Quantitative detection of human immunodeficiency virus type 1 (HIV-1) viral load by SYBR green real-time RT-PCR technique in HIV-1 seropositive patients

HIV-1 viral load represents a basic marker for evaluation of the rate and severity of HIV-1 related disease and to monitor the effectiveness of treatment. An SYBR green-based real-time RT-PCR (SYBR green real-time RT-PCR) revealed by Light Cycler technology was evaluated for quantitation of HIV-1 RNA viral load in plasma of HIV-1 seropositive patients. The performance of the SYBR green real-time PCR was assessed on 56 HIV-1 seropositive patients under highly active retroviral therapy (HAART) and 25 blood donors. The results demonstrated that this technique detected 50 HIV-1 RNA copies per millilitre of plasma. Moreover, we compared real-time RT-PCR with the b-DNA technique considered widely a reference technique for HIV-1 RNA viral load measurement. The parallel quantitative analysis of HIV-1 positive samples showed a high correlation (r=0.908) between the two methods. Although b-DNA and the real-time-based method gave similar sensitivity, the assay determined quantitatively HIV-1 RNA copies in 4 out of 16 samples shown as undetectable by b-DNA. The SYBR green real-time RT-PCR represents a good alternative to b-DNA assay in HIV-1 viral load determination especially during the monitoring of HAART treatment.     

Improved sensitivity of human immunodeficiency virus type 2 subtype B plasma viral load assay

We developed a new assay for human immunodeficiency virus type 2 plasma RNA quantification based on a previous format. The new version performed significantly better than the original regarding the detection of subtype B, allowing the detection of 14 out of 36 plasma RNAs in the subtype B-infected patients not detected with the original version.     

Use of Sno Strip filter-paper wicks for collection of genital-tract samples allows reproducible determination of human immunodeficiency virus type 1 (HIV-1) RNA viral load with a commercial HIV-1 viral load assay

To assess the reproducibility of measurements of cervical and vaginal human immunodeficiency virus (HIV) viral load, 92 duplicate cervical and 88 duplicate vaginal samples were collected from 13 HIV-infected women using Sno Strip filter-paper wicks. RNA was eluted from the strips, extracted, and assayed using a modified protocol for the Roche Cobas Amplicor HIV-1 Monitor assay. Pearson's correlation coefficient (R), coefficient of determination (D), and Bland-Altman plots (BA) were used to compare paired log10-transformed viral loads. Analysis of duplicate same-site samples showed good reproducibility (cervix: R = 0.72, D = 52%, BA = 89% within range; vagina: R = 0.72, D = 51%, BA = 87% within range); paired cervix/vagina measurements showed moderate correlation only (R = 0.56; D = 31.3%). Standardized sample collection and simple modification of the Roche Cobas Amplicor HIV-1 Monitor assay allows reproducible measurement of genital viral load.     

Evaluation of the Abbott LCx quantitative assay for measurement of human immunodeficiency virus RNA in plasma

Plasma human immunodeficiency virus RNA in 491 clinical specimens was measured by LCx. There was a strong correlation with the results provided by other methods (r(2) values of 0.93 with Cobas-Monitor version 1.5 and of 0.95 with Quantiplex version 3.0). However, values were uniformly higher with LCx than with Quantiplex when non-B subtypes were tested.     

Comparative evaluation of the automated Roche TaqMan real-time quantitative human immunodeficiency virus type 1 RNA PCR assay and the Roche AMPLICOR Version 1.5 conventional PCR assay

The need to evaluate antiviral treatment response and the emergence of resistance have made the human immunodeficiency virus (HIV) viral load assay a major feature of the diagnostic monitoring of HIV-infected individuals. The objective of this study was to evaluate the utility of the recently In Vitro Diagnostic Medical Devices Directive-approved Roche COBAS AmpliPrep/TaqMan96 real-time PCR assay by comparison with the existing Roche COBAS AmpliPrep/AMPLICOR MONITOR conventional PCR assay. EDTA-treated plasma samples from 191 HIV-1-infected individuals were tested for HIV-1 RNA by the AMPLICOR assay and the TaqMan assay. This was a prospective study using 191 pairs of samples from the same bleed per patient. The correlation coefficient of the assays was 98.08%. The mean difference between the assays was 0.05 log(10) copy/ml plasma, with a standard deviation (SD) of 0.27 log(10) copy/ml plasma. Thirteen samples gave results with variances greater than 0.5 log(10) copy/ml plasma, which is our clinical cutoff. Two samples were more than 3 SD different (0.81 log(10) copy/ml plasma). The TaqMan assay appeared to be slightly more sensitive at the lower end of the dynamic range. The assays correlated significantly (P > 0.95) with each other, and the regression analysis was also highly significant (R(2) > 0.95).     

Quantification of human immunodeficiency virus type 1 proviral load by a TaqMan real-time PCR assay

Proviral human immunodeficiency virus type 1 (HIV-1) DNA could be a useful marker for exploring viral reservoirs and monitoring antiretroviral treatment, particularly when HIV-1 RNA is undetectable in plasma. A new technique was developed to quantify proviral HIV-1 using a TaqMan real-time PCR assay. One copy of proviral HIV-1 DNA could be detected with 100% sensitivity for five copies and the assay had a range of 6 log(10). Reproducibility was evaluated in intra- and interassays using independent extractions of the 8E5 cell line harboring the HIV-1 proviral genome (coefficients of variation [CV], 13 and 27%, respectively) and peripheral blood mononuclear cells (PBMC) from a patient with a mean proviral load of 26 copies per 10(6) PBMC (CV, 46 and 56%, respectively). The median PBMC proviral load of 21 patients, measured in a cross-sectional study, was determined to be 215 copies per 10(6) PBMC (range, <10 to 8,381). In a longitudinal study, the proviral load of 15 out of 16 patients with primary infection fell significantly during 1 year of antiretroviral therapy (P = 0.004). In the remaining patient, proviral HIV-1 DNA was detectable but not quantifiable due to a point mutation at the 5' end of the TaqMan probe. No correlation was observed between proviral load and levels of CD4(+) cells or HIV-1 RNA in plasma. TaqMan PCR is sensitive and adaptable to a large series of samples. The full interest of monitoring proviral HIV-1 DNA can now be ascertained by its application to the routine monitoring of patients.     

Multicenter evaluation of the performance characteristics of the NucliSens HIV-1 QT assay used for quantitation of human immunodeficiency virus type 1 RNA

The analytical performance of the NucliSens HIV-1 QT assay, a highly sensitive test based on nucleic acid sequence-based amplification technology, was evaluated in a multicenter trial. Assay specificity was evaluated with 502 plasma (EDTA) specimens from human immunodeficiency virus type 1 (HIV-1)-seronegative volunteer donors. No HIV-1 RNA was reported in any of the donor specimens. Analytical sensitivity and reproducibility were estimated with panels prepared from a high-titer well-characterized HIV-1 RNA stock (5.84 x 10(8) RNA copies/ml). The assay's dynamic range was linear from 10(6) to 10(1) HIV-1 RNA copies, with a lower detectable limit of 25 copies/ml and a 95% detection rate of 176 copies/ml. Sensitivity of the assay to detect HIV-1 RNA in clinical specimens from patients (n = 101) and in commercially available or prepared panels (n = 24) was compared with NASBA HIV-1 RNA QT (an earlier version of NucliSens HIV-1 QT) and with the Food and Drug Administration-approved standard and ultrasensitive AMPLICOR HIV-1 MONITOR, version 1.0, assays. Detection of HIV-1 RNA was reproducible over a 5-log range (mean standard deviation = 0.15 log). The NucliSens and the standard AMPLICOR assays were equivalent in detection of HIV-1 RNA (concentration, 10(3) to 10(5) copies/ml) in 57 clinical specimens. The NucliSens assay was more sensitive in detecting HIV-1 RNA at lower concentrations (相似文献   

Validation of performance of the gen-probe human immunodeficiency virus type 1 viral load assay with genital swabs and breast milk samples

Human immunodeficiency type 1 (HIV-1) continues to spread at an alarming rate. The virus may be transmitted through blood, genital secretions, and breast milk, and higher levels of systemic virus in the index case, as measured by plasma RNA viral load, have been shown to correlate with increased risk of transmitting HIV-1 both vertically and sexually. Less is known about the correlation between transmission and HIV-1 levels in breast milk or genital secretions, in part because reliable quantitative assays to detect HIV-1 in these fluids are not available. Here we show that the Gen-Probe HIV-1 viral load assay can be used to accurately quantify viral load in expressed breast milk and in cervical and vaginal samples collected on swabs. Virus could be quantified from breast milk and swab samples spiked with known amounts of virus, including HIV-1 subtypes A, C, and D. As few as 10 copies of HIV-1 RNA could be detected above background threshold levels in > or =77% of assays performed with spiked breast milk supernatants and mock swabs. In genital swab samples from HIV-1-infected women, similar levels of HIV-1 RNA were consistently detected in duplicate swabs taken from the same woman on the same clinic visit, suggesting that the RNA values from a single swab sample can be used to measure genital viral load.     

Comparison of HIV-1 RNA measurements obtained by using plasma and dried blood spots in the automated abbott real-time viral load assay

Dried blood spots (DBS) may be a promising alternative specimen type to plasma for measuring the viral load (VL) in HIV-infected individuals in resource-limited settings. However, characterization of assay performance using DBS is incomplete. In this prospective study, the VL was measured in parallel using plasma and DBS specimens collected at the same time from 157 HIV-1-infected individuals. DBS were prepared by dispensing 50 μl of blood onto filter paper cards and were stored desiccated at -20°C. Nucleic acid extraction from plasma and DBS was performed automatically using the Abbott m2000sp instrument, and the VL was measured using the RealTime HIV-1 VL assay, which has a lower limit of detection of 40 HIV RNA copies/ml. The correlation between plasma and DBS results was good (R = 0.91; P < 0.001). The mean difference in the VL (DBS minus plasma) was 0.35 log copies (standard deviation [SD], 0.47 log copies). A total of 40 (26%) paired specimens had a difference of >0.5 log copy, and in 12 (7.8%) it was >1 log copy. the VL from DBS was measurable in 95.7% of specimens with a plasma VL of >2.74 log copies (550 HIV RNA copies/ml). In summary, the VL can reliably be measured using DBS with the Abbott RealTime HIV-1 assay. The estimated lower limit of detection of this automated methodology on DBS is 550 copies/ml, a threshold that may be acceptable for periodic VL monitoring in patients on antiretroviral therapy in resource-limited settings, where early detection of virologic treatment failure is often problematic.     

Clinical performance of an in-house real-time RT-PCR assay using a fluorogenic LUX primer for quantitation of human immunodeficiency virus type-1 (HIV-1)

The South African National Antiretroviral Treatment Guideline recommends the use of HIV viral load assays for routine monitoring of HIV-1 positive patients on Highly Active Antiretroviral Therapy (HAART). Approved commercial HIV-1 viral load assays are expensive for developing countries where a large number of patients are treated in the public sector. The evaluation of an in-house HIV-1 viral load assay (LUX assay) is described using 458 plasma specimens. Good specificity of the LUX assay was demonstrated using 50 seronegative plasma specimens. A group of 142 HIV-1 positive patients was used to assess the agreement between the LUX assay and the COBAS Amplicor assay. An intra class correlation (ICC) coefficient of 0.85 (CI 95%) indicated good agreement between the assays. The Bland-Altman model showed good agreement between the assays for approximately 87% of the results (mean 0.03 [-1.26; 1.32], CI 95%). In a cohort of 55 patients followed-up longitudinally the LUX assay showed similar declines in viral load to the COBAS Amplicor assay in response to therapy. Viral rebound was detected in 5 patients out of 55 by both assays. Thus, the LUX assay compares well to the gold standard and represents an affordable alternative for high volume testing in resource limited settings.     

Evaluation of automated sample preparation and quantitative PCR LCx assay for determination of human immunodeficiency virus type 1 RNA

Efforts have been made to achieve full automation of molecular assays for quantitative detection of human immunodeficiency virus type 1 (HIV-1). In the present study, the Abbott LCx HIV RNA Quantitative assay was evaluated in conjunction with automated HIV-1 RNA extraction on the MagNA Pure LC instrument and compared to the conventional LCx HIV RNA Quantitative assay, which uses a manual nucleic acid extraction protocol. Accuracy, linearity, and interassay and intra-assay variations were determined. The performance of the assay in a routine clinical laboratory was tested with a total of 105 clinical specimens. When the accuracy of the LCx HIV RNA Quantitative assay with the automated sample preparation protocol was tested, all results were found to be within +/- 0.5 log unit of the expected results. Determination of linearity resulted in a quasilinear curve over 3.5 log units. For determination of interassay variation, coefficients of variation were found to be between 21 and 66% for the LCx HIV RNA Quantitative assay with the automated sample preparation protocol and between 10 and 69% for the LCx HIV RNA Quantitative assay with manual sample preparation. For determination of intra-assay variation, coefficients of variation were found to be between 7 and 25% for the LCx HIV RNA Quantitative assay with the automated sample preparation protocol and between 7 and 19% for the LCx HIV RNA Quantitative assay with manual sample preparation. When clinical samples were tested by the LCx HIV RNA Quantitative assay with the automated sample preparation protocol and the results were compared with those of the LCx HIV RNA Quantitative assay with manual sample preparation, 95% of all positive results were found to be within +/- 0.5 log unit. In conclusion, the assay with automated sample preparation proved to be suitable for use in the routine diagnostic laboratory and required significantly less hands-on time.     

Comparison of three current viral load assays for the quantitation of human immunodeficiency virus type 1 RNA in plasma

The LCx HIV RNA quantitative assay (Abbott Laboratories, Delkenheim, Germany) was compared with the Versant HIV-1 RNA 3.0 (bDNA) assay (Bayer, Tarrytown, NY) and the COBAS Amplicor HIV-1 Monitor v1.5 test (Roche Diagnostics, Branchburg, NJ), using plasma samples of various viral load levels from HIV-1-infected patients. Considering the lower limit of the linear range of 50 copies/ml of both assays, the detection range of the LCx was 127/151 (84.1%) versus the 131/151 (86.8%) of the bDNA 3.0 assay, while overall agreement between the two assays was 93.4% (141/151). LCx and bDNA 3.0 results were found to be strongly correlated (r = 0.96). The fitted regression line was described by the equation log10(LCx copies/ml) = 0.05 + 1.06 x log10(bDNA 3.0 copies/ml) with 95% CI for the estimated slope and intercept at 1.01, 1.12 and -0.16, 0.26, respectively. Similarly, the detection range of the LCx was 115/148 (77.7%) versus the 128/148 (86.5%) of the Monitor v1.5 test. A 91.2% concordance (135/148) was observed between these two assays at a cut-off of 50 copies/ml. LCx and Monitor v1.5 results were highly correlated (r = 0.96). The fitted regression line was described by the equation log10(LCx copies/ml) = 0.06 + 1.03 x log(10)(Monitor v1.5 copies/ml) with 95% CI for the estimated slope and intercept at 0.97, 1.09 and -0.16, 0.28, respectively.     

Quantitative real-time PCR on Lightcycler for the detection of human immunodeficiency virus type 2 (HIV-2)

Similar to HIV-1, the viral load in HIV-2 is a marker of the evolution of infection and the success of therapy. No approved or commercially distributed assay exists to determine the plasma viral load of HIV-2. We therefore developed a quantitative real-time PCR to determine the plasma RNA viral load of HIV-2, based on the reference strains ROD and EHO, which represent subtypes A and B of HIV-2, respectively. After testing several pairs of primers, a set was chosen that recognised the 5'-LTR region of a subtypes A and B consensus sequence. The quantification of the PCR reaction was done using SYBR-Green I as the fluorescent dye in a Lightcycler system and relying on an external standard curve. The method was then optimised with reference strains, an validated further using patient samples and an inter-laboratory comparison. Good specificity was obtained for both subtypes of HIV-2, and a linear range between 10 and 10(6) copies of viral RNA. The limit of quantitation was 250 copies per millilitre of plasma. The coefficient of variation ranged from 0.7 to 5.6%, depending on the concentration of the target sequences.     

Chemokine receptors as fusion cofactors for human immunodeficiency virus type 1 (HIV-1)

CD4 is the primary cellular receptor for human immunodeficiency virus type 1 (HIV-1), but is not sufficient for entry of HIV-1 into cells. After a decade-long search, the cellular coreceptors that HIV-1 requires in conjunction with CD4 have been identified as members of the chemokine receptor family of seven-transmembrane G-protein coupled receptors. The discovery of distinct chemokine receptors that support entry of T-cell tropic (CXCR-4) and macrophage tropic HIV-1 strains (CCR-5) explains the differences in cell tropism between viral strains, the inability of HIV-1 to infect most nonprimate cells, and the resistance of a small percentage of the population to HIV-1 infection. Further understanding of the role of chemokine receptors in viral entry may also help explain the evolution of more pathogenic forms of the virus, viral transmission, and HIV-induced pathogenesis. These recent discoveries will aid the development of strategies for combating HIV-1 transmission and spread, the understanding of HIV-1 fusion mechanisms, and the possible development of small animal models for HIV-1 drug and vaccine testing.     

Multicenter performance evaluation of a new TaqMan PCR assay for monitoring human immunodeficiency virus RNA load

A TaqMan real-time PCR assay, the COBAS TaqMan human immunodeficiency virus (HIV) (HPS-CTMHIV) PCR assay, recently developed for the quantification of HIV type 1 RNA in plasma, was evaluated in comparison with the licensed COBAS AMPLICOR HIV-1 MONITOR (CAHIM) assay. In this study, we have analyzed the tests' sensitivities, precisions, and linearities using multiple replicates of a standard panel of HIV RNA covering a 7-logarithm range of concentrations, as well as serial threefold dilutions of high-titer clinical samples. The subtype inclusivity was also investigated, using a panel of subtypes A to H, while a collection of 160 clinical samples was analyzed to assess the tests' specificities and the systems' similarities. The results of these experiments showed that the HPS-CTMHIV assay has a sensitivity of 53 copies/ml (95% hit rate), 100% specificity, and good intra- and interassay precision. The results of the HPS-CTMHIV assay were linear in the 50- to 10(7)-copies/ml range, with a correlation coefficient (R) for expected versus observed results of 0.98. Compared to the CAHIM assay, the HPS-CTMHIV assay showed a high correlation (R=0.99) across the dynamic range of RNA concentrations that, for the CAHIM assay, requires two different sample preparations. Equivalent performances were also observed for the two systems in the detection and quantification of HIV subtypes A to H. These data indicate that the HPS-CTMHIV assay may be one of the tests of choice for monitoring viral load throughout the course of HIV infection and during highly active antiretroviral therapy.     

Comparative evaluation of the QUANTIPLEX HIV-1 RNA 2.0 and 3.0 (bDNA) assays and the AMPLICOR HIV-1 MONITOR v1.5 test for the quantitation of human immunodeficiency virus type 1 RNA in plasma

HIV-1 RNA measurements from 84 plasma specimens obtained with the QUANTIPLEX HIV-1 RNA 2.0 and 3.0 (bDNA) assays (Chiron Diagnostics, Emeryville, CA) and with the AMPLICOR HIV-1 MONITOR Test, version 1.5 with ultra-sensitive specimen preparation (Roche Diagnostic Systems, Inc., Branchburg, NJ) were compared. The absolute RNA values of tested specimens differed significantly between bDNA 2.0 and bDNA 3.0 or Monitor v1.5 measurements (Wilcoxon signed-rank test P<0.001). Results generated with bDNA 3.0 or with Monitor v1.5 were approximately twofold greater than those generated with bDNA 2.0, with smaller differences at higher HIV-1 RNA levels and greater differences at RNA levels below 1000 copies per ml. Although highly correlated (r=0.92 and 0.86, respectively), viral load data generated with bDNA 2.0 and either bDNA 3.0 or Monitor v1.5 were in poor agreement. Concordant results (difference in log(10) copies per ml <0.5) were found at frequencies of 80% for bDNA 2.0 and bDNA 3.0 and only at 58.5% for bDNA 2.0 and Monitor v1.5. In contrast, bDNA 3.0 and Monitor v1.5 measurements were highly correlated (r=0.96) and in good agreement (92.7%).