Comparative performance of three viral load assays on human immunodeficiency virus type 1 (HIV-1) isolates representing group M (subtypes A to G) and group O: LCx HIV RNA quantitative, AMPLICOR HIV-1 MONITOR version 1.5, and Quantiplex HIV-1 RNA version 3.0

The performance of the LCx HIV RNA Quantitative (LCx HIV), AMPLICOR HIV-1 MONITOR version 1.5 (MONITOR v1.5), and Quantiplex HIV-1 RNA version 3.0 (bDNA v3.0) viral load assays was evaluated with 39 viral isolates (3 A, 7 B, 6 C, 4 D, 8 E, 4 F, 1 G, 4 mosaic, and 2 group O). Quantitation across the assay dynamic ranges was assessed using serial fivefold dilutions of the viruses. In addition, sequences of gag-encoded p24 (gag p24), pol-encoded integrase, and env-encoded gp41 were analyzed to assign group and subtype and to assess nucleotide mismatches at primer and probe binding sites. For group M isolates, quantification was highly correlated among all three assays. In contrast, only the LCx HIV assay reliably quantified group O isolates. The bDNA v3.0 assay detected but consistently underquantified group O viruses, whereas the MONITOR v1.5 test failed to detect group O viruses. Analysis of target regions revealed fewer primer or probe mismatches in the LCx HIV assay than in the MONITOR v1.5 test. Consistent with the high level of nucleotide conservation is the ability of the LCx HIV assay to quantify efficiently human immunodeficiency virus type 1 group M and the genetically diverse group O.     

Quantitative assay for group M (subtype A-H) and group O HIV-1 RNA detection in plasma

A quantitative HIV-1 test is described based on a competitive RT-PCR assay combined with a sandwich hybridization as a detection system. The internal RNA standard (IS) was designed specifically to be competitive during the amplification and during the hybridization step. Sample viral load determination was carried out with one RT-PCR in the presence of 10(3) IS copies. The HIV-1 copy number was calculated by reference to an external standard curve performed on known and increasing amounts of the reference HIV-1 (Ref HIV-1) RNA co-amplified with a constant amount of the IS RNA. The assay had a linear range from 10(1) to 10(6) HIV-1 copies. HIV-1 strains belonging to the different subtypes from group M, but also group O, were all detected. Absolute quantification of purified HIV-1 RNA copies gave identical results as the AMPLICOR HIV-1 Monitor assay. The quantification of patient's samples was evaluated according to different criteria such as dynamic range, sensitivity, efficacy of material recovery, reproducibility and convenience of sample handling. The microplate format of the assay combined with the colorimetric detection provides a convenient tool and fulfills the requirement for routine molecular diagnostic laboratories.     

Evaluation of the new LCx HIV RNA quantitative assay: comparison with the Cobas Amplicor HIV Monitor assay

In the present study we evaluated the performance of the new LCx HIV RNA quantitative kit (Abbott Laboratories, Delkenheim, Germany) for the quantitative detection of HIV-1 RNA in human plasma in comparison to the Cobas Amplicor HIV-1 Monitor assay (Roche Diagnostics, Branchburg, N.J.), including samples containing a variety of HIV-1 subtypes. LCx and Cobas were compared using archived EDTA plasma samples collected from HIV-infected patients. Considering the lower limit of the linear range of 50 copies/ml, the detection rate of the LCx was 139 out of 174 (79.9%) versus 131 out of 174 (75.3%) of the Cobas. Overall agreement was 95.4% (166/174) at a cut-off of 50 copies. LCx and Cobas results on clinical samples were found linearly associated (r2 = 0.900) and strongly correlated (r = 0.949). The mean viral load in the 174 frozen patient samples was 3.25 log10 copies/ml by LCx compared to 2.71 log10 copies/ml by Cobas. Considering only samples with a viral load > or =50 copies/ml, the average difference was -0.132 log copy/ml. Using a panel consisting of 9 plasma samples spiked with 9 different HIV-1 cultured isolates (A-H, and O) LCx detected the 9 subtypes with a high degree of precision, i.e., 9-33% coefficient of variation. As expected, the Cobas failed to detect the group O isolates. The results of the remaining samples showed a higher degree of variation (when testing four replicates of the subtype panel) than the LCx of 14.2-40.3%. Nevertheless, the results were comparable with the LCx data.     

Performance attributes of the LCx HCV RNA quantitative assay

MedImmune Vaccines has created four, live, attenuated human cytomegalovirus (HCMV) vaccine candidates, each derived from defined portions of the parental strains, Towne and Toledo. To determine each candidate’s ability to induce HCMV specific immunity, a fluorescence-based microneutralization assay was developed using recombinants of Toledo and Towne which express enhanced green fluorescent protein (EGFP). Replication of the EGFP recombinants in cell culture was the same as the respective parental strains. Using the EGFP recombinants, this fluorescence-based microneutralization assay was compared with the traditional plaque reduction assay. Serum samples were analyzed by both the fluorescence microneutralization and plaque reduction assays and regression analysis showed a correlation of R² ≥ 0.90 between the two assays. As an alternative to measuring fluorescence, infected cells were examined microscopically and the number of green fluorescent cells was counted automatically. Regression lines between fluorescent cell counting and fluorescence in the well also showed a high correlation (R² ≥ 0.92). An excellent linear concordance in titers was observed between the two assays. Using the plaque reduction assay, serum samples were identified that preferentially neutralized the Toledo strain compared to the Towne strain. The same preferences were observed with the fluorescence-based microneutralization assay. This new assay is adaptable to rapid, automated collection of neutralization data and would therefore be suitable for the examination of large numbers of clinical serum samples.     

Clinical performance of the LCx HCV RNA quantitative assay

This study was conducted to assess the performance of the Abbott laboratories LCx HCV RNA Quantitative Assay (LCx assay) in the clinical setting. Four clinical laboratories measured LCx assay precision, specificity, and linearity. In addition, a method comparison was conducted between the LCx assay and the Roche HCV Amplicor Monitor, version 2.0 (Roche Monitor 2.0) and the Bayer VERSANT HCV RNA 3.0 Assay (Bayer bDNA 3.0) quantitative assays. For precision, the observed LCx assay intra-assay standard deviation (S.D.) was 0.060-0.117 log IU/ml, the inter-assay S.D. was 0.083-0.133 log IU/ml, the inter-lot S.D. was 0.105-0.177 log IU/ml, the inter-site S.D. was 0.099-0.190 log IU/ml, and the total S.D. was 0.113-0.190 log IU/ml. The specificity of the LCx assay was 99.4% (542/545; 95% CI, 98.4-99.9%). For linearity, the mean pooled LCx assay results were linear (r=0.994) over the range of the panel (2.54-5.15 log IU/ml). A method comparison demonstrated a correlation coefficient of 0.881 between the LCx assay and Roche Monitor 2.0, 0.872 between the LCx assay and Bayer bDNA 3.0, and 0.870 between Roche Monitor 2.0 and Bayer bDNA 3.0. The mean LCx assay result was 0.04 log IU/ml (95% CI, -0.08, 0.01) lower than the mean Roche Monitor 2.0 result, but 0.57 log IU/ml (95% CI, 0.53, 0.61) higher than the mean Bayer bDNA 3.0 result. The mean Roche Monitor 2.0 result was 0.60 log IU/ml (95% CI, 0.56, 0.65) higher than the mean Bayer bDNA 3.0 result. The LCx assay quantitated genotypes 1-4 with statistical equivalency. The vast majority (98.9%, 278/281) of paired LCx assay-Roche Monitor 2.0 specimen results were within 1 log IU/ml. Similarly, 86.6% (240/277) of paired LCx assay and Bayer bDNA 3.0 specimen results were within 1 log, as were 85.6% (237/277) of paired Roche Monitor 2.0 and Bayer specimen results. These data demonstrate that the LCx assay may be used for quantitation of HCV RNA in HCV-infected individuals.     

Plasma viral RNA assay in HIV-1 group O infection by real-time PCR

HIV-1 group O infections remains essentially restricted to central Africa, and particularly Cameroon, although isolated cases have been reported in Western countries. Genomic differences explain why commercial tests used to quantify HIV-1 group M plasma load are unsuitable for HIV-1 group O. This lack of a quantitative tool hinders the clinical management of HIV-O-infected patients. We have therefore developed a real-time PCR assay, based on LightCycler technology, to quantify HIV-1 group O RNA in plasma. The primers were selected in the LTR 3' region. Forty-eight plasma samples containing strains belonging to the different HIV-1 type O clades (O:A, O:B and O:C) were tested. RNA was quantifiable in 40 of these samples. RNA was always detected in samples from untreated patients, except for one patient infected by a highly divergent strain. The kinetics of plasma viral load were also examined in seven patients for whom clinical and immunologic follow-up data were available. HIV-1 group O plasma load was high in the absence of treatment and correlated negatively with the CD4 cell count. Serial samples obtained during treatment allowed us to compare viral load changes with immunologic outcome. Despite the high initial cost of acquiring the required cycling device, the per-sample cost of this real-time quantitative PCR assay for HIV-1 group O is low, making it suitable for use in endemic zones.     

Comparison of the COBAS TAQMAN HIV-1 HPS with VERSANT HIV-1 RNA 3.0 assay (bDNA) for plasma RNA quantitation in different HIV-1 subtypes

Quantitation of HIV-1 RNA levels in plasma has an undisputed prognostic value and is extremely important for evaluating response to antiretroviral therapy. The purpose of this study was to evaluate the performance of the real-time PCR COBAS TaqMan 48 analyser, comparing it to the existing VERSANT 3.0 (bDNA) for HIV-1 RNA quantitation in plasma of individuals infected with different HIV-1 subtypes (104 blood samples). A positive linear correlation between the two tests (r2 = 0.88) was found. Quantitation by the COBAS TaqMan assay was approximately 0.32log10 higher than by bDNA. The relationship between the two assays was similar within all subtypes with a Deming regression of <1 and <0 for the Bland-Altman plots. Overall, no significant differences were found in plasma viral load quantitation in different HIV-1 subtypes between both assays; therefore these assays are suitable for viral load quantitation of highly genetically diverse HIV-1 plasma samples.     

TaqMan RT-PCR and VERSANT HIV-1 RNA 3.0 (bDNA) assay Quantification of HIV-1 RNA viral load in breast milk.

BACKGROUND: Transmission of HIV via breast milk is a primary cause of pediatric HIV infection in developing countries. Reliable methods to detect breast milk viral load are important. OBJECTIVE: To correlate the ability of the VERSANT HIV 3.0 (bDNA) assay to real-time (RT) TaqMan PCR in quantifying breast milk HIV-1 RNA. STUDY DESIGN: Forty-six breast milk samples that had been spiked with cell-free HIV-1 and eight samples spiked with cell-associated HIV-1 were assayed for HIV-1 RNA by both VERSANT HIV 3.0 and TaqMan RNA assays. RESULTS: Only assays on the cell-free samples were statistically compared. Both a Deming regression slope and a Bland-Altman slope indicated a linear relationship between the two assays. TaqMan quantitations were on average 2.6 times higher than those of HIV 3.0. A linear relationship was observed between serial dilutions of spiked cell-free HIV-1 and both the VERSANT HIV 3.0 and the TaqMan RNA assays. CONCLUSION: The two methods correlated well although the VERSANT HIV 3.0 research protocol quantified HIV-1 RNA slightly lower than TaqMan.     

A genotypic assay for the amplification and sequencing of integrase from diverse HIV-1 group M subtypes

Recently, the Food and Drug Administration (FDA) of the USA approved the first integrase inhibitor for inclusion in treatment regimens of HIV-1 patients failing their current regimens with multi-drug resistant strains. However, treatment failure has been observed during integrase inhibitor-containing therapy. Several mutational pathways have been described with signature mutations at integrase positions 66, 92, 148 and 155. Therefore, a genotypic assay for the amplification and sequencing of HIV-1 integrase was developed. The assay displayed a detection limit of 10 HIV-1 III(B) RNA copies/ml plasma. As the HIV-1 pandemic is characterised by a large genetic diversity, the new assay was evaluated on a panel of 74 genetically divergent samples belonging to the following genetic forms A, B, C, D, F, G, J, CRF01-AE, CRF02-AG, CRFF03-AB, CRF12-BF and CRF13-cpx. Their viral load ranged from 178 until >500,000 RNA copies/ml. The amplification and sequencing was successful for 70 samples (a success rate of 95%). The four failures were most probably due to low viral load or poor quality of RNA and not to subtype issues. Some of the sequences obtained from integrase inhibitor-na?ve patients displayed polymorphisms at integrase positions associated with resistance: 74IV, 138D, 151I, 157Q and 163AE. The relevance of these polymorphisms in the absence of the signature mutations remains unclear.     

Comparison of the LCx human immunodeficiency virus (HIV) RNA quantitative, RealTime HIV, and COBAS AmpliPrep-COBAS TaqMan assays for quantitation of HIV type 1 RNA in plasma

The LCx human immunodeficiency virus (HIV) RNA Quantitative, RealTime HIV, and COBAS AmpliPrep-COBAS TaqMan assays for HIV type 1 (HIV-1) were compared for their abilities to quantitate HIV-1 RNA in plasma. High degrees of correlation and agreement were observed between the assays. Differences in HIV-1 RNA levels according to HIV-1 subtypes did not reach statistical significance.     

Impact of human immunodeficiency virus type 1 (HIV-1) genetic diversity on performance of four commercial viral load assays: LCx HIV RNA Quantitative, AMPLICOR HIV-1 MONITOR v1.5, VERSANT HIV-1 RNA 3.0, and NucliSens HIV-1 QT

Human immunodeficiency virus type 1 (HIV-1) evolution and changing strain distribution present a challenge to nucleic acid-based assays. Reliable patient monitoring of viral loads requires the detection and accurate quantification of genetically diverse HIV-1. A panel of 97 HIV-1-seropositive plasma samples collected from Cameroon, Brazil, and South Africa was used to compare the performance of four commercially available HIV RNA quantitative tests: Abbott LCx HIV RNA Quantitative assay (LCx), Bayer Versant HIV-1 RNA 3.0 (bDNA), Roche AMPLICOR HIV-1 MONITOR v1.5 (Monitor v1.5), and bioMérieux NucliSens HIV-1 QT (NucliSens). The panel included group M, group O, and recombinant viruses based on sequence analysis of gag p24, pol integrase, and env gp41. The LCx HIV assay quantified viral RNA in 97 (100%) of the samples. In comparison, bDNA, Monitor v1.5, and NucliSens quantified viral RNA in 96.9%, 94.8%, and 88.6% of the samples, respectively. The two group O specimens were quantified only by the LCx HIV assay. Analysis of nucleotide mismatches at the primer/probe binding sites for Monitor v1.5, NucliSens, and LCx assays revealed that performance characteristics reflected differences in the level of genetic conservation within the target regions.     

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.     

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.     

Performance of two commercially available sequence-based HIV-1 genotyping systems for the detection of drug resistance against HIV type 1 group M subtypes

The use of genotyping assays for the detection and evaluation of drug resistance mutations within the polymerase gene of human immunodeficiency virus type 1 (HIV-1) is becoming increasingly relevant in the clinical management of HIV-1 infection. However, genotypic resistance assays available currently have been optimised for genetic subtype B strains of the virus and many clinical centres are presented with strains from subtypes A, C, and D. In the present report, we compare the performance of two sequence-based commercially available kits, the ViroSeq Genotyping System (Applied Biosystems, Foster City, CA) and the TruGene HIV-1 Genotyping Kit (Visible Genetics, Toronto, Ontario) against a panel of 35 virus isolates from HIV-1 Group M (subtypes A-J). Full-length consensus sequences were generated by the ViroSeq genotyping system for 26 of 31 (83.8%) of the isolates tested, in contrast to the TruGene genotyping system, which generated 16 of 30 (53%) usable sequences overall. Overall, subtype B isolates were sequenced with a greater degree of success than non-subtype B isolates. Discrepancies were found between the consensus sequences reported by each system for each sample (mean difference 1.0%; range 0.0-3.2%), but these appeared to be random and did not affect interpretation of the major resistance codons. In addition, both systems were able to amplify template RNA from low copy viral load plasma samples (10(2)-10(3) RNA copies/ml) taken from a random selection of patient samples encompassing subtypes A-C. While the availability of these genotyping systems should facilitate studies of HIV-1 drug resistance in countries in which these subtypes are prevalent, the performance against subtypes other than B needs to be improved.     

Evaluation of performance across the dynamic range of the Abbott RealTime HIV-1 assay as compared to VERSANT HIV-1 RNA 3.0 and AMPLICOR HIV-1 MONITOR v1.5 using serial dilutions of 39 group M and O viruses

Performance of the Abbott m2000 instrument system and the Abbott RealTime HIV-1 assay was evaluated using a panel of 37 group M (subtypes A-D, F, G, CRF01_AE, CRF02_AG and unique recombinant forms) and 2 group O virus isolates. Testing was performed on 273 sample dilutions and compared to VERSANT HIV-1 RNA 3.0 (bDNA) and AMPLICOR HIV-1 MONITOR v1.5 (Monitor v1.5) test results. RealTime HIV-1, bDNA, and Monitor v1.5 tests quantified 87%, 78%, and 81% of samples, respectively. RealTime HIV-1 detected an additional 31 samples at < 40 copies/mL. For group M, RealTime HIV-1 dilution profiles and viral loads were highly correlated with bDNA and Monitor v1.5 values; 87% and 89% of values were within 0.5 log(10) copies/mL. In contrast, the group O viruses were not detected by Monitor v1.5 and were substantially underquantified by approximately 2 log(10) copies/mL in bDNA relative to the RealTime HIV-1 assay. Sequence analysis revealed that RealTime HIV-1 primer/probe binding sites are highly conserved and exhibit fewer nucleotide mismatches relative to Monitor v1.5. The automated m2000 system and RealTime HIV-1 assay offer the advantages of efficient sample processing and throughput with reduced "hands-on" time while providing improved sensitivity, expanded dynamic range and reliable quantification of genetically diverse HIV-1 strains.     

Multicenter evaluation of the NucliSens EasyQ HIV-1 v1.1 assay for the quantitative detection of HIV-1 RNA in plasma

The Nuclisens EasyQ HIV-1 v1.1 assay (Biomerieux) is a real-time detection method combined with NASBA technology designed to measure plasma HIV-RNA. Its performance was assessed in 1008 clinical specimens collected from individuals infected with clade B (774) and non-B (234) HIV-1 variants at four European laboratories. The results were compared with those obtained using three other commercial viral load assays: Cobas Amplicor Monitor HIV-1 v1.5 (Roche), Versant HIV-1 RNA assay (Bayer) and Nuclisens HIV-1 QT (Biomerieux). Overall, the linearity, specificity and reproducibility of the EasyQ assay was comparable with that from the other tests. The correlation coefficient (R) between methodologies was 0.85 for Amplicor; 0.87 for Versant; and 0.91 for Nuclisens. The specificity of the assay was 99.4%. Of note, Versant missed 17% of specimens with non-B subtypes which could be detected by EasyQ, while Amplicor provided similar results than EasyQ. HIV-1 group O specimens were only detected by the EasyQ assay. In conclusion, the performance of the EasyQ assay seems to be similar to that of other HIV-1 viral load tests currently on the market, but it is more sensitive than Versant for HIV-1 non-B subtypes and shows a wider dynamic range than Amplicor. Moreover, as it incorporates the advantage of real-time detection procedures, it facilitates high throughput and short turnaround time.