Highly Sensitive Multiplex Assay for Detection of Human Immunodeficiency Virus Type 1 and Hepatitis C Virus RNA

Giachetti, Cristina; Linnen, Jeffrey M.; Kolk, Daniel P.; Dockter, Janel; Gillotte‐Taylor, Kristin; Park, M.; Ho-Sing-Loy, Marcy; McCormick, Mary Kay; Mimms, Larry; McDonough, Sherrol H.

doi:10.1128/jcm.40.7.2408-2419.2002

Cited by 105 publications

(130 citation statements)

References 42 publications

Supporting

Mentioning

122

Contrasting

Unclassified

Order By: Relevance

“…The HCV RNA TMA test used (Procleix HIV-1/HCV assay, Gen-Probe) has a 95% detection sensitivity of 30 HCV RNA copies/mL, using an input of 0.5 mL of plasma, and detects all 6 HCV genotypes. [23][24][25] The sensitivity of the TMA assay for PBMC-associated HCV RNA was first measured with total cellular RNA extracted from the PBMC of 3 viremic blood donors. HCV RNA levels in these PBMC were first measured with a commercial qPCR assay and then serially diluted to 1000 to 0.1 copies of HCV RNA (see the Patients and Methods section).…”

Section: Resultsmentioning

confidence: 99%

“…The beads are rinsed, and the HCV RNA is amplified by isothermal TMA. [23][24][25] Blood donations that were HCV EIA 3.0 -seropositive (specificity confirmed with RIBA 3.0) were originally tested for HCV RNA with TMA of plasma minipools of 16 or 24 donations, as is customary for all blood donations. 26,27 Seropositive index donations that were TMA minipool-positive were resolved to a single donor by TMA testing of the individual donations using the residual plasma, and donors were notified of their infection status.…”

Section: Methodsmentioning

confidence: 99%

“…The presence of plasma viremia was tested for using a highly sensitive transcription-mediated amplification (TMA) assay. [23][24][25] After determining that this TMA assay could also be used to sensitively test for HCV RNA in PBMC and confirming our results using reverse transcription-nested polymerase chain reaction (RT-nPCR), we investigated whether clearance of plasma viremia was associated with clearance of viral RNA from PBMC. We have found that clearance of plasma viremia is tightly associated with clearance of HCV RNA from PBMC.…”

mentioning

confidence: 91%

See 2 more Smart Citations

Clearance of hepatitis C virus RNA from the peripheral blood mononuclear cells of blood donors who spontaneously or therapeutically control their plasma viremia

et al. 2008

View full text Add to dashboard Cite

We determined whether hepatitis C virus (HCV) RNA could be detected associated with peripheral blood mononuclear cells (PBMC) of seropositive blood donors who had spontaneously or therapeutically cleared their plasma viremia. Blood donor plasma viremia status was first determined with a highly sensitive transcription-mediated amplification (TMA) test performed in duplicate assays. PBMC from 69 aviremic and 56 viremic blood donors were then analyzed for the presence of HCV RNA with TMA adapted to detect viral RNA in PBMC and with a reverse transcription-nested polymerase chain reaction assay. PBMCassociated HCV RNA was detected in none of the 69 aviremic donors, including all 6 subjects with a sustained viral response following antiviral therapy. PBMC-associated HCV RNA was detected in 43 of the 56 viremic donors. The 13 viremic donors with no detectable PBMCassociated HCV RNA all had very low viral loads (6 positive only in 1 of 2 duplicate plasma TMA assays, 6 with viral loads below 100 HCV RNA copies/mL, and 1 with a viremia of 2700 HCV RNA copies/mL). The absence of detectable PBMC HCV RNA detection in all 69 aviremic donors reported here contrasts with prior studies, possibly as a result of the higher sensitivity of the TMA assay used to test for plasma viremia. Conclusion: Our results indicate that PBMC are unlikely to serve as a long-lived reservoir of HCV in aviremic subjects.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 91%

See 1 more Smart Citation

Clearance of hepatitis C virus RNA from the peripheral blood mononuclear cells of blood donors who spontaneously or therapeutically control their plasma viremia

et al. 2008

View full text Add to dashboard Cite

show abstract

“…Sera nonreactive by RT-PCR were further evaluated by use of an HCV transcription-mediated amplification (TMA) assay (Gen-Probe Inc., San Diego, CA). 12 Only one anti-HCV-positive serum was nonreactive for RNA by both RT-PCR and TMA but was associated with posttransfusion acquisition of passive anti-HCV, RNA reactivity, and later active seroconversion.…”

mentioning

confidence: 95%

Viral and Host Factors in Early Hepatitis C Virus Infection *

et al. 2005

View full text Add to dashboard Cite

Since 1980, the Transfusion-transmitted Viruses Study (TTVS) (1974)(1975)(1976)(1977)(1978)(1979)(1980)) has continued to maintain its computerized database and stored sera to enable ongoing study of new transfusion events since the 1970s. Most recently, we have used this resource to study parameters of acute hepatitis C virus (HCV) infection among 94 donor-recipient pairs in which there was transmission. In addition, frequent recipient observations permitted further characterization of the early phase of the infection's course. Donor RNA load ranged from 3.7 to 3,160,000 IU/mL. Onset of recipient viremia was judged from a total of 67 sera collected during the 4th through 8th days posttransfusion; only 2 of the 67 sera were still RNA nonreactive by that time. The recipients' latent periods to an alanine aminotransferase (ALT) elevation of >90 IU/L ranged from 6 to 112 days (median, 46 days) and was shorter with higher donor RNA levels. Descriptors of the recipient's illness showed several strongly positive and negative correlations. The latent period tended to be shorter in the 37% of cases that were clinically overt. Attributes of donors with genotypes 1 and non-1 and subtypes 1a and 1b did not differ significantly. Recipients with genotype 1 strains had shorter latent intervals than non-1 strains. On multivariate analysis, latent period was significantly associated (negatively) only with the highest ALT level during the first 120 days of follow-up (P ‫؍‬ .014). In conclusion, host factors are more important determinants of acute HCV infection dynamics than virus-associated factors. ( I n the past, the most clearly defined cases for study of acute hepatitis C virus (HCV) infection resulted from blood-borne transfusion transmission in populations followed prospectively. This approach identified not only overt cases but also the more numerous subclinical infections. In the last decade, however, screening of blood donors for antibody to HCV (anti-HCV) and then hepatitis C viremia has almost eliminated this route of infection. 1 Fortunately, databases and specimens for several past groups of prospectively followed transfusion recipients are still available. 2 One of these is the Transfusion-transmitted Viruses Study (TTVS), from which it is still possible to obtain new information about HCV infections as increasingly informative laboratory assays have been developed. [3][4][5][6][7] We have identified and further characterized 94 acute transfusion-transmitted HCV cases in which an anti-HCV-negative recipient was infected by a single seropositive donor. We have examined the idea that there could be correlations between donor and recipient parameters, reflecting the replicative capacity or pathogenetic potential of the strain shared between donor and recipient. We have also attempted a thorough description of recipient events during the first 4 months of the infection. Patients and MethodsPopulation. Many details of TTVS have been previously reported. [8][9][10] Briefly, from July 1974 through Octo-

show abstract

“…In our case, we used an extensively validated plasma HCV RNA assay used for blooddonation screening (Transcription mediated amplification, Procleix human immunodeficieny virus-1/HCV assay, Gen-probe, with a 95% detection sensitivity of 30 HCV RNA/mL). [11][12][13] This TMA assay was used in duplicate assays (consuming a total of 1 mL plasma) to identify a total of 69 plasma aviremic blood donors, none of whom possessed detectable PBMC-associated HCV RNA. 1 Pham et al analyzed and found HCV RNA in the PBMCs of two aviremic subjects (so classified using their RT-nPCR-NAH).…”

mentioning

confidence: 99%

Reply

Delwart

2008

Hepatology

View full text Add to dashboard Cite

cells facilitated HCV RNA detection in as much as 75% of initially HCV-negative cases. 7 Further, in up to 20% of cases, the HCV genome can be found in stimulated PBMCs but not in parallel serum. The simultaneous detection of an HCV RNA replicative strand, HCV protein, and unique HCV variants served to authenticate active virus replication in PBMCs, irrespective of serum HCV RNA status, as our recent study reassured. 6 The above approach to HCV RNA identification was not employed by Bernardin and colleagues. 1 Thus, as they alluded, it remains a distinct possibility that ex vivo stimulation of PBMCs might have augmented HCV detection in their study. Nevertheless, we recently demonstrated that replication of HCV can be confined to particular immune cell subsets, while total PBMCs appear nonreactive, 6 suggesting that PBMC negativity does not necessarily exclude low-level HCV infection in this compartment.In summary, substantial differences in the methodology by which PBMCs and RNA were prepared, in the amount of template analyzed, and in the overall assay sensitivity are the most probable reasons behind the contradictory findings described by Bernardin et al. 1 and others. [2][3][4][5][6] As such, standardization of methods for HCV RNA detection in circulating immune cells based on the most sensitive approaches uncovered should be considered in future studies on this subject. TRAM N.Q.

show abstract

Highly Sensitive Multiplex Assay for Detection of Human Immunodeficiency Virus Type 1 and Hepatitis C Virus RNA

Cited by 105 publications

References 42 publications

Clearance of hepatitis C virus RNA from the peripheral blood mononuclear cells of blood donors who spontaneously or therapeutically control their plasma viremia

Clearance of hepatitis C virus RNA from the peripheral blood mononuclear cells of blood donors who spontaneously or therapeutically control their plasma viremia

Viral and Host Factors in Early Hepatitis C Virus Infection *

Reply

Contact Info

Product

Resources

About