A more precise HIV integration assay designed to detect small differences finds lower levels of integrated DNA in HAART treated patients

Yu, Jianqing; Wu, Te Lang; Liszewski, M. Kathryn; Dai, Jihong; Swiggard, William J.; Baytop, Clifford; Frank, Ian; Levine, Bruce L.; Yang, Wei; Theodosopoulos, Theodore V.; O’Doherty, Una

doi:10.1016/j.virol.2008.05.030

Cited by 73 publications

(101 citation statements)

References 49 publications

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“…Sorting of green fluorescent protein (GFP)-positive cells resulted in a population of cells with 1 proviral copy per cell (15)(16)(17). Peripheral blood mononuclear cells were isolated from 56 HIVinfected patients by density gradient centrifugation and frozen to Ϫ80°C at a cooling rate of 1°C/min in 90% fetal calf serum with 10% DMSO.…”

Section: Methodsmentioning

confidence: 99%

“…Over several years, the assay was further optimized, enabling its use in patient samples (7,8,(15)(16)(17)(18)(19)(20).…”

confidence: 99%

“…1). This PCR is used to set the threshold quantification cycle (Cq) value above which integrated copies can no longer be discerned from unintegrated copies (14,17 ). This solution only partially overcomes the problem, since very high concentrations of unintegrated HIV DNA may still lead to overestimation of proviral copies (17 ).…”

confidence: 99%

“…The percentage of positive reactions is compared to the mean percentage of positive reactions from a serial dilution of the standard curve run at an equal number of replicate reactions. This relation is nearly linear when Ͻ30% of the replicates are positive (17 ). If this threshold is surpassed, then the natural logarithm of the mean Cq values (lnCq) method is used.…”

confidence: 99%

“…Alu-HIV PCR was performed as described earlier (15,17 ) by use of 42 replicate reactions of a mix with gag-reverse HIV primer and Alu-forward primer as well as 42 replicates by use of only primers specific to HIV gag (HIV-only PCR). Samples were diluted to 2 or 10 g DNA/mL and distributed in replicate PCR reactions containing 25 L sample combined with 25 L master mix, resulting in an equivalent of approximately 7500 cells (1 ng/L PCR mix) or approximately 37 500 cells (5 ng/L PCR mix) per 50 L PCR replicate.…”

Section: Alu-hiv Pcrmentioning

confidence: 99%

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Quantification of Integrated HIV DNA by Repetitive-Sampling Alu-HIV PCR on the Basis of Poisson Statistics

Spiegelaere¹,

Malatinková²,

Lynch

et al. 2014

Clinical Chemistry

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BACKGROUND Quantification of integrated proviral HIV DNA by repetitive-sampling Alu-HIV PCR is a candidate virological tool to monitor the HIV reservoir in patients. However, the experimental procedures and data analysis of the assay are complex and hinder its widespread use. Here, we provide an improved and simplified data analysis method by adopting binomial and Poisson statistics. METHODS A modified analysis method on the basis of Poisson statistics was used to analyze the binomial data of positive and negative reactions from a 42-replicate Alu-HIV PCR by use of dilutions of an integration standard and on samples of 57 HIV-infected patients. Results were compared with the quantitative output of the previously described Alu-HIV PCR method. RESULTS Poisson-based quantification of the Alu-HIV PCR was linearly correlated with the standard dilution series, indicating that absolute quantification with the Poisson method is a valid alternative for data analysis of repetitive-sampling Alu-HIV PCR data. Quantitative outputs of patient samples assessed by the Poisson method correlated with the previously described Alu-HIV PCR analysis, indicating that this method is a valid alternative for quantifying integrated HIV DNA. CONCLUSIONS Poisson-based analysis of the Alu-HIV PCR data enables absolute quantification without the need of a standard dilution curve. Implementation of the CI estimation permits improved qualitative analysis of the data and provides a statistical basis for the required minimal number of technical replicates.

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Section: Methodsmentioning

confidence: 99%

“…Over several years, the assay was further optimized, enabling its use in patient samples (7,8,(15)(16)(17)(18)(19)(20).…”

confidence: 99%

Section: Alu-hiv Pcrmentioning

confidence: 99%

See 3 more Smart Citations

Quantification of Integrated HIV DNA by Repetitive-Sampling Alu-HIV PCR on the Basis of Poisson Statistics

Spiegelaere¹,

Malatinková²,

Lynch

et al. 2014

Clinical Chemistry

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Assays to Measure Latency, Reservoirs, and Reactivation

Siliciano

2017

Current Topics in Microbiology and Immunology

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HIV-1 persists even in patients who are successfully treated with combination antiretroviral therapy. The major barrier to cure is a small pool of latently infected resting CD4 T cells carrying an integrated copy of the viral genome that is not expressed while the cells remain in a resting state. Targeting this latent reservoir is a major focus of HIV-1 cure research, and the development of a rapid and scalable assay for the reservoir is a rate-limiting step in the search for a cure. The most commonly used assays are standard PCR assays targeting conserved regions of the HIV-1 genome. However, because the vast majority of HIV-1 proviruses are defective, such assays may not accurately capture changes in the minor subset of proviruses that are replication-competent and that pose a barrier to cure. On the other hand, the viral outgrowth assay that was used to initially define the latent reservoir may underestimate reservoir size because not all replication-competent proviruses are induced by a single round of T cell activation in this assay. Therefore, this assay is best regarded as a definitive minimal estimate of reservoir size. The best approach may be to measure all of the proviruses with the potential to cause viral rebound. A variety of novel assays have recently been described. Ultimately, the assay that best predicts time to viral rebound will be the most useful to the cure effort.

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HIV antibodies level as a marker of HIV persistence: the role of hepatitis C virus coinfection

Arca-Suárez

Cuevas

Pascual-Pérez

et al. 2020

Eur J Clin Microbiol Infect Dis

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A more precise HIV integration assay designed to detect small differences finds lower levels of integrated DNA in HAART treated patients

Cited by 73 publications

References 49 publications

Quantification of Integrated HIV DNA by Repetitive-Sampling Alu-HIV PCR on the Basis of Poisson Statistics

Quantification of Integrated HIV DNA by Repetitive-Sampling Alu-HIV PCR on the Basis of Poisson Statistics

Assays to Measure Latency, Reservoirs, and Reactivation

HIV antibodies level as a marker of HIV persistence: the role of hepatitis C virus coinfection

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