50 CFUs per 1 ml of amniotic fluid. Conclusions:, This method can be an adequate alternative for the identification of L. ivanovii and for complete diagnosis of animal and human listeriosis. Significance and Impact of the Study:, We present an alternative for the detection of another pathogenic member of Listeria genus, which can help to distinguish from Listeria monocytogenes and therefore facilitates the establishment of preventive and prophylactic measures in food and farm environments. [source] Time course of hepatitis A viremia and viral load in the blood of human hepatitis A patientsJOURNAL OF MEDICAL VIROLOGY, Issue 1 2004Andrea Normann Abstract The hepatitis A virus (HAV) is the most common etiological cause of acute hepatitis infections in humans in industrialized countries. Investigations into the viral load during HAV viremia, however, are rare. Therefore, correlation studies between viral load, biochemical, and specific serological markers have been undertaken. The group of sera comprised a series of multiple consecutive blood samples drawn from 11 patients at different times after onset of the disease. During the period up to 70 days after the onset of icterus, the individual range was at 1×103 to 3×104 HAV genome equivalents/ml. From day 75 until 120 after onset of the disease, the levels traced were at 103. In one case, it was possible to trace 1.25×104 genome equivalents/ml up to 180 days after onset of icterus and in two cases even up to 408 and 490 days viral load levels of 5×103 and 4×104 were detected, respectively. The same sera were used to measure IgM class antibodies to hepatitis A virus and the total anti-HAV. The results demonstrate that a direct correlation to peak levels of viral load exists with peak serum transaminase levels, but neither with peak anti-HAV IgM levels nor with total anti-HAV. Decreasing amounts of anti-HAV IgM tend to occur with decreasing amounts of HAV genome equivalents; and, vice versa, increasing amounts of total anti-HAV are accompanied by decreasing amounts of HAV genome equivalents. The longest duration of viremia was found in patients infected with HAV genotype IA. J. Med. Virol. 72:10,16, 2004. © 2004 Wiley-Liss, Inc. [source] Kinetics of HBV DNA and HBsAg in acute hepatitis B patients with and without coinfection by other hepatitis virusesJOURNAL OF MEDICAL VIROLOGY, Issue 3 2003Vladimir P. Chulanov Abstract The kinetics of hepatitis B virus (HBV) and its surface antigen (HBsAg) during acute hepatitis has not yet been studied accurately in a representative number of patients. The influence of coinfecting hepatitis viruses during the acute phase of infection is not known. Three to four serum samples from 21 patients with acute HBV monoinfection and 27 with coinfection were taken at intervals of 6,10 days and analyzed for the number of HBV genome equivalents (ge) by real time polymerase chain reaction (PCR) and for HBsAg quantity using Laurell electrophoresis. Log HBV ge/ml decreased during the follow-up from 6.8,±,1.1 to 5.1,±,1.0 to 4.2,±,0.8 to 3.3,±,1.1 (mean,±,SD). The half-life times of HBV ge increased from 1.6 days at the beginning to 4 days at the end. HBsAg decreased much slower: from 38 to 23 to 12 to3.8 ,g/ml. Half-life time was around 8 days at the beginning and 5.7 days at the end, but 11 patients showed a rapid elimination of HBsAg and HBV DNA. Hepatitis C virus (HCV) coinfection did not change the kinetics of HBV ge and HBsAg significantly. A moderate but significant suppression of HBV ge levels was observed in hepatitis D virus (HDV) coinfected patients. HBsAg levels were, however, enhanced in this cohort. In conclusion, the data suggest that expression and elimination of HBV is in most patients with acute hepatitis B not altered by coinfecting hepatitis viruses. The initial decrease of HBV ge and HBsAg in serum appears to be caused by decay or non-specific removal in the absence of replacement. J. Med. Virol. 69:313,323, 2003. © 2003 Wiley-Liss, Inc. [source] Parvovirus B19 infection in pregnancy: Quantitative viral DNA analysis using a kinetic fluorescence detection system (TaqMan PCR)JOURNAL OF MEDICAL VIROLOGY, Issue 2 2002Antje Knöll M.D. Abstract Human parvovirus B19 infections are common in the general population, and infection during pregnancy may cause hydrops fetalis and fetal death. To initiate adequate treatment, accurate laboratory diagnosis is essential. The most sensitive tests are nested PCR systems, but these assays provide semiquantitative results at best. A parvovirus B19 DNA assay was developed based on the real time TaqMan PCR. This method was calibrated on the basis of serial plasmid dilutions and tested with an international parvovirus B19 standard. The assay was capable of quantifying parvovirus B19 DNA from one to about 5,×,107 genome equivalents per reaction (corresponding to 100 to 5,×,109 genome equivalents per ml serum). Samples from 51 pregnant women with suspected acute parvovirus B19 infection were tested, and positive PCR results were obtained in at least one of the materials investigated in 41 cases. The median viral DNA load in maternal blood samples was 1.3,×,104 copies/ml (range 7.2,×,102,2.6,×,107). Maternal virus DNA concentration was not associated with the presence of maternal symptoms and/or fetal complications. As the stage of infection was not known in the majority of cases, our data do not exclude an association between peak levels of parvovirus B19 DNA and the development of complications. Maternal sera and corresponding fetal material were available for concurrent testing from 15 DNA-positive cases: in most fetal samples, viral DNA concentrations were several orders of magnitude higher (up to 2.1,×,1012 copies/ml) compared to the corresponding maternal blood samples. J. Med. Virol. 67:259,266, 2002. © 2002 Wiley-Liss, Inc. [source] Chronic hepatitis C infection: Influence of the viral load, genotypes, and GBV-C/HGV coinfection on the severity of the disease in a Brazilian populationJOURNAL OF MEDICAL VIROLOGY, Issue 1 2002Leila M.M.B. Pereira Abstract The distributions of the different genotypes of the hepatitis C virus (HCV) and GBV-C virus (GBV-C/HGV) vary geographically and information worldwide is still incomplete. In particular, there are few data on the distribution of genotypes (and their relationship to the severity of liver disease) in South America. Findings are described in 114 consecutive patients from Northeast Brazil (median age 52 years, range 18,72 years) who had abnormal levels of serum aminotransferases and seropositivity for HCV RNA. The patients were recruited from an outpatient clinic between November 1997 and April 1998. Quantitative HCV RNA and GBV-C/HGV RNA estimations were carried out by double-nested polymerase chain reaction (PCR) using primers from the 5,-untranslated regions (UTRs) of the genomes. HCV genotypes were determined by restriction fragment length polymorphism (RFLP) analysis with 5,-UTR primers and by PCR with type-specific 5,-UTR primers. GBV-C/HGV-RNA genotypes were determined by RFLP with specific 5,-UTR primers and phylogenetic trees were constructed using the Neighbour-Joining and Drawtree programs. Histological features were graded and staged according to international criteria. Of the 114 patients, 35 (30.7%) patients had cirrhosis and 22 (27.8%) had mild, 51 (64.6%) had moderate, and 6 (7.6%) had severe chronic hepatitis. Median HCV viral load was 106 genome equivalents per millilitre (range 104,109/ml). Frequencies of genotypes were 5.3% type 1a, 44.7% type 1b, 3.5% type 2, 41.2% type 3, and 5.3% mixed types. GBV-C/HGV-RNA was detected in the sera of 12 (10.5%) patients and was distributed among three phylogenetic groups. There were no significant differences between patients with the predominant HCV genotypes (1b and 3) with respect to gender, age group, viral load, severity of liver disease, or coinfection with GBV-C/HGV. J. Med. Virol. 67:27,32, 2002. © 2002 Wiley-Liss, Inc. [source] Smoking in pregnancy is associated with increased total maternal serum cell-free DNA levelsPRENATAL DIAGNOSIS, Issue 3 2008Adam C. Urato Abstract Objective Cell-free DNA is a marker of cellular apoptosis and necrosis. We wished to determine if maternal smoking affects maternal and fetal serum cell-free DNA levels. Methods Case,control sets of stored second-trimester serum-screening samples from 27 smoking and 90 nonsmoking pregnant women were developed. Smoking status was confirmed by measuring serum cotinine levels. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and DYS1 levels were determined using real-time polymerase chain reaction (PCR) to measure total and fetal cell-free DNA, respectively. At delivery, medical records were reviewed to confirm gender and determine other factors that could affect DNA values. Results Smoking was associated with significantly elevated GAPDH levels compared with nonsmokers (median: 97 662 genome equivalents (GE)/mL vs 38 217 GE/mL; p = 0.018). DYS1 levels were not statistically significantly elevated in smokers (p = 0.29). Other factors that affected DYS1 levels included maternal age in nonsmokers only (r2 = 0.30, p = 0.013) and maternal Synthroid use (p = 0.0045) Conclusion Pregnant smokers have threefold higher levels of total cell-free DNA compared with pregnant nonsmokers. Maternal age and Synthroid exposure may also affect circulating cell-free fetal DNA levels. Copyright © 2008 John Wiley & Sons, Ltd. [source] Construction and evaluation of a porcine bacterial artificial chromosome libraryANIMAL GENETICS, Issue 1 2000K Suzuki Summary A porcine bacterial artificial chromosome (BAC) library consisting of 103 488 clones has been constructed. The average insert size in the BAC vector was calculated to be 133 kb based on the examination of 189 randomly selected clones, indicating that the library contained 4.4 genome equivalents. The library can be screened by two-step PCR. The first screening step is performed on 22 superpools, each containing 4704 clones (49×96 well plates). In the second screening step, 49 plates comprising a superpool are arrayed in a 7×7 matrix and 4D-PCR is performed. Screening of the library superpools by PCR for 125 marker sequences selected from different regions of swine genome revealed 123 sequences, indicating that the library is not biased. Subsequent screenings (4D-PCR) were successfully applied for identification of clones containing each marker sequence. This porcine BAC library and the PCR screening system are useful for isolation of genomic DNA fragments containing desired sequences. [source] Molecular diagnosis of lyssaviruses and sequence comparison of Australian bat lyssavirus samplesAUSTRALIAN VETERINARY JOURNAL, Issue 7 2006AJ FOORD Objective To evaluate and implement molecular diagnostic tests for the detection of lyssaviruses in Australia. Design A published hemi-nested reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of all lyssavirus genotypes was modified to a fully nested RT-PCR format and compared with the original assay. TaqMan assays for the detection of Australian bat lyssavirus (ABLV) were compared with both the nested and hemi-nested RT-PCR assays. The sequences of RT-PCR products were determined to assess sequence variations of the target region (nucleocapsid gene) in samples of ABLV originating from different regions. Results The nested RT-PCR assay was highly analytically specific, and at least as analytically sensitive as the hemi-nested assay. The TaqMan assays were highly analytically specific and more analytically sensitive than either RT-PCR assay, with a detection level of approximately 10 genome equivalents per µl. Sequence of the first 544 nucleotides of the nucleocapsid protein coding sequence was obtained from all samples of ABLV received at Australian Animal Health Laboratory during the study period. Conclusion The nested RT-PCR provided a means for molecular diagnosis of all tested genotypes of lyssavirus including classical rabies virus and Australian bat lyssavirus. The published TaqMan assay proved to be superior to the RT-PCR assays for the detection of ABLV in terms of analytical sensitivity. The TaqMan assay would also be faster and cross contamination is less likely. Nucleotide sequence analyses of samples of ABLV from a wide geographical range in Australia demonstrated the conserved nature of this region of the genome and therefore the suitability of this region for molecular diagnosis. [source] | |||||||||||||