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

ABSTRACT: 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.

SUBMITTER: Rutsaert S

PROVIDER: S-EPMC6250682 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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

Rutsaert Sofie S De Spiegelaere Ward W Van Hecke Clarissa C De Scheerder Marie-Angélique MA Kiselinova Maja M Vervisch Karen K Trypsteen Wim W Vandekerckhove Linos L

Scientific reports 20181122 1

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 ...[more]

PMID: 30467426

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Project description:IntroductionThe latent reservoir is the main barrier on the road to HIV cure, and clinical approaches towards eradication are often evaluated by their effect on proviral DNA. To ensure inclusiveness and representativeness in HIV cure studies, proviral DNA quantification assays that are able to detect all common circulating HIV clades are urgently needed. Here, three HIV DNA assays targeting three different genomic regions were evaluated for their sensitivity and subtype-tolerance using digital PCR.MethodsA subtype-B-specific assay targeting gag (GAG) and two assays targeting conserved sequences in ltr and pol (LTR and JO) were assessed for their sensitivity and subtype-tolerance in digital PCR (Bio-Rad QX200), using a panel of serially diluted subtype reference plasmids as well as a panel of clinical isolates. Both panels represent subtypes A, B, C, D, F, G and circulating recombinant forms (CRFs) AE and AG, which together are responsible for 94% of HIV infections worldwide.ResultsHIV subtype was observed to greatly affect HIV DNA quantification results. Robust regression analysis of the serially diluted plasmid panel showed that the GAG assay was only able to linearly quantify subtype B, D and G isolates (4/13 reference plasmids, average R2 = 0.99), whereas LTR and JO were able to quantify all tested isolates (13/13 reference plasmids, respective average R2 = 0.99 and 0.98). In the clinical isolates panel, isolates were considered detectable if all replicates produced a positive result. The GAG assay could detect HIV DNA in four out of five subtype B and one out of two subtype D isolates, whereas the LTR and JO assays detected HIV DNA in all twenty-nine tested isolates. LTR and JO results were found to be equally precise but more precise than GAG.ConclusionsThe results demonstrate the need for a careful validation of proviral reservoir quantification assays prior to investigations into non-B subtype reservoirs. The LTR and JO assays can sensitively and reliably quantify HIV DNA in a panel that represents the worldwide most prevalent subtypes and CRFs (A, B, C, D, AE, F, G and AG), justifying their application in future trials aimed at global HIV cure.

Dataset Information

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

Publications

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

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