607 Detection of Minority Drug-resistant Variants of HIV-1 During Virologic Failure of Lamivudine, Indinavir, and Zidovudine or Stavudine in ACTG 320 C. Dykes*1, J. Najjar1, R. Bosch2, M. Wantman2, M. Furtado3, S. Hart4, S. Hammer5, L. M. Demeter1 1Univ of Rochester Sch of Med and Dentistry, NY; 2Harvard Sch of Public Hlth, Boston, MA; 3Celera Diagnostics/ABI, Foster City, CA; 4Frontier Sci, Buffalo, NY; and 5Columbia Univ, New York, NY
Background: HIV-1 isolates obtained early during failure on a regimen of Indinavir (IDV), Lamivudine (3TC), and a 2nd nRTI commonly have the 3TC-R mutation (mutn) M184V, but rarely have mutns conferring Indinavir-resistance (IDV-R). We sought to determine whether IDV-R minority variants exist in plasma from patients (pts) who were failing an IDV- and 3TC-containing regimen.
Methods: Pts enrolled in ACTG 320 had CD4 < 200 and were highly nRTI-experienced but 3TC- and PI-naive at entry. Pts were randomized to receive IDV+3TC+ (Stavudine [d4T] or Zidovudine [AZT]) vs 2 nRTIs alone. Plasma viral load (VL) was measured at wks 0, 4, 8, 24, and 40 after the conclusion of the study. Stored plasma samples were obtained from 10 pts randomized to the IDV arm, who had a wk 0 VL of > 10,000 c/mL; developed VL suppression to < 1,000 copies/mL, followed by a subsequent increase; and had sufficient sample volume stored for additional testing. Four (4) out of 10 pts had an IDV-R mutation on bulk sequencing (M46I or V82A) at the failure visit, and were excluded from subsequent clonal analyses. HIV RNA was extracted from pt plasma, and a region encompassing PR and RT was amplified by RT-PCR using a modification of the ViroSeq HIV Genotyping kit that allows propagation of amplified DNA in E. coli without degradation by bacterial uracil N glycosylase. Duplicate RT-PCRs from each sample were pooled. Half was used for cloning (> 30 clones/specimen) and half for bulk sequencing. The PR region of purified DNA from each clone was sequenced using the ViroSeq kit reagents. Two-sided 95% CIs were used to assess the frequencies of minority variants and Fisher's exact test to compare time points.
Results: One (1) of 6 pts without IDV-R on bulk sequencing had the IDV-R mutn,V82A, in 9/30 (30%, 95% CI: 15%-49%) clones at the first documented failure visit (wk 24). V 82A was not present at wk 0 (0/32 clones, CI: 0%-11%, p < 0.001 c/t wk 24). This minority variant was still present at wk 40 (6/36, 17%, CI: 6%-33%, p = 0.25 c/t wk 24), although it had not increased significantly in frequency despite continued treatment with IDV.
Conclusions: Drug-resistant minority variants can be present in pts failing a regimen containing IDV, 3TC and either d4T or AZT, but may not be detected by bulk sequencing. This technical limitation should be considered when using resistance testing to assist in selecting a salvage antiretroviral regimen. Further study is needed to determine whether such minority variants significantly impact treatment outcome.
