Viral RNA In Blood Research Articles

T-cell responses and previous exposure to hepatitis C virus in indeterminate blood donors

Blood donors are routinely screened for hepatitis C virus infection. Some individuals have weak or restricted virus-specific antibody responses, and are classed as indeterminate. Such donors are almost always negative for viral RNA in blood. We postulated that previous transient virus exposure might account for some of these cases. With sensitive ex-vivo analyses of T-cell responses, we identified virus-specific responses in 15 of 30 indeterminate blood donors tested, compared with none in controls (p=0·0013). Additionally, these responses were typically focused on core-derived peptides. These findings suggest previous exposure to the virus in many indeterminate blood donors. Blood donors are routinely screened for hepatitis C virus infection. Some individuals have weak or restricted virus-specific antibody responses, and are classed as indeterminate. Such donors are almost always negative for viral RNA in blood. We postulated that previous transient virus exposure might account for some of these cases. With sensitive ex-vivo analyses of T-cell responses, we identified virus-specific responses in 15 of 30 indeterminate blood donors tested, compared with none in controls (p=0·0013). Additionally, these responses were typically focused on core-derived peptides. These findings suggest previous exposure to the virus in many indeterminate blood donors. Hepatitis C virus in blood donationThe natural history of infection by hepatitis C virus (HCV) in the northern hemisphere indicates that 80% or more of infected individuals become chronic carriers.1 Infection is currently defined by the presence of specific antibodies identified by the association of a reactive screening assay and confirmation by recombinant immunoblot assay (RIBA), with or without the presence of detectable viral RNA. Confirmed positivity in RIBA is defined as reactivity with at least two of the four viral recombinant antigens, separately lined in the assay. Full-Text PDF

T-cell responses and previous exposure to hepatitis C virus in indeterminate blood donors

Blood donors are routinely screened for hepatitis C virus infection. Some individuals have weak or restricted virus-specific antibody responses, and are classed as indeterminate. Such donors are almost always negative for viral RNA in blood. We postulated that previous transient virus exposure might account for some of these cases. With sensitive ex-vivo analyses of T-cell responses, we identified virus-specific responses in 15 of 30 indeterminate blood donors tested, compared with none in controls (p=0.0013). Additionally, these responses were typically focused on core-derived peptides. These findings suggest previous exposure to the virus in many indeterminate blood donors.

Rapidly Progressed Acquired Immunodeficiency Syndrome Dementia Complex as an Initial Manifestation

We report a patient with acquired immunodeficiency syndrome dementia complex (ADC) that presented human immunodeficiency virus infection as an initial manifestation. A 34-year-old man developed disturbance of consciousness and severe abulia over 3 months. The CD4 lymphocyte count was 7.9/microl, while human immunodeficiency virus RNA in blood amounted to 4.2 x 10(4) copies/ml. T2-weighted magnetic resonance imaging showed diffusely high signal intensity in the deep white matter of both cerebral hemispheres. On the 20th hospital day, the patient died of sepsis caused by methicillin-resistant Staphylococcus aureus. Autopsy findings in the brain included increased glial cells and multinucleated giant cells in cerebral white matter and subcortical gray matter. These features were compatible with ADC.

Experimental infection of rhesus macaques with West Nile virus: level and duration of viremia and kinetics of the antibody response after infection.

Reports of transfusion-associated cases of West Nile virus (WNV) infection indicate the need for sensitive screening methods to identify WNV-infected blood products. We experimentally infected 5 rhesus macaques with WNV, to determine the level and duration of viremia, the kinetics of the humoral immune response, and the sensitivity of various assay systems for detecting WNV in blood. All macaques developed subclinical infections with low levels of viremia; nested reverse-transcription polymerase chain reaction was the most sensitive method for detecting virus or viral RNA in blood. Specific WNV antibodies appeared during the second week of infection; the results of an IgM enzyme-linked immunosorbent assay became positive on the ninth or tenth day after infection, followed in 1-2 days by hemagglutination-inhibiting and neutralizing antibodies. Our results suggest that both nucleic acid and serological testing may be needed to determine exposure to WNV and to identify potentially infected blood donors.

Low levels of hepatitis C virus RNA in blood of infected patients under maintenance haemodialysis with high-biocompatibility, high-permeability filters

Background. Patients undergoing maintenance haemodialysis are often infected with hepatitis C virus, yet the clinical course of liver disease is usually mild. We investigated whether the hepatitis C virus viral load is influenced by the haemodialytic procedure and the type of dialyser. Methods. Hepatitis C virus-RNA levels were measured using a quantitative polymerase chain reaction assay in predialysis blood from 51 hepatitis C virus-infected patients dialysed with different membranes. Paired pre- and post-dialysis measurements were also obtained in 18 patients. Results. Patients dialysed using cuprammonium-regenerated cellulose membranes with low (cuprofan) or intermediate (cellulose acetate or diacetate) biocompatibility had higher pre-dialysis hepatitis C virus-RNA levels (p<0.05) compared to those dialysed with synthetic high-biocompatibility, high permeability polymeric membranes (polyacrylonitrile, polysulfone, polymethylmethacrylate or polycarbonate). Hepatitis C virus-RNA levels were unrelated to the duration of haemodialysis and the presence of abnormal transaminases. A significant reduction (p=0.04) of serum hepatitis C virus-RNA levels was observed after a single haemodialysis, particularly in patients with high pre-dialysis viral load. Patients with low pre-dialysis hepatitis C virus-RNA levels [<2.5×10 3 copies/ml) exhibited only minimal changes following the procedure. Four patients with medium-high basal viral load switched from a low-biocompatibility/low-permeability to a high-biocompatibility/high permeability filter had a marked reduction of viraemia after three weeks, in one case followed by a new increase after return to the original filter. Conclusions. Haemodialysis with high-biocompatibility/high-permeability filters in hepatitis C virus-infected patients is associated with low blood levels of hepatitis C virus-RNA. This finding may be of clinical relevance, especially in patients listed for kidney transplantation.

Measuring infectious bursal disease virus RNA in blood by multiplex real-time quantitative RT-PCR

A quantitative reverse transcription polymerase chain reaction (RT-PCR) protocol for assessing infectious bursal disease virus (IBDV) RNA levels in blood was developed using the ABI PRISM™ 7700 Sequence Detection System coupled with TaqMan® chemistry. To control for variations in sampling and processing between samples 28S rRNA was co-amplified in a multiplex reaction and used to quantify total RNA. Relative quantification and standardisation was achieved using a log 10 dilution series of RNA extracted from IBDV stock. A linear relationship was observed between input RNA and cycle threshold values ( C T) over 5 log 10 dilutions for the IBDV-specific product and 6 log 10 dilutions for the 28S rRNA-specific product. As a test of the assay it was used to determine whether differences in susceptibility to IBDV observed between inbred lines of chickens could be detected at the level of viral load in the blood. Viral RNA levels peaked 2 days post-infection when there was significantly less viral RNA in the blood of resistant line 6 1 chickens compared with the more susceptible Brown Leghorns ( P=0.01). These results demonstrate that the course of IBDV infection can be monitored by quantifying IBDV RNA extracted from blood of infected chickens using TaqMan® technology.

Detection of respiratory syncytial virus RNA in blood of neonates by polymerase chain reaction.

During the winter season of 1994/1995, nasopharyngeal aspirates and blood samples of neonates who were admitted to the Neonatal Intensive Care Unit (NICU) (group 1) and infants with respiratory tract disease (group 2) were examined prospectively for the presence of respiratory syncytial virus (RSV). Examination of nasal washes were done by antigen detection and blood samples were tested by nested reverse transcription and polymerase chain reaction (RT-PCR). The results of the 41 neonates studied were as follows: 14/41 were positive for RSV antigen in nasal washes and for RSV-RNA in blood, 5/41 were only RSV antigen positive, 13/41 neonates had negative nasal washes; 6 had positive RT-PCR results in blood. In 9/41 cases only blood samples were available. Five of these were positive by RT-PCR testing. Group 2 included 20 infants hospitalized with respiratory tract disease, e.g., pneumonia, bronchiolitis, or Upper Respiratory Tract Infection (URTI). Eleven out of twenty were positive for RSV antigen in nasal washes and 6/20 were also positive for RSV-RNA in blood. The conclusion is that viremia may be a frequent occurrence in neonates and young children.

Improved methods for quantification of human immunodeficiency virus type 1 RNA and hepatitis C virus RNA in blood using spin column technology and chemiluminescent assays of PCR products

The quantification of human immunodeficiency virus type 1 (HIV-1) RNA or hepatitis C virus (HCV) RNA has been facilitated by adapting a spin column procedure for sample preparation and the use of chemiluminescent detection of polymerase chain reaction (PCR) products in microtiter plate format. All materials were commercially available and relatively inexpensive. By making a single dilution prior to amplification, concentrations of 500 copies to 2.5 million HIV-1 1 RNA copies per mL and 1,000 copies to 50 million HCV RNA copies per mL could be determined on 140-microL samples. Between-run imprecision employing the improved procedure for HIV-1 RNA was 23%. Correlation of HIV-1 RNA concentrations obtained using chemiluminescent detection with values obtained by colorimetric assay of PCR products was 0.98. Correlation of HCV RNA concentration determined by the spin column-chemiluminescent assay procedure with those obtained by branched DNA methodology was 0.91. Spin columns could be used with serum or plasma containing acid-citrate-dextrose or heparin anticoagulant, but heparinized samples required treatment with heparinase prior to amplification.

Improved methods for quantification of human immunodeficiency virus type 1 RNA and hepatitis C virus RNA in blood using spin column technology and chemiluminescent assays of PCR products

The quantification of human immunodeficiency virus type 1 (HIV-1) RNA or hepatitis C virus (HCV) RNA has been facilitated by adapting a spin column procedure for sample preparation and the use of chemiluminescent detection of polymerase chain reaction (PCR) products in microtiter plate format. All materials were commercially available and relatively inexpensive. By making a single dilution prior to amplification, concentrations of 500 copies to 2.5 million HIV-1 1 RNA copies per mL and 1,000 copies to 50 million HCV RNA copies per mL could be determined on 140-μL samples. Between-run imprecision employing the improved procedure for HIV-1 RNA was 23%. Correlation of HIV-1 RNA concentrations obtained using chemiluminescent detection with values obtained by colorimetric assay of PCR products was 0.98. Correlation of HCV RNA concentration determined by the spin column-chemiluminescent assay procedure with those obtained by branched DNA methodology was 0.91. Spin columns could be used with serum or plasma containing acid-citrate-dextrose or heparin anticoagulant, but heparinized samples required treatment with heparinase prior to amplification. J Med Virol 51:56–63, 1997. © 1997 Wiley-Liss, Inc.

Identification of tick-borne encephalitis virus ribonucleic acid in tick suspensions and in clinical specimens by a reverse transcription-nested polymerase chain reaction assay

Background: Tick-borne encephalitis virus (TBEV) is a major human pathogenic flavivirus. Sensitive assays for the detection of viral RNA may be valuable both for the identification of virus in ticks as well as for diagnostic purposes. Objectives: (1) The development of a sensitive polymerase chain reaction (PCR) test system for the detection of TBEV-RNA and its application to the identification of infected ticks; and (2) evaluation of the PCR assay for diagnostic purposes, i.e., detection of TBE virus RNA in blood and in cerebrospinal fluid (CSF) of TBE patients. Study design: (1) Establishment of a TBEV-specific reverse transcription (RT)-nested PCR assay and evaluation of its sensitivity; (2) comparison of the PCR assay with that of virus isolation from tick suspensions; and (3) investigation of 105; serum and CSF samples from patients with serologically confirmed TBE by RT-nested PCR. Results: An RT-nested PCR assay was established with a detection limit of 100–1000 copies of TBEV RNA. All tick suspensions from which the virus could be isolated by inoculation of suckling mice also screened positive in the PCR assay. Of the 105 clinical samples investigated, only one serum and one CSF sample were positive by PCR assay, and these were both obtained very early in the course of the disease. Conclusions: The PCR assay described is valuable for the detection of TBEV in tick suspensions and can substitute for the usual virus isolation procedure in which suckling mice are inoculated. Its application for diagnostic purposes, however, does not seem to provide a significant improvement over serological diagnosis. Only in very rare cases, when a sample is drawn extremely early in the course of disease, may TBEV RNA be detected in serum or CSF before the appearance of specific IgM antibodies and thus allow an earlier diagnosis.