Instability of 8E5 calibration standard revealed by digital PCR risks inaccurate quantification of HIV DNA in clinical samples by qPCR

Busby, Eloise; Whale, Alexandra S.; Ferns, R. Bridget; Grant, Paul; Morley, Gary; Campbell, Jonathan E.; Foy, Carole A.; Nastouli, Eleni; Huggett, Jim F.; Garson, Jeremy A.

doi:10.1038/s41598-017-01221-5

Cited by 34 publications

(35 citation statements)

References 34 publications

(46 reference statements)

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“…Of note, RNA quantification represents cDNA molecules and should therefore be corrected for cDNA synthesis efficiency [ 27 ]. Accurate quantification by qPCR is based on the quality of the standard curve: instability of the standard curve can lead to inaccurate HIV DNA quantification [ 28 ]. Additionally, Cq values in qPCR that arise from the standard and the samples are based on amplification efficiencies, and several factors may confound their correct interpretation.…”

Section: Challenges and Benefits Of Droplet Digital Pcrmentioning

confidence: 99%

Digital PCR as a tool to measure HIV persistence

et al. 2018

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Although antiretroviral therapy is able to suppress HIV replication in infected patients, the virus persists and rebounds when treatment is stopped. In order to find a cure that can eradicate the latent reservoir, one must be able to quantify the persisting virus. Traditionally, HIV persistence studies have used real-time PCR (qPCR) to measure the viral reservoir represented by HIV DNA and RNA. Most recently, digital PCR is gaining popularity as a novel approach to nucleic acid quantification as it allows for absolute target quantification. Various commercial digital PCR platforms are nowadays available that implement the principle of digital PCR, of which Bio-Rad's QX200 ddPCR is currently the most used platform in HIV research. Quantification of HIV by digital PCR is proving to be a valuable improvement over qPCR as it is argued to have a higher robustness to mismatches between the primers-probe set and heterogeneous HIV, and forfeits the need for a standard curve, both of which are known to complicate reliable quantification. However, currently available digital PCR platforms occasionally struggle with unexplained false-positive partitions, and reliable segregation between positive and negative droplets remains disputed. Future developments and advancements of the digital PCR technology are promising to aid in the accurate quantification and characterization of the persistent HIV reservoir.

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Section: Challenges and Benefits Of Droplet Digital Pcrmentioning

confidence: 99%

Digital PCR as a tool to measure HIV persistence

et al. 2018

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“…ddPCR utilizes absolute quantification rather than relative quantification based off extrapolating from a standard curve in traditional qPCR. Eliminating the error from user generated or instable standard curves enables ddPCR to be more accurate than qPCR [ 105 ]. Furthermore, PCR inhibition is limited since the bulk PCR reaction is partitioned into circa 20,000 individual reactions.…”

Section: Dynamics Of Hiv Infected Populationsmentioning

confidence: 99%

The role of integration and clonal expansion in HIV infection: live long and prosper

Anderson¹,

Maldarelli²

2018

Retrovirology

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Integration of viral DNA into the host genome is a central event in the replication cycle and the pathogenesis of retroviruses, including HIV. Although most cells infected with HIV are rapidly eliminated in vivo, HIV also infects long-lived cells that persist during combination antiretroviral therapy (cART). Cells with replication competent HIV proviruses form a reservoir that persists despite cART and such reservoirs are at the center of efforts to eradicate or control infection without cART. The mechanisms of persistence of these chronically infected long-lived cells is uncertain, but recent research has demonstrated that the presence of the HIV provirus has enduring effects on infected cells. Cells with integrated proviruses may persist for many years, undergo clonal expansion, and produce replication competent HIV. Even proviruses with defective genomes can produce HIV RNA and may contribute to ongoing HIV pathogenesis. New analyses of HIV infected cells suggest that over time on cART, there is a shift in the composition of the population of HIV infected cells, with the infected cells that persist over prolonged periods having proviruses integrated in genes associated with regulation of cell growth. In several cases, strong evidence indicates the presence of the provirus in specific genes may determine persistence, proliferation, or both. These data have raised the intriguing possibility that after cART is introduced, a selection process enriches for cells with proviruses integrated in genes associated with cell growth regulation. The dynamic nature of populations of cells infected with HIV during cART is not well understood, but is likely to have a profound influence on the composition of the HIV reservoir with critical consequences for HIV eradication and control strategies. As such, integration studies will shed light on understanding viral persistence and inform eradication and control strategies. Here we review the process of HIV integration, the role that integration plays in persistence, clonal expansion of the HIV reservoir, and highlight current challenges and outstanding questions for future research.

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“…Overall, this study highlights the limitation of comparing results between studies using different assays for HIV DNA quantification and the importance of identifying a validated HIV-1 DNA assay that accurately measures total HIV-1 DNA for its further implementation in HIV-1 clinical trials. Since PCR-based HIV DNA quantification has a good inter-laboratory reproducibility 3 , 24 , a validated assay will allow the comparison of results between different clinical studies and different laboratories but good standards will be necessary for validation of the method, especially for qPCR which requires an stable external calibrator 25 . Lastly, a precise quantification of HIV-1 DNA can guide the selection of patients participants in HIV-1 cure studies and early diagnosis of infants of seropositive mothers.…”

Section: Discussionmentioning

confidence: 99%

In-depth validation of total HIV-1 DNA assays for quantification of various HIV-1 subtypes

Rutsaert

Spiegelaere

Hecke

et al. 2018

Sci Rep

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HIV-1 DNA quantification serves as an important reservoir biomarker in HIV cure trials. However, the high genetic diversity of HIV-1 represented by different subtypes may bring inaccuracy in quantifying HIV-1 DNA and a sensitive and validated assay covering diverse HIV-1 subtypes is lacking. Therefore, we cross-validated total HIV-1 DNA assays described in literature using a three-step comparative analysis. First, a bioinformatics tool was developed in-house to perform an in silico evaluation of 67 HIV-1 DNA assays. Secondly, these selected assays were in vitro validated using a panel of different HIV-1 subtypes and, finally, ex vivo assessed on selected patient samples with different HIV-1 subtypes. Our results show that quantification of HIV-1 DNA substantially differs between assays and we advise five best performing HIV-1 DNA assays for ddPCR and qPCR (Schvachsa_2007, Viard_2004, Heeregrave_2009, Van_der_Sluis_2013, Yu_2008 and Yun_2002). This in-depth analysis of published HIV-1 DNA assays indicates that not all assays guarantee an optimal measurement of HIV-1 DNA, especially when looking across subtypes. Using an in-depth cross-validation, we were able to validate HIV-1 DNA assays that are suitable for quantification of HIV-1 DNA in a wide variety of HIV-1 infected patients.

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Instability of 8E5 calibration standard revealed by digital PCR risks inaccurate quantification of HIV DNA in clinical samples by qPCR

Cited by 34 publications

References 34 publications

Digital PCR as a tool to measure HIV persistence

Digital PCR as a tool to measure HIV persistence

The role of integration and clonal expansion in HIV infection: live long and prosper

In-depth validation of total HIV-1 DNA assays for quantification of various HIV-1 subtypes

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