The Importance of Testing Genotypic Resistance in Proviral DNA of Patients Fully Responding to Highly Active Antiretroviral Therapy

To the Editor: In the September first issue of Journal of Acquired Immune Deficiency Syndrome, RS Diaz et al1 report the detection of resistance mutations in the proviral DNA of patients with plasma viremia levels below 400 copies per milliliter at the end of a clinical trial of indinavir-based highly active antiretroviral therapy (HAART). Because the limit of detection of the assay used for measuring HIV RNA was 400 copies per milliliter, the authors could not rule out the possibility that low-level viremia between 50 and 400 copies in these subjects was a cause of viral evolution and accumulation of resistance. In addition, in the absence of follow-up, they could not associate the presence of mutations in proviral DNA with subsequent virologic failure. In conducting a secondary analysis of the Istituto Superiore di Sanità-Pulsed Antiretroviral Therapy (ISS-PART), a randomized controlled clinical trial evaluating intermittent versus continuous HAART in patients with persistent suppression of viral replication,2 we made some interesting observations that confirm and expand Diaz's hypothesis. Briefly, we measured drug resistance mutations in proviral DNA at the beginning of the study and after 2-year follow-up in 36 subjects of the control arm, who were randomized to continuing their HAART regimen. Proviral DNA was extracted and amplified from peripheral blood mononuclear cells and sequenced by the TruGene assay (Bayer HealthCare, Tarrytown, NY). Resistance-associated mutations were classified according to the 2007 International AIDS Society USA classification. The main characteristics of patient population were as follows: females, 27.8%; median age, 37 years; median HAART duration, 28.5 months (range, 11-46 months); median CD4 cell count, 748 (range 347-1377) per μl; median HIV-1 DNA 65 (range 10-1497) copies per 106 peripheral blood mononuclear cells. All subjects had plasma viremia <50 copies per milliliter (median value by an ultra-ultrasensitive assay: 2.5, range 2.5-82 copies/mL). According to the ISS PART inclusion criteria, all were on first line HAART; however, they could have changed their regimen once for toxicity or adherence reasons. HAART regimens at study entry included unboosted protease inhibitors in 5 of 36 and nonnucleosidic inhibitors of reverse transcriptase in 31 of 36 subjects. Baseline genotypic analysis of proviral DNA showed the presence of 14 resistance-associated mutations (13 in the reverse transcriptase and 1 in the protease gene) in 10 of 36 subjects (27%). The most frequent mutation was the M41L (4 cases), followed by the M184V/I (3 cases), the V108I (2 cases), and the V118I, L210W, K70R, T215Y, and M46L, each in single cases. During the following 24 months, virologic failure, defined as the detection of HIV-1 RNA values >400 copies per milliliter on at least 1 occasion, occurred in 5 subjects at different times. The baseline characteristics of these subjects (Table 1) were not different respect to those of the 31 subjects who maintained undetectable HIV-1 RNA values. However, by dividing them according to the presence of mutations in proviral DNA, failure occurred in 4 of 10 subjects with mutations and in 1 of 26 with a wild-type genotype (Fisher exact test, P = 0.0152).TABLE 1: Characteristics of Patients Experiencing Virologic Failure During 2-Year Follow-UpIn summary, in a population of HIV+ subjects fully responder to their first line HAART, we observed an association between the presence of mutations in proviral DNA and the occurrence of virologic failure in the subsequent 2 years. Although plasma HIV-1 RNA remains the material of choice for assessing antiretroviral drug resistance and guiding therapeutic decisions,3 several studies, mostly conducted in untreated or failing patients, have underlined the value of proviral DNA as an additional source of information on the total burden of resistance in an individual.4,5 Our data, stemming from a chance observation, support the hypothesis of Diaz et al1 that in HAART responder subjects, where plasma HIV-1 RNA cannot be sequenced by the commercially available assays, proviral DNA may represent an alternative and easy to obtain source to investigate the genetic evolution of the predominant viral species in an individual. Prospective studies should be conducted to verify the predictive capacity of HIV-1 DNA genotype and its value in clinical practice. Lucia Palmisano, MD Clementina M. Galluzzo, BSc Marina Giuliano, MD Department of Therapeutic Research and Medicines Evaluation Istituto Superiore di Sanità Rome, Italy