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At least 667 records · Page 37Linked to original sources
PubMed2003

Detection of a healthy carrier of HCV with no evidence of antibodies for over four years.

BACKGROUND: Posttransfusion HCV has been notably reduced over recent years as a result of the systematic testing for antibodies to HCV in blood donors. However, the risk of transfusing blood-derived components from virus-carrying donors still remains. A diagnosis is reported here of HCV in a regular blood donor who had no antibodies during the entire time she was followed up. CASE REPORT: The pharmaceutical company responsible for fractioning the plasma detected a donor who was a carrier of HCV, confirmed by PCR, but whose tests to detect anti-HCV were systematically negative. The donor had given blood on five previous occasions, from which 14 components were manufactured. Of the 11 components traced, six had been transfused, and in the two cases in which study of the anti-HCV was possible in the recipients, the result was positive. It was possible to check the blood samples from the donor from May 1997 to March 2002 (58 months). The tests to detect anti-HCV were all negative, while the PCR and core antigen tests were positive. CONCLUSION: The incorporation of RNA detection or HCV core antigen techniques in blood banks may reduce the residual risk of contracting posttransfusion HCV. Measures such as the correct traceability of the components, the existence of a specimen bank, or follow up of the recipients of blood-derived components would help to improve the quality of blood banking with percentage of survivability and case investigations.

Biopsy↗
PubMed2026

Amplification of RNA for identification of Zika and HCV in whole blood.

Direct RNA amplification from whole blood is fundamentally limited by rapid enzymatic degradation and inhibitory matrix effects. Here, we present a blood drying protocol that enables sensitive and robust RNA detection without the need for extraction, purification, or cold-chain logistics. Using whole blood, the platform achieves high detection sensitivity, down to 10 copies per microliter for Zika virus and 1 international unit per microliter for hepatitis C virus (HCV). We further demonstrate that the protocol can be scaled to larger blood volumes and achieve single-copy sensitivity without any sample loss. This is accomplished through thermal treatments of the sample combined with a primer-limited reverse transcription step, which together stabilize RNA within a dried blood matrix and permit spatially resolved enzymatic amplification. The system supports multiplexed detection from a single sample, enabling simultaneous identification of multiple targets. Separately, we introduce a concept wherein the very few copies of the preserved RNA within the matrix can be accessed repeatedly for molecular analysis. Furthermore, we demonstrated the detection of Zika and HCV using a portable fluorometer for point-of-care (POC) uses. With lyophilized reagents and minimal instrumentation such as a heater and an inexpensive portable fluorometer, this platform enables robust, reusable, and field-deployable diagnostics, advancing toward truly accessible on-site RNA testing in urgent care or low-resource settings from whole blood.

Humans↗
PubMed2001

Detection of viable Cryptosporidium parvum using DNA-modified liposomes in a microfluidic chip.

This paper describes a microfluidic chip that enables the detection of viable Cryptosporidium parvum by detecting RNA amplified by nucleic-acid-sequence-based amplification (NASBA). The mRNA serving as the template for NASBA is produced by viable C. parvum as a response to heat shock. The chip utilizes sandwich hybridization by hybridizing the NASBA-generated amplicon between capture probes and reporter probes in a microfluidic channel. The reporter probes are tagged with carboxyfluorescein-filled liposomes. These liposomes, which generate fluorescence intensities not obtainable from single fluorophores, allow the detection of very low concentrations of targets. The limit of detection of the chip is 5 fmol of amplicon in 12.5 microL of sample solution. Samples of C. parvum that underwent heat shock, extraction, and amplification by NASBA were successfully detected and clearly distinguishable from controls. This was accomplished without having to separate the amplified RNA from the NASBA mixture. The microfluidic chip can easily be modified to detect other pathogens. We envision its use in mu-total analysis systems (mu-TAS) and in DNA-array chips utilized for environmental monitoring of pathogens.

Animals↗
PubMed2002

Prospective evaluation of genetic abnormalities and telomerase expression in exfoliated urinary cells for bladder cancer detection.

PURPOSE: To evaluate alternative procedures to cytoscopic examination we prospectively compared noninvasive procedures for detecting bladder cancer namely cytology, loss of heterozygosity (LOH), microsatellite instability and human telomerase catalytic subunit reverse transcriptase (hTERT) messenger (m) RNA detection. MATERIALS AND METHODS: Specificity and cutoff values were established in the blood and urine sediment of 50 controls. Sensitivity was analyzed in the urine and tissue samples of 50 patients with bladder cancer. The diagnosis was established by cystoscopic and histological examination. Genomic alterations were studied using a panel of 24 microsatellite markers to detect LOH events, while 3 additional mononucleotide repeats were analyzed for microsatellite instability detection. Telomerase expression was detected in urinary cells by nested RT-polymerase chain reaction amplification of hTERT mRNA. All techniques were compared by cytological examination. RESULTS: Sensitivity and specificity were 31% and 100% for cytological testing, 96% and 100% for LOH, and 75% and 69% for RT-polymerase chain reaction of hTERT, respectively. No alteration was detected on microsatellite instability analysis in urine or tumor tissue cells. Using only the 5 markers most strongly associated with bladder cancer selected by logistic regression analysis, namely ABL1, IFNa, D9S12, MJD58 and D18S364, LOH test sensitivity slightly decreased to 90%. CONCLUSIONS: Urinary LOH analysis was the most sensitive and specific method for bladder cancer detection and it appeared less dependent on urine sediment quality. The logistic regression score may be an interesting complement to cystoscopy. The specificity of hTERT mRNA detection was incomplete since false-positives were observed in 31% of cases. Absent microsatellite instability in our cohort showed that these genomic alterations are not present at the early step of bladder cancer.

Catalytic Domain↗
PubMed2026

Protocol to improve isoform-level quantification of low-abundance transcripts via STALARD pre-amplification.

STALARD (selective target amplification for low-abundance RNA detection) enables isoform-level quantification of low-abundance RNAs using conventional laboratory equipment. Here, we describe steps for RNA isolation, primer design, reverse transcription, selective target amplification, and downstream analysis. The protocol couples selective pre-amplification with a quantitative reverse-transcription PCR (RT-qPCR) readout and optional nanopore sequencing. Using 1 μg input RNA and 12 pre-amplification cycles, STALARD reduces Cq values by approximately 10-12 cycles, bringing the target into a reliably quantifiable range. For complete details on the use and execution of this protocol, please refer to Jeong et al.1.

Gene Expression↗
PubMed1999

Comparison of genetic heterogeneity of hepatitis C viral RNA in liver tissue and serum.

OBJECTIVE: Hepatitis C virus (HCV) is known to be heterogeneous and to circulate as a group of closely related quasispecies in individual patients, although hepatic viral genetic characteristics have not been well documented. METHODS: Matched serum and liver samples were tested by reverse transcription polymerase chain reaction amplification and single stranded conformation polymorphism analysis of the hypervariable portion of the E2/NS1 region of the HCV genome. The number of quasispecies was compared with the amount of HCV RNA, HCV genotyping, and infection with the hepatitis G virus. RESULTS: Sixteen of 40 patients had HCV RNA detectable in serum and liver. The HCV genotype was identical in serum and liver of all but one case. HCV RNA levels were approximately 10-fold higher in liver than serum. The number of HCV quasispecies in serum ranged between two and six (median 3.0) and in the liver between 2 and 19 (median 3.5, mean liver/serum ratio 1 to 6.3, median 1.8). The number of quasispecies in liver was equal to or greater than that in serum in all cases. HGV infection was found in 14 cases and did not influence serum or hepatic levels of HCV RNA. CONCLUSIONS: The number of hepatic HCV quasispecies usually exceeds that in serum, independent of the amount of HCV RNA and HCV genotype. This finding is compatible with clearance of some quasispecies from serum, but not liver, by putative neutralizing antibodies.

Adult↗
PubMed2001

[Patient with West Nile fever in the Netherlands].

A 45-year-old patient was admitted with high fever and inadequate behaviour. She had recently stayed in Israel. Extensive investigation suggested a viral infection, but the cause was not established. Her partner suggested the possibility of West Nile virus infection, based upon information he had found on the Internet. Serology was performed and specific IgM and IgG antibodies were demonstrated, but a paired serum sample was not available. The patient recovered spontaneously. The West Nile virus is endemic in Israel, Africa, Asia and the Balkans. It is transmitted by mosquitoes. The incubation time is 1-3 weeks. The disease is characterised by fever, malaise, maculopapular exanthema and lymphadenopathy. Complications are encephalitis, pancreatitis, hepatitis and myocarditis. However, these symptoms are present in less than 5% of all infections. A full-blown encephalitis has a mortality rate of 50%. There is no specific therapy, but prevention by means of controlling the mosquito population is feasible. The diagnosis is made by the detection of West Nile virus-specific IgM antibodies in serum or cerebrospinal fluid. However, in the acute stage viral RNA detection by a polymerase chain reaction in the serum is also possible.

Acute Disease↗
PubMed2006

[Late spontaneous elimination of HCV-RNA from mononuclear cells derived from peripheral blood in patients with chronic hepatitis C].

THE AIM: To follow persistence of HCV-RNA in PBMC in patients with chronic hepatitis C (CHC). To estimate the influence of this phenomenon on the cellular immune response of peripheral blood lymphocytes. MATERIAL AND METHODS: 8 HCV-RNA in PBMC positive children, with undetectable serum HCV-RNA after antiviral treatment, have been examined every 2-3 years. The amount of IFN-gamma, IL-12 and IL-18 secreted by PBMC obtained from the children after stimulation with phytohemagglutinin (PHA) was measured. RESULTS: Spontaneous elimination of HCV-RNA from PBMC in 2 to 6 years after treatment was found in all children. In two children HCV-RNA detectable both in serum and in PBMC, without recurrence of hepatitis, was found in single examination. PBMC containing HCV-RNA secreted more IFN-gamma than PBMC lacking it (1221 +/- 458 pg/ml vs. 651 +/- 147 pg/ml; p=0.009, similar correlation was revealed with the regard of IL-12: 21.8 +/- 12.3 pg/ml vs. 5,6 +/- 3,3 pg/ml respectively; p=0.009. Production and release of IL-18 were not correlated with HCV-RNA persistence (p=0.12). CONCLUSION: Patients with CHC and persistence of HCV-RNA in PBMC require longitudinal follow-up in the respect of possible reseroconversion. PBMC containing HCV-RNA reveal enhanced cellular immune response, which most probably effects in spontaneous elimination of the virus.

Adolescent↗
PubMed2004

The biology and diagnostic applications of plasma RNA.

The detection of cell-free RNA in the plasma and serum of human subjects has opened up new investigational opportunities. Tumor-derived RNA was detected in the plasma and serum of patients with various forms of cancer. Through the development of robust extraction and quantification protocols for circulating RNA, new information regarding its biology and characteristics has been obtained. Plasma RNA was shown to be particle associated and to be surprisingly stable. Recently, fetal RNA was also demonstrated in maternal plasma. The placenta was shown to be an important source of such circulating fetal RNA. This discovery has created new prenatal diagnostic and monitoring possibilities. It is expected that circulating RNA will find application in many other clinical scenarios, including emergency medicine.

Biology↗
PubMed2006

Microarray analysis of RNA processing and modification.

Most RNAs are processed from precursors by mechanisms that include covalent modifications, as well as the removal of flanking and intervening sequences. Traditional methods to detect RNA processing, such as Northern blotting, reverse-transcribed polymerase chain reaction and primer extension assays, are difficult to apply on a large scale. This chapter outlines several methods for analysis of the processing and modification of RNA using microarrays. These encompass protocols for the application of homemade microarrays and custom-designed commercial inkjet microarrays and are tailored for the large-scale analysis of processing of mRNA, including alternative splicing, as well as for the analysis of processing and modification of noncoding RNA. This chapter also describes practical aspects of microarray design, sample preparation, hybridization, and data analysis.

Animals↗
PubMed2005

Evaluation of COBAS AmpliPrep nucleic acid extraction in conjunction with COBAS AmpliScreen HBV DNA, HCV RNA and HIV-1 RNA amplification and detection.

BACKGROUND AND OBJECTIVES: This report describes the evaluation of the COBAS AmpliPrep instrument for fully automated generic nucleic acid extraction in conjunction with hepatitis B virus (HBV) DNA, hepatitis C virus (HCV) RNA, and human immunodeficiency virus (HIV)-1 RNA COBAS AmpliScreen amplification and detection using serial dilutions of the WHO international standards (IS) and the PeliCheck reference panels. MATERIALS AND METHODS: Serial diluted samples of the WHO IS and the PeliCheck reference panels were tested 24 times to determine the HBV DNA, HCV RNA and HIV-1 RNA detection limits by Probit analysis. The existence and extent of cross-contamination were assessed by testing alternating high titre HBV DNA-positive and -negative samples. The specificity of the AmpliPrep-AmpliScreen test for HBV was determined by testing 232 minipools consisting of six donations, all negative for HCV/HIV-1 nucleic acid testing (NAT) and HBsAg. In addition, a HBV genotypes A-G panel was tested. RESULTS: The respective 95% detection limits (and 95% CI) on the WHO IS and on the PeliCheck reference panels were 6.7 (4.3-13) IU/ml and 123 (68-301) gEq/ml for HBV DNA, 23 (11-106) IU/ml and 126 (84-233) gEq/ml for HCV RNA, and 187 (108-422) IU/ml and 183 (108-434) gEq/ml for HIV-1 RNA. Based on the WHO IS and the PeliCheck reference panels, no significant differences in sensitivity for HBV and HCV were found between AmpliPrep and the licensed MultiPrep extraction method. The sensitivity of AmpliPrep-AmpliScreen for HIV-1 was probably twofold lower as compared to the MultiPrep-AmpliScreen method. No cross contamination was observed. All 232 minipools were HBV NAT-negative. The AmpliPrep-AmpliScreen test for HBV detected HBV genotypes A-G with equal sensitivity. CONCLUSIONS: The AmpliPrep instrument combined with the AmpliScreen assays for HBV, HCV and HIV-1 is robust and suitable for NAT donor screening. The sensitivity criteria for HIV-1 and HCV as defined by the Paul Ehrlich Institute and the Food and Drug Administration for minipool NAT screening are met by this system. SINGLE SENTENCE SUMMARY: Generic COBAS AmpliPrep nucleic acid extraction in conjunction with COBAS AmpliScreen detection for HBV, HCV and HIV-1.

DNA, Viral↗
PubMed2005

Identification of a functioning mitochondrial uncoupling protein 1 in thymus.

We present evidence that rat and mouse thymi contain mitochondrial uncoupling protein (UCP 1). Reverse transcriptase-PCR detected RNA transcripts for UCP 1 in whole thymus and in thymocytes. Furthermore, using antibodies to UCP 1 the protein was also detected in mitochondria isolated from whole thymus and thymocytes but not in thymus mitochondria from UCP 1 knock-out mice. Evidence for functional UCP 1 in thymus mitochondria was obtained by a comparative analysis with the kinetics of GDP binding in mitochondria from brown adipose tissue. Both tissues showed equivalent B(max) and K(D) values. In addition, a large component of the nonphosphorylating oxygen consumption by thymus mitochondria was inhibited by GDP and subsequently stimulated by addition of nanomolar concentrations of palmitate. UCP 1 was purified from thymus mitochondria by hydroxyapatite chromatography. The isolated protein was identified by peptide mass mapping and tandem mass spectrometry by using MALDI-TOF and LC-MS/MS, respectively. We conclude that the thymus contains a functioning UCP 1 that has the capacity to regulate metabolic flux and production of reactive oxygen-containing molecules in the thymus.

Amino Acid Sequence↗
PubMed1996

Use of NASBA RNA amplification for detection of Mycobacterium leprae in skin biopsies from untreated and treated leprosy patients.

This study was performed to assess the value of NASBA RNA amplification of a 16S rRNA target for the detection of presumably viable Mycobacterium leprae in sections of skin biopsies from leprosy patients. The NASBA positivity rate was 90.4% (84/93) for untreated multibacillary (MB) patients [bacterial index (BI) > or = 2] and 16.7% (8/48) for the untreated paucibacillary (PB) patients (BI < 2). NASBA positivity showed a good concordance with the presence of solidly stained M. leprae [morphological index (MI)] in skin biopsies from leprosy patients, but no relationship could be demonstrated between the strength of the NASBA signals and the BI. Furthermore, the usefulness of the detection of 16S rRNA by NASBA to monitor the efficacy of leprosy treatment was investigated using an additional 154 biopsy specimens analyzed from 80 MB patients during the course of treatment. The NASBA positivity rate declined during treatment. A significant decrease was observed after only 1-3 months. These results favor the view that detection of RNA by NASBA may reflect the viability of M. leprae.

Biopsy↗
PubMedDate not supplied

Evaluation of an enzyme immunoassay for hepatitis C virus antibody detection using a recombinant protein derived from the core region of hepatitis C virus genome.

This study was undertaken to evaluate an enzyme immunoassay (EIA) for hepatitis C virus antibody detection (anti-HCV), using just one antigen. Anti-HCV EIA was designed to detect anti-HCV IgG using on the solid-phase a recombinant C22 antigen localized at the N-terminal end of the core region of HCV genome, produced by BioMérieux. The serum samples diluted in phosphate buffer saline were added to wells coated with the C22, and incubated. After washings, the wells were loaded with conjugated anti-IgG, and read in a microtiter plate reader (492 nm). Serum samples of 145 patients were divided in two groups: a control group of 39 patients with non-C hepatitis (10 acute hepatitis A, 10 acute hepatitis B, 9 chronic hepatitis B, and 10 autoimmune hepatitis) and a study group consisting of 106 patients with chronic HCV hepatitis. In the study group all patients had anti-HCV detected by a commercially available EIA (Abbott), specific for HCV structural and nonstructural polypeptides, alanine aminotransferase elevation or positive serum HCV-RNA detected by nested-PCR. They also had a liver biopsy compatible with chronic hepatitis. The test was positive in 101 of the 106 (95%) sera from patients in the study group and negative in 38 of the 39 (97%) sera from those in the control group, showing an accuracy of 96%. According to these results, our EIA could be used to detect anti-HCV in the serum of patients infected with hepatitis C virus.

Adolescent↗
PubMed2006

Achieving specific RNA cleavage activity by an inactive splicing endonuclease subunit through engineered oligomerization.

Protein-protein interaction is a common strategy exploited by enzymes to control substrate specificity and catalytic activities. RNA endonucleases, which are involved in many RNA processing and regulation processes, are prime examples of this. How the activities of RNA endonucleases are tightly controlled such that they act on specific RNA is of general interest. We demonstrate here that an inactive RNA splicing endonuclease subunit can be switched "on" solely by oligomerization. Furthermore, we show that the mode of assembly correlates with different RNA specificities. The recently identified splicing endonuclease homolog from Sulfolobus solfataricus, despite possessing all of the putatively catalytic residues, has no detectable RNA cleavage activity on its own but is active upon mixing with its structural subunit. Guided by the previously determined three-dimensional structure of the catalytic subunit, we altered its sequence such that it could potentially self-assemble thereby enabling its catalytic activity. We present the evidence for the specific RNA cleavage activity of the engineered catalytic subunit and for its formation of a functional tetramer. We also identify a higher order oligomer species that possesses distinct RNA cleavage specificity from that of previously characterized RNA splicing endonucleases.

Amino Acid Sequence↗
PubMed2006

[Use of combined detection of hepatitis C virus core antigen and antibodies to reduce the serological window-phase].

OBJECTIVES: In this study, we aimed at evaluating the performances of a combined assay for the detection of hepatitis C virus core antigen and antibodies and comparing this test with conventional third generation Elisa. MATERIAL AND METHODS: Two hundred forty-one samples were included in this study and tested by Monolisa HCV Ag-Ab ULTRA, Biorad and compared to Monolisa Anti-HCV Plus. A comparative study was performed on a HCV seroconversion panel (Monolisa anti-HCV Plus, Biorad; Innotest HCV Ab IV, Innogenetics and Murex anti-HCV, Abbott). False positive samples were detected with western blot assay (INNO-LIA HCV Ab III, Innogenetics). Two anti-HCV negative haemodialysis patients with rise in ALT have been tested for RNA detection (Amplicor v2.0, Roche). RESULTS: Results obtained with Biorad Ag-Ab were in agreement with third generation ELISA on HCV seroconversion panel. From anti-HCV negative patients, four samples were found low positive with HCV Ag-Ab. Two anti-HCV negative haemodialysis patients/HCV RNA positive were also negative with HCV Ag-Ab and 13 low positive samples with Biorad Ab were found negative with Ag-Ab. CONCLUSION: The HCV Ag-Ab assay has a high specificity and sensitivity comparatively to conventional ELISA; but in our study we don't prove the reduction of the "serologic window" for detection of anti-HCV antibodies.

Early Diagnosis↗
PubMed1994

[Summary and evaluation of genome and antibody diagnosis in hepatitis C virus infection].

Hepatitis C virus (HCV) infection can be diagnosed by antibody assay systems using recombinant antigens since the HCV genome has been identified. It is still impossible to detect viral antigens associated with HCV. Therefore, the polymerase chain reaction (PCR) has been widely used in practical laboratory examinations. Because HCV is an RNA virus and the reverse transcription process is required prior to the PCR reaction, the process of HCV RNA detection has a risk of contamination and the detection rate may differ with the PCR condition. There are several genome diagnostic methods for HCV infection; that is, genome detection by nested PCR, HCV subtyping using mixed primer, quantitation of HCV genome using competitive PCR, Quant-Amp and branched DNA probe. To clarify what factors are responsible, the efficacy of interferon therapy against chronic hepatitis C was examined. The normalization rate of serum ALT level and clearance rate of HCV RNA in serum at six months after the end of the treatment were correlated to the titer of HCV RNA genome and the HCV subtype. The patients with HCV RNA titers of less than 10(4) copies/50 microliters and with subtype group III showed a high response to interferon administration. On the other hand, the results of quantitation of the HCV core antibody titer were closely associated with the presence of HCV RNA using core protein with the recombinant vaccinia expression system or recombinant E. coli system (JCC-2). Especially, in the group of high responders to interferon treatment, the antibody titer against core protein was apparently decreased after the end of interferon therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Genome, Viral↗