RNA In Plasma Samples Research Articles

Genotype Dependence of Hepatitis C Virus Load Measurement in Commercially Available Quantitative Assays

Standardization and genotype independence of methods used to quantify hepatitis C virus (HCV) RNA in clinical specimens are necessary for accurate assessment of the role of HCV quantitation as a prognostic marker for HCV infection and monitoring of the response to antiviral treatment. Commercially available methods used to measure HCV loads include PCR-based (Roche Monitor) and hybridization-based (Quantiplex bDNA-2) methods. Recently, a new version of the Roche Monitor assay (version 2.0) has become available; it has been modified to achieve more equal quantitation of different HCV genotypes. Consistent with previous reports, Roche Monitor version 1.0 substantially underestimated concentrations of RNA transcripts of types 2b, 3a, 4a, 5a, and 6a and virus loads in individuals infected with genotypes 2 to 6 relative to reference tests. However, version 2.0 achieved equivalent quantitation of each genotype over a narrow quantitative range (10(3) to 5 x 10(5) copies of RNA/ml) but significantly underestimated RNA concentrations above this range. The assay showed an equivalent inability to quantify high levels of HCV RNA in plasma samples, and this was responsible for the falsely narrow range of virus loads detected in HCV-infected individuals. In contrast, the Chiron bDNA-2 assay could only measure RNA concentrations in the upper quantitative range (2 x 10(5) to 5 x 10(7) copies of RNA/ml) but showed equivalent sensitivity for genotypes 1 to 5; however, concentrations of type 6a RNA transcripts and virus loads in clinical specimens from individuals infected with type 6a were underestimated by a factor of 2 to 4. Differences were observed between PCR- and hybridization-based assays in their relative quantitation of HCV RNA transcripts and HCV genomic RNA, which may cause problems with the use of transcripts for interassay calibration.

MSRV IN JAPANESE PATIENTS WITH MULTIPLE SCLEROSIS.

P207 Objective: Perron et al have recently identified a novel human endogenous retrovirus (MSRV) in patients with multiple sclerosis (MS). We examined whether MSRV RNA can also be detected in Japanese MS patients. Methods: RNA was isolated from plasma of Japanese MS patients. After treatment with DNase I, RNA samples were used for RT-PCR which amplifies the pol region of MSRV. Genomic DNA, which was isolated from neutrophils, was used for PCR as control. PCR products were run on an agarose gel, transferred by Southern blotting and hybridized with the probe of MSRV pol region. Results: The pol region of MSRV was amplified from genomic DNA by PCR from all the individuals tested. On the other hand, we could not amplify the MSRV sequence by RT-PCR, from plasma RNA samples in MS patients. Conclusion: Further studies are necessary to clarify the role of MSRV in the pathogenesis of multiple sclerosis in Japanese populations.

Clinical Evaluation of an In-House Reverse Transcription-Competitive PCR for Quantitation of Human Immunodeficiency Virus Type 1 RNA in Plasma

An in-house reverse transcription (RT)-competitive PCR (RT-cPCR) for the quantitation of human immunodeficiency virus type 1 (HIV-1) RNA in plasma samples was developed and validated. The procedure involves (i) extraction of RNA with spin columns, (ii) ready-to-use bead-mediated RT, (iii) competitive PCR in a microtiter plate, (iv) agarose gel electrophoresis of the reaction products, and (v) densitometric analysis of the digitized image of the gel. Quadruplicate tests and dilution studies showed that the sensitivity and intertest coefficient of variability of the RT-cPCR are comparable to those of the reference AMPLICOR HIV-1 MONITOR test. The results obtained by the two assays with a panel of 45 clinical samples were in good agreement (mean difference, 0.36 +/- 0.25 log units). Analysis of 1,982 clinical samples by the in-house RT-cPCR yielded the typical range of plasma HIV-1 RNA levels with the expected inverse correlation between CD4 counts and HIV-1 RNA titers. In addition, testing of plasma from 36 subjects at weeks 0 and 4 with respect to the time of initiation of protease inhibitor therapy detected a significant decrease in HIV-1 viremia. The mean reduction in the HIV-1 RNA level was 0.914 log unit for those receiving saquinavir (P = 0.0210), 1.584 log units for those receiving indinavir (P = 0.0047), and 1.904 log units for those receiving ritonavir (P < 0.0001). The in-house RT-cPCR assay is simple to develop and perform and allows quantitation of HIV-1 RNA in 100 to 200 samples per operator per week. Since the cost is 1/8 to 1/10 of those of reference commercial assays, this procedure could be conveniently used in medium-scale laboratories.

Analysis of human immunodeficiency virus type 1 env and gag sequence variants derived from a mother and two vertically infected children provides evidence for the transmission of multiple sequence variants.

In order to investigate the transmission of human immunodeficiency virus type 1 (HIV-1) from mother-to-child we have examined serial plasma RNA samples obtained from a mother over an eight year period spanning four pregnancies. Child 1 and 2 (born January 1987 and June 1990) were uninfected whilst child 3 and 4 (born July 1992 and February 1994) were HIV positive. Genetic variation was examined within the viral population of the mother and her two infected children for both the V3 loop and flanking regions of the env gene and the p17 region of the gag gene. In one child (child 4) a highly homogeneous virus population was observed within both env and gag in contrast to the more heterogeneous virus population observed within the mother. Viral sequences of child 4 clustered within a single branch within the reconstructed phylogenetic tree. This is consistent with the transmission of a single maternal variant to the child in this case, which may indicate a selective process. By contrast, child 3 showed substantial genetic heterogeneity even within the first samples obtained shortly after birth. Sequences of child 3 clustered in two distinct groups within the phylogenetic tree and were separated by sequences of the mother. These results are not consistent with the selective transmission of a single maternal variant to the child in this case and we therefore propose that the infection within child 3 is the result of the transmission of multiple sequence variants to the child. All transmitted sequence variants were predicted to be of the macrophage-tropic, nonsyncytium-inducing (NSI) phenotype.

Detection of HCV and GBV-C/HGV infection by multiplex PCR in plasma samples of transfused subjects

A multiplex polymerase chain reaction (PCR) was applied to clinical samples for simultaneous detection of hepatitis C virus (HCV) and GBV-C/HGV genome. With both RNA viruses, the amplification was performed with primers of the 5′ UTR region starting from the single viral RNA reverse transcripted (cDNA) with random hexanucleotide primer mix. GBV-C/HGV RNA was detected in plasma sample of seven out of 50 transfused patients (14%). The multiplex PCR demonstrated a sensitivity up to 7.8×10 2 copies/ml respectively for GBV-C/HGV and HCV RNA in plasma samples of 5/50 patients with GBV-C/HGV/HCV co-infection and in patients with HCV (27/50) or GBV-C/HGV infection alone (2/50).

Detection of human immunodeficiency virus type 1 RNA in plasma samples from high-risk pediatric patients by using the self-sustained sequence replication reaction.

There is an urgent need for rapid and sensitive methods to assess human immunodeficiency virus (HIV) infection in infants and children. We evaluated an approach by using the self-sustained sequence replication reaction (3SR) to amplify HIV type 1 (HIV-1) RNA directly. The amplified RNA product was then detected by bead-based sandwich oligonucleotide capture hybridization and rare earth metal chelate time-resolved fluorescence. The sensitivity of this technology was determined to be less than 12 HIV-1 RNA copies with an amplification level of 10(10)-fold with purified HIV-1 RNA. Plasma samples from 19 high-risk pediatric patients younger than 5 years of age were examined, and results were compared with viral culture of patient plasma. Results from plasma culture and 3SR amplification agreed for 14 of these patients and disagreed for 5. Of the five samples which did not agree, four were positive by 3SR and negative by culture and one was positive by culture and negative by 3SR but became positive by 3SR at a subsequent testing. We conclude that 3SR amplification coupled with time-resolved fluorescence is a promising technology for investigating the relationship between the presence of HIV-1 RNA in plasma and progression of disease in HIV-infected pediatric patients. This technology should be important in the assessment of HIV-1 infection, in evaluating drug therapies, and in understanding the pathogenesis and transmission of the virus.

Detection of human immunodeficiency virus type 1 genomic RNA in plasma samples by reverse‐transcription polymerase chain reaction

An application of the polymerase chain reaction (PCR) to the direct detection of human immunodeficiency virus type 1 (HIV-1) viremia is described. The amplification of specific HIV-1 sequences of gag and env viral genes was carried out after the reverse-transcription of plasma samples (plasma RT-PCR) from seropositive subjects. The assay is faster and cheaper than detection of specific HIV-1 transcripts from peripheral blood mononuclear cells by RT-PCR. The data suggest that HIV-1 viremia is detectable by plasma RT-PCR in a large proportion of seropositive individuals.