HIV-1 Resistance, Proviral DNA (RTI, PI, Integrase Inhibitors) and HIV-1 Resistance and Coreceptor Tropism, Proviral DNA

In infected cells, HIV-1 RNA is reverse-transcribed to DNA. The viral integrase enzyme then mediates the integration of the viral DNA into the chromosomes of the host cell. This integrated HIV DNA, known as proviral DNA, may remain latent or may be transcribed to produce new viral particles. Proviral DNA represents an archive of viral mutations that emerged through the course of a patient’s infection and may provide a historical record of the patient’s drug resistance mutations.

For patients with virologic suppression or low-level viremia (eg, less than 1,000 copies/mL), routine plasma RNA drug resistance testing may not be successful, because too little viral RNA is present to allow for amplification and sequencing. In contrast to HIV RNA, HIV proviral DNA remains present in a patient's cells even during virological suppression.

In certain cases, when RNA-based genotypic resistance testing is not possible, proviral DNA may be used to assess genotypic resistance. Several studies have presented data showing good concordance between proviral DNA resistance mutations and mutations detected in plasma RNA genotypes and/or related to previously administered antiretroviral drugs prior to virologic suppression.

Lübke et al investigated the concordance between viral plasma RNA and proviral DNA mutations in 48 treatment-naïve and 30 treatment-experienced patients.¹ Among all drug resistance-associated mutations detected by either method, 75% were detected in both RNA and DNA, 23% were found exclusively in RNA, and 2% were found exclusively in proviral DNA.

Porter et al investigated the clinical utility of proviral DNA resistance analysis for virologically suppressed patients in the SPIRIT study to evaluate the safety and efficacy of switching to a rilpivirine/emtricitabine/tenofovir regimen.² They found that 89% of occurrences of NRTI and NNRTI resistance-associated mutations reported by historical genotype were also detectable in proviral DNA.

Zaccarelli et al investigated proviral DNA resistance mutations in 149 virologically suppressed patients with 2 or more plasma RNA genotype tests performed prior to virologic suppression.³ They found that 51% of patients had proviral DNA primary resistance mutations that were previously identified in plasma RNA. Primary resistance mutations that were mostly detected in proviral DNA were found to be related to previously administered antiretrovirals and/or to drugs with a low genetic barrier to resistance. Thus, proviral DNA resistance testing may be useful for detecting archived resistance mutations in the setting of low viral load when prior plasma RNA genotypes are not available.

Allavena et al performed proviral DNA resistance testing on 69 virologically suppressed patients with no history of virologic failure.⁴ They found that 88% of proviral protease mutations and 76% of proviral reverse transcriptase mutations were concordant with plasma RNA genotypes obtained prior to initiation of therapy.

Villalobos et al investigated the use of proviral DNA resistance testing in patients with persistent low-level viremia for whom plasma RNA genotyping was unsuccessful and found that 11 of 19 patients harbored resistance-associated mutations.⁵

In some situations, yes. The 2024 International Antiviral Society USA (IAS USA) guidelines recommend using proviral DNA genotyping for virologically suppressed patients being evaluated for a switch to a long-acting cabotegravir plus long-acting rilpivirine regimen. “If rilpivirine-associated mutations are present on genotypic testing, long-acting cabotegravir plus long-acting rilpivirine should be avoided (evidence rating: AIa).”⁶

The DHHS guidelines indicate that proviral DNA genotyping using next-generation sequencing may be considered in virologically suppressed patients; however, these assays have not been fully clinically validated, and they may miss some or all previous drug resistance mutations.⁷

Proviral DNA resistance tests should only be used in the setting of a low viral load. The DHHS guidelines and the 2024 IAS USA guidelines recommend obtaining a plasma RNA genotype in patients failing antiretroviral therapy.^6,7 However, both guideline documents note that genotypic testing may be unsuccessful at low viral loads of between 500 and 1,000 HIV RNA copies/mL.

The DHHS guidelines suggest that if HIV RNA genotypic drug-resistance testing is unsuccessful or unavailable due to low HIV RNA levels, a proviral DNA resistance test can be considered.⁶

DNA is extracted from EDTA whole blood, and the entire pol gene—including the protease (codons 1-99), reverse transcriptase (codons 1-560), and integrase (codons 1-288) regions—is amplified by polymerase chain reaction (PCR). The PCR product is then sequenced by next-generation sequencing (NGS), followed by bioinformatic analysis to filter out nonviable viral species and to identify resistance-associated mutations present in 10% or more of the viral species. 

Predicted or probable resistance is reported for each NRTI, NNRTI, PI, and integrase inhibitor (INI). Resistance-associated mutations present in 10% or more of the viral species sequenced are reported. The HIV-1 viral subtype is also reported. 

Yes. In this circumstance use test code 94810(X), which provides a panel that includes both tests. Three mL whole blood (minimum: 1.2 mL) is required. See the HIV-1 Coreceptor Tropism Proviral DNA | Quest Diagnostics FAQ for more information on the Quest proviral DNA tropism test.

Please collect 2 mL (minimum 0.6 mL) whole blood in a lavender-top EDTA tube. If also ordering proviral DNA tropism testing as part of panel test code 94810(X), collect 3 mL (minimum 1.2 mL) whole blood.

• The PCR primers used for proviral DNA resistance testing are able to amplify HIV-1 subtype B and many major non-B subtypes including A, C, BF, D, AE, AG, and H. Other non-B subtypes or HIV-1 strains with uncommon mutations may not be amplified in the assay.
• Data on the clinical performance of proviral DNA resistance testing are still limited, and the clinical utility has yet to be fully established.^6,7
  • Some resistance mutations previously found in plasma viral RNA may no longer be detectable in proviral DNA.^3,8
  • Some archived resistance mutations may only be detectable in proviral DNA, and their clinical significance is not known.^3,9,10,11 Although the proviral DNA compartment archives many resistance mutations that emerged throughout the course of infection, this archive should not be considered comprehensive. Cumulative plasma RNA genotypes may provide substantial additional information.¹⁰