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HIV-1 Resistance, Proviral DNA (RTI, PI, Integrase Inhibitors)Test code(s) 94807(X), 94810(X)
Question 1. What is 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 (e.g., 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 genotypes 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.
Question 3. Can proviral DNA testing be used to guide regimen switches in virologically suppressed patients?
In some situations, yes. As noted in the 2016 International Antiviral Society USA (IAS USA) guidelines, “a proviral DNA genotype test may be helpful” for virologically suppressed patients being considered for a regimen change if their treatment and drug resistance history is not available.6
Several lines of evidence support this use. Proviral DNA tropism testing in virologically suppressed patients being evaluated for a switch to a CCR5 antagonist, which has been evaluated in a number of studies and used clinically for a number of years, is described here: https://education.questdiagnostics.com/faq/FAQ87. Studies have also shown good concordance of proviral DNA resistance testing with plasma RNA-based testing, as described in FAQ question 2 above.
In addition, Singh et al conducted a retrospective study of 140 patients who had access to a commercially available proviral DNA resistance test.5 Concordance with historical RNA genotypes was 72.5% for NRTI, 72.5% for NNRTI, and 76.8% for PI resistance-associated mutations. Of the 140 patients, 79 underwent an antiretroviral regimen change as a result of the proviral DNA test result. Supporting the utility of this approach, 85% of these patients maintained an undetectable viral load at 3 months post-therapy switch.
Question 4. Should proviral DNA resistance tests be used in patients with virologic failure?
Proviral DNA resistance tests should only be used in the setting of a low viral load. The DHHS guidelines and the 2016 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 IAS USA guidelines suggest that if an RNA genotype cannot be obtained in this situation, “proviral DNA assays to estimate archived resistance may be considered.”6
Question 5. How do you perform proviral DNA resistance testing?
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.
Question 6. How is predicted HIV drug resistance, based on proviral DNA, reported?
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.
Question 7. Can I order both proviral DNA resistance and proviral DNA tropism testing?
Yes. In this circumstance use test code 94810(X), which provides a panel that includes both tests. 3 mL whole blood (minimum: 1.2 ml) is required. See https://education.questdiagnostics.com/faq/FAQ87 for more information on our proviral DNA tropism test.
Question 8. What are the sample requirements for proviral DNA resistance testing?
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.
Question 9. What are some of the limitations of the proviral DNA resistance test?
- 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.3,8,9
- Some archived resistance mutations may only be detectable in proviral DNA, and their clinical significance is not known.3,9,10 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.11
- Lubke N, Di Cristanziano V, Sierra S, et al. Proviral DNA as a target for HIV-1 resistance analysis. Intervirology. 2015;58:184-189.
- Porter DP, Daeumer M, Thielen A, et al. Emergent HIV-1 drug resistance mutations were not present at low-frequency at baseline in non-nucleoside reverse transcriptase inhibitor-treated subjects in the STaR study. Viruses. 2015;7:6360-6370.
- Zaccarelli M, Santoro MM, Armenia D, et al. Genotypic resistance test in proviral DNA can identify resistance mutations never detected in historical genotypic test in patients with low level or undetectable HIV-RNA. J Clin Virol. 2016;82:94-100.
- Allavena C, Rodallec A, Leplat A, et al. Interest of proviral HIV-1 DNA genotypic resistance testing in virologically suppressed patients candidate for maintenance therapy. J Virol Methods. 2018;251:106-110.
- Singh H, Jones S, Vaamonde C, Wilkin T. Application of GenoSure archive in clinical practice. Open Forum Infect Dis. 2016;3(Suppl 1):1507.
- Gunthard HF, Saag MS, Benson CA, et al. Antiretroviral drugs for treatment and prevention of HIV infection in adults: 2016 recommendations of the International Antiviral Society-USA Panel. JAMA. 2016;316:191-210.
- Panel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the use of antiretroviral agents in adults and adolescents living with HIV. Department of Health and Human Services. http://aidsinfo.nih.gov/contentfiles/lvguidelines/AdultandAdolescentGL.pdf. Accessed 12/26/2017.
- Delaugerre C, Braun J, Charreau I, et al. Comparison of resistance mutation patterns in historical plasma HIV RNA genotypes with those in current proviral HIV DNA genotypes among extensively treated patients with suppressed replication. HIV Medicine. 2012;13:517-525.
- Gantner P, Morand-Joubert L, Sueur C, et al. Drug resistance and tropism as markers of the dynamics of HIV-1 DNA quasispecies in blood cells of heavily pretreated patients who achieved sustained virological suppression. J Antimicrob Chemother. 2016;71:751-761.
- Falasca F, Montagna C, Maida P, et al. Analysis of intracellular human immunodeficiency virus (HIV)-1 drug resistance mutations in multi-failed HIV-1-infected patients treated with a salvage regimen: 72-week follow-up. Clin Microbiol Infect. 2013;19:E318-321.
- Wirden M, Soulie C, Valantin MA, et al. Historical HIV-RNA resistance test results are more informative than proviral DNA genotyping in cases of suppressed or residual viraemia. J Antimicrob Chemother. 2011;66:709-712.