Evaluation of an ultrasensitive p24 antigen assay as a potential alternative to human immunodeficiency virus type 1 RNA viral load assay in resource-limited settings

An inexpensive enzyme-linked immunosorbent assay method for human immunodeficiency virus type 1 quantitation, ultrasensitive p24 antigen assay (Up24), was compared with RNA viral load assay (VL). Up24 had 100% sensitivity of detection at a viral load of >/=30,000, with sensitivity of 46.4% at a viral load of <30,000 (232 specimens from 65 seropositive subjects). The assay was highly reproducible, with excellent correlation between duplicates and among three laboratories.     

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.     

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.     

Quantitation of human immunodeficiency virus type 1 in breast milk

The distribution and stability of human immunodeficiency virus type 1 (HIV-1) in breast milk (BM) components remain largely unknown. Inhibitory effects, if any, of BM on HIV RNA and DNA PCR amplification are poorly understood. We have addressed these issues by using virus-spiked BM samples from HIV-negative women. BM samples from HIV-negative women were spiked with HIV-1 virions or cells containing a single integrated copy of HIV DNA (8E5/LAV). After incubation under different experimental conditions, viral RNA was detected by the Roche Amplicor UltraSensitive assay in whole-milk, skim milk, and lipid fractions. We found excellent correlation between HIV-1 input copy and recovery in whole milk (r = 0.965, P < 0.0001), skim milk (r = 0.972, P < 0.0001), and the lipid fraction (r = 0.905, P < 0.001). PCR inhibition was observed in less than 10% of the spiked samples. Similar levels of inhibition were noted in BM samples collected from HIV-infected women. HIV proviral DNA was detected in BM samples using real-time PCR (linear correlation between the threshold cycle versus log DNA copy number, >0.982). The effects of incubation duration and temperature and repeated freeze-thaw cycles on HIV RNA recovery were analyzed. HIV RNA levels were remarkably stable in whole milk after three freeze-thaw cycles and for up to 30 h at room temperature. Our findings improve the understanding of the dynamics of HIV detection in BM and the conditions for BM sample collection, storage, and processing.     

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.     

Plasma RNA viral load in human immunodeficiency virus type 2 subtype A and subtype B infections

Human immunodeficiency virus type 2 (HIV-2) is much less pathogenic than HIV-1, and HIV-2 infection is associated with plasma viral loads significantly lower than those found in HIV-1 infection. We have developed a real-time quantitative PCR method for measuring the HIV-2 RNA load that covers the range of genetic diversity of HIV-2 isolates and that detects extremely low viral loads. Samples from 49 patients were studied. Proviral DNA was first detected and quantified. The strains that were detected were then genotyped: 21 patients were infected with HIV-2 subtype A and 15 patients were infected with HIV-2 subtype B; 1 patient was infected with a highly divergent strain. Env PCR failed for the remaining 12 patients, so subtypes could not be determined. For viral RNA quantification, a stock of HIV-2 strain NIHZ, which was counted by electron microscopy, was used as the standard. Several primer sets targeting the highly conserved gag region were evaluated. Various primer combinations failed to amplify subtype B strains. With the final primer pair selected, which detected both subtype A and subtype B strains, the sensitivity of the assay was 100% at a viral load of 250 copies/ml and 66% at a viral load of 125 copies/ml. We found a correlation between the CD4(+)-cell count, the clinical stage, and the plasma HIV-2 RNA level. The median plasma HIV-2 RNA value for the 33 asymptomatic patients was 2.14 log(10), whereas it was 3.1 log(10) for the 16 patients with AIDS (P < 0.01). Proviral DNA was detectable in 18 symptom-free patients with high CD4(+)-cell counts, in whom viral RNA was undetectable.     

Quantitation of human immunodeficiency virus type 1 DNA and RNA by a novel internally controlled PCR assay.

A novel internally controlled PCR (ICPCR) assay was developed to accurately quantitate human immunodeficiency virus type 1 (HIV-1) DNA and RNA in peripheral blood mononuclear cells and plasma. The ICPCR assay was sensitive and reproducible within a linear range of amplification of 10(0) to 10(3) copies for HIV-1 DNA and 10(1) to 10(4) copies for HIV-1 RNA. The assay detected HIV-1 RNA in plasma and peripheral blood mononuclear cells from all HIV-1 subjects regardless of disease stage. ICPCR was compared with a branched-DNA signal amplification assay for subjects beginning antiretroviral therapy. The reductions in plasma HIV-1 RNA in response to therapy were comparable with the two assays. The ICPCR assay should be useful in monitoring HIV-1 RNA levels both in natural history studies and in clinical trials of antiretroviral agents.     

Quantitation of human immunodeficiency virus type 1 DNA by competitive nested PCR assay

A quantitative competitive nested PCR assay was developed for quantifying HIV-1 proviral DNA in clinical samples. A competitor DNA was constructed from a conserved region of the HIV-1 gag gene by deleting a sequence of 18 base pairs. We quantitated HIV-1 proviral DNA copy number in clinical samples. Peripheral blood mononuclear cells (PBMCs) from 35 HIV-infected patients with a CD4 count range of 4-728 cell/mm3 were analyzed by this method. The copy numbers of HIV-1 DNA detected ranged between 518 to 67,340 copies per 10(6) CD4+ T-cells. The copy numbers correlated inversely with the CD4 counts.     

Quantitation of human immunodeficiency virus type 1 in the blood of infected persons

We used end-point-dilution cultures to measure the level of infectious human immunodeficiency virus type 1 (HIV-1) in peripheral-blood mononuclear cells (PBMC) and plasma of 54 infected patients who were not receiving antiviral chemotherapy. HIV-1 was recovered from the plasma and PBMC of every seropositive patient, but from none of 22 seronegative control subjects. The mean titers in plasma were 30, 3500, and 3200 tissue-culture-infective doses (TCID) per milliliter for patients with asymptomatic infection, the acquired immunodeficiency syndrome (AIDS), and the AIDS-related complex, respectively. In PBMC, the mean titers were significantly higher for symptomatic patients (AIDS, 2200, and AIDS-related complex, 2700 TCID per 10(6) PBMC) than asymptomatic patients (20 TCID per 10(6) PBMC). The values for the symptomatic patients were considered to indicate that at least 1 in 400 circulating mononuclear cells harbored HIV-1. The HIV-1 titers of seven patients with AIDS or AIDS-related complex treated with zidovudine for four weeks decreased significantly in plasma but not in PBMC. In addition, the mean titer in the plasma of 20 patients receiving long-term zidovudine treatment (130 TCID per milliliter) was 25-fold lower than the mean for comparable untreated patients with AIDS or AIDS-related complex. We conclude that the levels of HIV-1 in plasma and PBMC are much higher than previous estimates. This high degree of HIV-1 viremia raises the possibility that the direct cytopathic effect of this retrovirus alone may be sufficient to explain much of the pathogenesis of AIDS.     

Use of calibrated viral load standards for group M subtypes of human immunodeficiency virus type 1 to assess the performance of viral RNA quantitation tests

Optimal management of human immunodeficiency virus type 1 (HIV-1) disease requires accurate quantitation of viral RNA concentrations in plasma. Evidence for increasing geographic intermixing of HIV-1 subtypes makes equivalent quantitation of all subtypes essential. The performances of six quantitative viral RNA tests are described, for the first time, with calibrated viral isolates of diverse subtypes.     

Rapid and sensitive viral culture method for human immunodeficiency virus type 1.

Peripheral blood mononuclear cells from 142 consecutive patients with antibodies to human immunodeficiency virus type 1 (HIV-1) were cultured for HIV-1. All 72 patients with symptoms of HIV-1 infection were culture positive, as were 69 of 70 asymptomatic patients. Of the 142 patients, 132 (93%) were culture positive within 10 days after initiation of the culture.     

Quantitative and cost comparison of ultrasensitive human immunodeficiency virus type 1 RNA viral load assays: Bayer bDNA quantiplex versions 3.0 and 2.0 and Roche PCR Amplicor monitor version 1.5

Quantification of human immunodeficiency virus type 1 (HIV-1) RNA as a measure of viral load has greatly improved the monitoring of therapies for infected individuals. With the significant reductions in viral load now observed in individuals treated with highly active anti-retroviral therapy (HAART), viral load assays have been adapted to achieve greater sensitivity. Two commercially available ultrasensitive assays, the Bayer Quantiplex HIV-1 bDNA version 3.0 (bDNA 3.0) assay and the Roche Amplicor HIV-1 Monitor Ultrasensitive version 1.5 (Amplicor 1.5) assay, are now being used to monitor HIV-1-infected individuals. Both of these ultrasensitive assays have a reported lower limit of 50 HIV-1 RNA copies/ml and were developed from corresponding older generation assays with lower limits of 400 to 500 copies/ml. However, the comparability of viral load data generated by these ultrasensitive assays and the relative costs of labor, disposables, and biohazardous wastes were not determined in most cases. In this study, we used matched clinical plasma samples to compare the quantification of the newer bDNA 3.0 assay with that of the older bDNA 2.0 assay and to compare the quantification and costs of the bDNA 3.0 assay and the Amplicor 1.5 assay. We found that quantification by the bDNA 3.0 assay was approximately twofold higher than that by the bDNA 2.0 assay and was highly correlated to that by the Amplicor 1.5 assay. Moreover, cost analysis based on labor, disposables, and biohazardous wastes showed significant savings with the bDNA 3.0 assay as compared to the costs of the Amplicor 1.5 assay.     

Improvement of AMPLICOR human immunodeficiency virus type 1 viral load test (version 1.5) by addition of a coprecipitant during the RNA isolation step

The effect of the addition of a coprecipitant during the RNA isolation step on the analytical performance of the COBAS AMPLICOR human immunodeficiency virus type 1 (HIV-1) Monitor (version 1.5; Roche) viral load test was tested. Thirty-six specimens including patient samples, positive control samples, and negative control samples were processed in the presence and absence of the Pellet Paint coprecipitant. Specimens processed without the coprecipitant had lower RNA yields, as evidenced by a lower signal for the quantitation standard (QS). In addition, the results for all samples processed with the coprecipitant were acceptable on the basis of the optical density (OD) reading for the QS, whereas the result for one specimen processed without the coprecipitant was unacceptable on the basis of the OD reading for the QS, which required the assay to be repeated. Furthermore, the use of the coprecipitant improved the overall precision of the assay.     

Automated procedure for improving the RNA isolation step in viral load testing for human immunodeficiency virus

An automated RNA isolation procedure on the Qiagen BioRobot is described for performing viral load tests for human immunodeficiency virus (HIV) with the Amplicor HIV type 1 test. The new procedure improves the precision of the assay and requires significantly less labor than the presently used manual RNA isolation procedure.