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At least 217 records · Page 12Linked to original sources
PubMed1996

Factors influencing human immunodeficiency virus type 1 transmission by blood transfusion. Transfusion Safety Study Group.

One hundred thirty-two recipients of blood components that retrospectively tested positive for antibody to human immunodeficiency virus type 1 (anti-HIV-1) were identified. Fourteen (11%) remained seronegative throughout follow-up. Donor and recipient characteristics that could have influenced transmission were examined. Attributes did not differ for infected and uninfected recipients. Peripheral blood mononuclear cells (PBMC) from uninfected recipients were HIV-1-negative by DNA amplification and culture but were susceptible to in vitro infection. Transmitting and nontransmitting donors at donation differed only for HIV-1 RNA positivity. By immunocapture reverse transcriptase-polymerase chain reaction, 6 of 11 transmitters and 0 of 11 nontransmitters tested RNA-positive (P = .02). A more sensitive quantitative RNA assay detected RNA in all donation sera, but median levels were higher in transmitting than nontransmitting sera (P = .01). Median CD4 cell counts were lower for transmitting than nontransmitting donors at enrollment (P = .02). Level of viremia is an important determinant of HIV infection by blood transfusion.

Acquired Immunodeficiency Syndrome↗
PubMed1999

High Prevalence of Hepatitis C Virus Infection in Chronic Hemodialysis Patients.

Hepatitis C virus (HCV) infection is very common among hemodialysis (HD) patients. Transmission of infection in this setting has been related to the number of blood transfusions, the duration of hemodialysis and to nosocomial transmission of virus in the dialysis unit. We conducted a study of 74 HD patients to determine the frequency of HCV at a single point in time (cross-sectional analysis), and to evaluate the association between HCV infection and patients' demographic, clinical and biochemical features. Serum samples were tested for anti-HCV antibodies using a third-generation enzyme-linked immunosorbent assay (ELISA). In the case of a positive result, third-generation recombinant immunoblot and HCV RNA detection by polymerase chain reaction (PCR) tests were performed. Collected data included the patient's age, gender, time on HD, number of blood transfusions and serum alanine aminotransferase (ALT) activity. Twenty-nine patients (29/74.4%) were found to be HCV positive using a third-generation ELISA assay. Of these 29 patients, 27 were also positive by recombinant immunoblot assay and 2 patients had indeterminate results. In the anti-HCV ELISA-positive subgroup, 20 (69%) of the 29 patients had detectable HCV RNA. The HCV RNA-positive patients had received more blood transfusions (15&plusm;3 vs. 5&plusm;1 units of packed red blood cells, p<0.0001) and had been on HD for a longer period of time than the HCV RNA-negative patients (65&plusm;32 vs. 32&plusm;29 months, p<0.0001). Mean serum ALT levels were significantly higher in the HCV RNA positive group (30&plusm;18 vs. 15&plusm;9, p<0.0001). We were unable to determine the most likely mode of transmission in our unit, but these results emphasize the need for strict adherence to blood collecting and handling precautions, careful attention to hygiene in the dialysis units, and sterilization of dialysis machines in order to properly combat this frequent infection.

Journal Article↗
PubMed1994

Isolation and purification of U7 and snRNP particles.

A procedure was developed for the isolation of U7 small nuclear ribonucleoprotein particles involved in the processing of histone pre-mRNAs. U7 snRNP particles were fractionated from the rest of the snRNPs by means of a series of gel filtration and affinity chromatography steps. The isolated assembly of U7 snRNP particles appeared to be intact and pure as judged by gel electrophoresis of their RNA. No detectable RNA species were monitored other than U7 RNA.

Cell Fractionation↗
PubMed1991

Nonradioactive, sequence-specific detection of RNA in situ by primed in situ labeling (PRINS).

A new method for the detection of RNA in situ is presented. It is based on sequence-dependent annealing of unlabeled specific oligonucleotide primers to intracellular RNA and subsequent chain elongation catalyzed by reverse transcriptase. Under the conditions described, biotin-labeled nucleotides can be incorporated and the cDNA synthesized in situ can thus be detected using fluorescein-conjugated avidin. Compared to traditional in situ hybridization the use of short oligonucleotide primers has the potential advantage of being better to discriminate between closely related RNA transcripts. Compared to in situ transcription with radioactive precursors we find it more attractive to use fluorescein-conjugated avidin as detection system because it allows a more detailed study of cell and signal simultaneously.

Animals↗
PubMed1998

Molecular beacon probes combined with amplification by NASBA enable homogeneous, real-time detection of RNA.

Molecular beacon probes can be employed in a NASBA amplicon detection system to generate a specific fluorescent signal concomitantly with amplification. A molecular beacon, designed to hybridize within the target sequence, was introduced into NASBA reactions that amplify the genomic RNA of potato leafroll virus (PLRV). During amplification, the probe anneals to the antisense RNA amplicon generated by NASBA, producing a specific fluorescent signal that can be monitored in real-time. The assay is rapid, sensitive and specific. As RNA amplification and detection can be carried out in unopened vessels, it minimizes the risk of carry-over contaminations. Robustness has been verified on real-world samples. This homogeneous assay, called AmpliDet RNA, is a significant improvement over current detection methods for NASBA amplicons and is suitable for one-tube applications ranging from high-throughput diagnostics to in vivo studies of biological activities.

Base Sequence↗
PubMed1986

Detection of RNA transcripts in normal lymphoid and myeloid colonies.

A procedure is described for the routine detection of RNA transcripts in small numbers of hematopoietic cells growing in semi-solid agar. It is suggested that hybridization depends upon RNA expression and that as few as 2500 mRNA molecules per colony are easily detected. Applications of this technique are described in three diverse experimental systems; immunoglobulin gene expression in B cell colonies; neo expression in normal and transformed B cell clones derived from multipotent stem cells infected with a neo-containing retrovirus; and c-myc expression in factor-dependent myeloid colonies.

B-Lymphocytes↗
PubMed2000

The cost-effectiveness of elective Cesarean delivery for HIV-infected women with detectable HIV RNA during pregnancy.

OBJECTIVES: To determine the net health consequences, costs, and cost-effectiveness of alternative delivery strategies for HIV-infected pregnant women with detectable HIV RNA in the USA. DESIGN: Cost-effectiveness analysis using a probabilistic decision model. METHODS: The model compared two strategies: elective Cesarean section and vaginal delivery. Data for HIV transmission rate, maternal death rate, health-related quality of life and costs were obtained from the literature, national databases, and a tertiary hospital's cost accounting system. Model outcomes included total lifetime costs, quality-adjusted life expectancy, maternal death rate, HIV transmission rate, and incremental cost-effectiveness ratios. RESULTS: Elective Cesarean section resulted in a vertical HIV transmission rate of 34.9 per 1000 births compared with 62.3 per 1000 births for vaginal delivery. Elective Cesarean section was more effective (38.7 quality adjusted life years per mother and child pair) and less costly ($10600 per delivery) than trial of labor (38.2 combined quality adjusted life years at a cost of $14500 per delivery). However, elective Cesarean section increased maternal mortality by 2.4 deaths per 100000 deliveries. The results were consistent over a wide range of the variables, but were sensitive to the risk of HIV transmission with vaginal delivery and the relative risk of HIV transmission with elective Cesarean section. CONCLUSIONS: In pregnant HIV-infected women with detectable HIV RNA, elective Cesarean section would reduce total costs and increase overall quality-adjusted life expectancy for the mother-child pair, albeit at a slight loss of quality adjusted life expectancy to the mother.

Adult↗
PubMed1996

Microwave treatment enhances detection of RNA and DNA by in situ hybridization.

The use of oligonucleotide probes with nonisotopic detection systems has made in situ hybridization (ISH) more accessible for use in diagnostic pathology; however, ISH using formalin-fixed, paraffin-embedded tissues remains much more sensitive when performed with radioactively labeled probes compared with nonisotopic reporter systems, especially using oligonucleotide probes. We investigated the effects of microwave pretreatment on the detection of RNA and DNA in formalin-fixed, paraffin-embedded tissues in an attempt to improve the sensitivity of ISH with digoxigenin-labeled probes. Titration of normal tissues with oligonucleotide probe cocktails for albumin, prolactin, chromogranin (Cg) A and B mRNAs and a cDNA probe for JC virus showed a significant increase in sensitivity with the use of microwave treatment step rather than heating at 70 degrees C in 2 x SSC for 30 min. Analysis of various solutions used for microwaving showed that 10 mM citrate buffer was more effective than 2 x SSC, phosphate-buffered saline, tissue unmasking fluid, or water. A 15- to 20-min period of microwaving in an 800-W oven produced optimum results. Analysis of a group of tumors for albumin and CgA and B mRNAs and infected brain biopsies for JC virus DNA showed increased sensitivity of the microwave technique using digoxigenin-labeled probes. These results show that microwave treatment can enhance the detection of mRNA and DNA in formalin-fixed, paraffin-embedded tissues.

DNA↗
PubMed2003

The effect of storage at different temperatures on the stability of Hepatitis C virus RNA in plasma samples.

One important issue related to Hepatitis C virus (HCV) RNA nucleic acid amplification testing (NAT) is the storage conditions of plasma samples in order to obtain reliable results. Many authors have reported that the storage conditions could affect the RNA stability and, hence, HCV RNA detection. We have studied HCV RNA stability in plasma samples after storage at different temperatures (-70, -20, 5 and 25 degrees C). Samples containing different HCV titres were stored and analysed by qualitative or quantitative NAT techniques at defined time points. At -20 degrees C, samples containing high HCV RNA titres were followed-up during approximately 2.6-2.7 years, samples with intermediate concentrations during approximately 1 year and samples with 100 International Units/millilitre (IU/ml) during 2.5 years. Independently of the HCV RNA concentration, the results show absence of decay in HCV RNA detectability. Samples stored at 25 degrees C maintain their HCV RNA titre during 14 days and samples at 5 degrees C were stable for at least 3 months.

Hepacivirus↗
PubMed1999

Detectable HIV-1 RNA at levels below quantifiable limits by amplicor HIV-1 monitor is associated with virologic relapse on antiretroviral therapy.

OBJECTIVE: To assess the clinical significance of HIV-1 RNA levels detectable using the Amplicor HIV-1 Monitor method but < 400 copies/ml versus levels undetectable by this method. DESIGN: Retrospective cohort study. METHODS: All plasma HIV-1 RNA results over 13 months in our institution were reviewed. The study population comprised all individuals that achieved an HIV-1 RNA level < 400 copies/ml and remained on stable antiretroviral therapy. Results of < 400 copies/ml were stratified as 'below quantifiable limits' (BQL) or 'below detectable limits' (BDL). We examined the incidence of virologic relapse, defined as an HIV-1 RNA level > 400 copies/ml, for individuals with viral loads of BQL or BDL. Cox proportional hazards regression analyses were performed to control for baseline CD4 cell count, the number of antiretroviral medications, and the use of protease inhibitors (PI) and/or non-nucleoside reverse transcriptase inhibitors (NNRTI). RESULTS: Virologic relapse occurred in 52 of 168 individuals over 29,576 person-days overall (incidence rate 1.8 cases/1,000 person-days). The relapse rate was three times greater following HIV-1 RNA levels of BQL rather than BDL [crude rate ratio 3.2; 95% confidence interval (CI) 1.8-5.8]. After adjusting for baseline CD4 cell count, number of antiretroviral medications, and use of PI and/or NNRTI, the rate of relapse was nearly four times greater for individuals with HIV-1 RNA levels of BQL (hazard ratio 3.7; 95% CI 2.0-6.7). CONCLUSIONS: In a large clinic population, low-level HIV-1 RNA detected in plasma below the 400 copies/ml limit of quantifiability for Amplicor HIV-1 Monitor was associated with an increased rate of virologic relapse on therapy.

Adult↗
PubMed2004

Detection of RNA in purified cytomegalovirus virions.

Five viral RNA transcripts have recently been detected in purified virions of human cytomegalovirus (HCMV) strain AD169, a well-characterized member of the family Herpesviridae [Science 288 (2000) 2373]. While the function of these transcripts and/or the proteins they encode remains to be elucidated, it is not known whether these transcripts are unique to strain AD169 or are present in other HCMV strains. The objective of this study was to determine if these RNAs are present in other HCMV laboratory strains (Towne and Davis), and a low passage clinical isolate (CL203). These strains of CMV were purified by sequential ultracentrifugation through 20% D-sorbitol and glycerol-potassium tartarate gradients and the morphology and infectivity of the virions confirmed by electron microscopy and inoculation into cell culture. When RNA extracted from the purified virions was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) the UL 21.5 and TRL/IRL 2-5 transcripts were detected in virions of HCMV strains AD169, Davis, Towne and CL203. The presence of the UL 21.5 and TRL/IRL 2-5 RNA transcripts in all strains tested demonstrates that the packaged transcripts occurs in all strains of HCMV suggesting that they may have a relevant role in the biology of this virus.

Cytomegalovirus↗
PubMed2003

Detection of RNA viruses in sudden infant death (SID).

Continuing previous work, reverse transcriptase polymerase chain reaction approaches for the detection of RNA viruses (influenza A+B, parainfluenza virus 3, respiratory syncytial virus) were developed. A total of 118 infant deaths, including 13 cases of non-natural death (nND), 78 cases of natural death (ND) without morphological signs of interstitial pneumonia (IP), 27 cases showing IP (ND+IP), were investigated using frozen lung tissue ( N=100) and paraffin-embedded material ( N=18). In five of the autopsy cases (ND+IP) the influenza B virus genome could be detected and the other types of viruses were completely negative. Together with previous results (detection of adenoviruses and cytomegaloviruses) in the same groups, the frequency of virus detection in the cases with IP was 48% compared with 14% in the ND without IP and 7% in the nND. Significant differences in the frequency of virus detection were also obtained when the cases were divided in SIDS and non-SIDS. The results obtained indicate an association between IP and some viruses and support the hypothesis that respiratory virus infections could act as trigger in sudden infant death.

Autopsy↗
PubMed2001

Simultaneous detection of RNA and protein by in situ hybridization and immunological staining.

Proteinase K is widely used in methods for detection of transcripts in biological specimens by in situ hybridization (ISH). However, treatment with proteinase K hampers detection of RNA and protein simultaneously. We have developed a method for double staining of transcripts and proteins by ISH and IHC staining in imaginal discs and embryos of Drosophila. Instead of treatment with proteinase K, samples are treated with ethanol plus xylene and with acetone. Acetone renders cell membranes permeable to probes and antibodies without damaging tissue integrity, whereas treatment with proteinase K sometimes damages tissues. Treatment of samples with acetone allows hybridization of probe with transcripts in tissue. It is also effective for immunological staining of samples after ISH with a riboprobe. Thus, our method allows detection not only of transcripts but also of specific proteins in relatively intact single samples. (J Histochem Cytochem 49:1177-1182, 2001)

Acetone↗
PubMed2005

Hepatitis C virus in the semen of men coinfected with HIV-1: prevalence and origin.

OBJECTIVE: To compare the prevalence of hepatitis C (HCV) RNA in semen from men infected with HCV and those coinfected with HIV-1/HCV and to study the origin of HCV shed in semen. DESIGN: Two prospective studies (HC EP09 and BINECO) included 120 HCV-positive men, 82 coinfected with HIV-1; all had positive HCV RNA detection in blood. METHODS: Paired blood and semen samples were collected for HCV RNA detection and quantification in seminal plasma and in blood serum; repeated semen samples were obtained for 45 men. HCV RNA was sought in spermatozoa and non-sperm cells. Phylogenetic analysis of the HVR-1 region of HCV compared the quasispecies in blood serum and seminal plasma of two men. RESULTS: HCV RNA was more frequently found in the semen of men coinfected with HIV-1 (37.8%) than in those with only HCV infection (18.4%) (P = 0.033). HCV RNA detection in semen was intermittent and was positive in at least one semen sample of 42.8% of HIV-1/HCV-coinfected men who provided repeated samples. Men with HCV-positive semen had significantly higher HCV load in blood than men with HCV-negative semen (P = 0.038). Phylogenetic comparison of HCV quasispecies in blood and in semen showed no evidence of HCV replication in genital leukocytes; however, a phenetic structure was observed between compartments (P < 0.001). CONCLUSIONS: HCV particles in semen originate from passive passage from blood, with preferential transfer of some variants. Nearly half of HIV-1/HCV-coinfected men may intermittently harbour HCV in their semen. Recommendations of protected sex for HIV-infected individuals should be reinforced.

Adult↗
PubMed1991

Computer-aided quantification of RNA levels detected by in situ hybridization of tissue sections.

A semi-automatic program, designed for non-computer scientists, was developed for quantification of RNA levels detected by in situ hybridization in heterogeneous tissues. A video camera was used to acquire microscopic images of autoradiographed tissue sections which are then digitized on a video monitor for semi-automated quantification of silver grains. We describe a data entry and analysis procedure for systematic quantification of RNA levels in which about 300 cells per tissue sample can be analysed within 10 min. When compared with visual counting, computer-aided quantification was found to be more objective and reliable, with the highest variation coefficient between individuals being 7.5% using computer-aided quantification, compared to 24% with manual counting of the same section areas. A comparative study of c-myc oncogene expression in 11 mammary adenocarcinomas from 3 independent experiments showed the good reproducibility of results using the computer-aided method, with an 18% maximum variation between experiments. The program, with its simple user-interface, reliability and rapidity, is convenient for measuring specific genetic expression levels in clinical studies requiring large numbers of specimens.

Autoradiography↗
PubMed1990

Fluorescent in-situ hybridization to detect cellular RNA by flow cytometry and confocal microscopy.

A fluorescent in-situ hybridization procedure was developed which is suitable for detection of specific cellular RNA in cells fixed in suspension. The procedure was originally developed for analysis of single-cell suspensions by flow cytometry. The resulting fluorescent cells proved to have their 3-D morphology perfectly preserved. The spatial distribution of specific ribosomal RNA and messenger RNA could then be analysed by confocal microscopy of individual cells. In the hybridization procedure, biotinylated single-stranded RNA probes were used that were produced by transcription from cloned DNA fragments. Detection of poly-(A)+ RNA was performed with a poly-biotin-d(U)-tailed oligo-d(T) probe. Bound probe was detected using streptavidin-fluorescein isothiocyanate. For flow cytometry, nuclear DNA was counterstained with di-amidine-2-phenyl indol. Multi-parameter flow cytometry was used to quantify the fluorescence intensity, i.e. the hybridization signal, of thousands of cells. Confocal scanning microscopy on individual cells revealed the intracellular distribution of the target RNA. The distribution of ribosomal RNA and poly-(A)+ RNA mRNA in mouse bone marrow and the human leukaemia cell line HL60 cells was investigated. Ribosomal RNA was confined to the cytoplasm of the cells, although in many cells the nucleoli could also be distinguished. Poly-(A)+ RNA in HL60 cells was found both in the cytoplasm and nucleus. A granular cytoplasmic distribution was seen in part of these cells.

Animals↗
PubMed2006

Multicenter comparison of nucleic acid extraction methods for detection of severe acute respiratory syndrome coronavirus RNA in stool specimens.

The emergence of a novel coronavirus (CoV) as the cause of severe acute respiratory syndrome (SARS) catalyzed the development of rapid diagnostic tests. Stool samples have been shown to be appropriate for diagnostic testing for SARS CoV, although it has been recognized to be a heterogeneous and difficult sample that contains amplification inhibitors. Limited information on the efficiency of extraction methods for the purification and concentration of SARS CoV RNA from stool samples is available. Our study objectives were to determine the optimal extraction method for SARS CoV RNA detection and to examine the effect of increased specimen volume for the detection of SARS CoV RNA in stool specimens. We conducted a multicenter evaluation of four automated and four manual extraction methods using dilutions of viral lysate in replicate mock stool samples, followed by quantitation of SARS CoV RNA using real-time reverse transcriptase PCR. The sensitivities of the manual methods ranged from 50% to 100%, with the Cortex Biochem Magazorb method, a magnetic bead isolation method, allowing detection of all 12 positive samples. The sensitivities of the automated methods ranged from 75% to 100%. The bioMérieux NucliSens automated extractor and miniMag extraction methods each had a sensitivity of 100%. Examination of the copy numbers detected and the generation of 10-fold dilutions of the extracted material indicated that a number of extraction methods retained inhibitory substances that prevented optimal amplification. Increasing the volume of sample input did improve detection. This information could be useful for the extraction of other RNA viruses from stool samples and demonstrates the need to evaluate extraction methods for different specimen types.

Feces↗