Use of a High Resolution Melting (HRM) Assay to Compare Gag, Pol, and Env Diversity in Adults with Different Stages of HIV Infection

Abstract

BackgroundCross-sectional assessment of HIV incidence relies on laboratory methods to discriminate between recent and non-recent HIV infection. Because HIV diversifies over time in infected individuals, HIV diversity may serve as a biomarker for assessing HIV incidence. We used a high resolution melting (HRM) diversity assay to compare HIV diversity in adults with different stages of HIV infection. This assay provides a single numeric HRM score that reflects the level of genetic diversity of HIV in a sample from an infected individual.MethodsHIV diversity was measured in 203 adults: 20 with acute HIV infection (RNA positive, antibody negative), 116 with recent HIV infection (tested a median of 189 days after a previous negative HIV test, range 14–540 days), and 67 with non-recent HIV infection (HIV infected >2 years). HRM scores were generated for two regions in gag, one region in pol, and three regions in env.ResultsMedian HRM scores were higher in non-recent infection than in recent infection for all six regions tested. In multivariate models, higher HRM scores in three of the six regions were independently associated with non-recent HIV infection.ConclusionsThe HRM diversity assay provides a simple, scalable method for measuring HIV diversity. HRM scores, which reflect the genetic diversity in a viral population, may be useful biomarkers for evaluation of HIV incidence, particularly if multiple regions of the HIV genome are examined.

HIV Diversity as a Biomarker for HIV Incidence Estimation: Including a High-Resolution Melting Diversity Assay in a Multiassay Algorithm

“…The HRM diversity assay was performed as previously described (12,16) using the LightScanner HR-1 (BioFire Diagnostics, Inc., Salt Lake City, UT). Briefly, a region of the HIV genome is amplified, followed by nested amplification of a smaller region in the presence of a fluorescent duplex-dependent DNA dye (LCGreen Plus, BioFire Diagnostics, Inc.).…”

“…Because the genetic diversity of HIV changes over the course of disease, it may be a useful biomarker for estimating HIV incidence (12). A variety of methods for assessing HIV diversity are being explored as tools for estimating HIV incidence, including the quantification of ambiguous nucleotide calls and other computational approaches based on analyses of data from traditional or next-generation sequencing (13,14).…”

“…A variety of methods for assessing HIV diversity are being explored as tools for estimating HIV incidence, including the quantification of ambiguous nucleotide calls and other computational approaches based on analyses of data from traditional or next-generation sequencing (13,14). We recently developed an assay that quantifies the level of genetic diversity in HIV populations without the use of sequencing (12,15). This assay is based on high-resolution melting (HRM) and can be used to measure HIV diversity in different regions of the HIV genome.…”

“…This assay is based on high-resolution melting (HRM) and can be used to measure HIV diversity in different regions of the HIV genome. The level of genetic diversity in each region is reported as a single numeric HRM score that corresponds to the width of the DNA melting peak (12,(15)(16)(17). HRM scores correlate highly with diversity measures obtained from next-generation sequencing data (18).…”

“…HRM scores correlate highly with diversity measures obtained from next-generation sequencing data (18). Previous studies have shown that the HRM diversity assay is useful for discriminating between recent and nonrecent infections (12,19). In this report, we evaluated whether the HRM diversity assay can be used in place of CD4 cell count testing as part of a 4-assay MAA without compromising the performance of the testing algorithm.…”