Simple PCR assays improve the sensitivity of HIV-1 subtype B drug resistance testing and allow linking of resistance mutations

Abstract

Background: The success of antiretroviral therapy is known to be compromised by drug-resistant HIV-1 at frequencies detectable by conventional bulk sequencing. Currently, there is a need to assess the clinical consequences of low-frequency drug resistant variants occurring below the detection limit of conventional genotyping. Sensitive detection of drug-resistant subpopulations, however, requires simple and practical methods for routine testing.

Methodology: We developed highly-sensitive and simple real-time PCR assays for nine key drug resistance mutations and show that these tests overcome substantial sequence heterogeneity in HIV-1 clinical specimens. We specifically used early wildtype virus samples from the pre-antiretroviral drug era to measure background reactivity and were able to define highly-specific screening cut-offs that are up to 67-fold more sensitive than conventional genotyping. We also demonstrate that sequencing the mutation-specific PCR products provided a direct and novel strategy to further detect and link associated resistance mutations, allowing easy identification of multi-drug-resistant variants. Resistance mutation associations revealed in mutation-specific amplicon sequences were verified by clonal sequencing.

Significance: Combined, sensitive real-time PCR testing and mutation-specific amplicon sequencing provides a powerful and simple approach that allows for improved detection and evaluation of HIV-1 drug resistance mutations.

Figure 1. Principle of the real-time PCR assay.

A. HIV-1 template generated from RT-PCR of viral RNA is subjected to both total copy and mutation-specific real-time reactions. B. The difference in the total copy and mutation-specific reactions (ΔCT) is used to differentiate mutant and wildtype specimens. In this example, the experimental cutoff is a ΔCT of 10.5 cycles. A mutation-specific CT within 10.5 cycles of the total copy reaction CT would indicate the presence of mutant virus.

Figure 2. Mutation-specific assay reactivity on plasmids.

Cloned L90M (A.) and K103N (B.) mutant virus sequence was diluted 10-fold, from 100% to 0.001%, in backgrounds of wildtype sequence to determine assay detection limits. Plotted are the mean ΔCT versus log₁₀ of the mutant dilution series (•), and the mean ΔCT for wildtype sequence alone (▪). The lower detection limit (lower dotted line) was placed at the ΔCT equivalent to 0.5 log₁₀ below (0.5-log greater reactivity than) the wildtype ΔCT. Dilutions that fall outside the linear range are not considered. For comparison, the mutant virus frequency equivalences for the established clinical cutoffs are also shown (dashed line).

Figure 3. Assay reactivities with clinical samples having sequence-detectable mutations and with pre-antiretroviral wildtype virus samples.

The range of reactivity for each assay is shown for wildtype and mutant samples. The upper and lower ΔCT and the mean (hash) for each group are indicated. Assay cutoffs (horizontal line) were established to exclude all wildtype viruses from the pre-antiretroviral era.

Figure 4. Detection of other associated resistance mutations in mutation-specific amplicons.

A. The undecipherable codon 215 in the bulk sequence of this sample was resolved (positive) with the T215Y test. The sequence of the T215Y-positive amplicon showed that the mutations present in the bulk sequence were linked. B. The low-frequency K103N amplicon sequence from this sample uncovered another previously undetected mutation, M184V. 215X, undecipherable codon 215.