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Standard Genotype Tests May Not Detect Low Level Resistance; new Super-Sensitive Resistance Tests
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Reported for NATAP by David Margolis, MD, University of Texas Southwestern MC
11th Annual Retrovirus Conference
Feb 8-11, 2004, San Francisco
New "Super-Sensitive" Test Detects Low Level NNRTI ResistanceThe persistence of drug-resistant HIV-1 variants is important for designing optimal antiretroviral treatment and prevention strategies. Currently clinically available methods of analyzing the genetic sequence of HIV, and thereby predicting the sensitivity of that virus to various antivirals (genotyping), detect only the predominant species of virus circulating in the blood. Minor variants comprising less than 10 to 25% of the viral population are not detected. Palmer and colleagues at the HIV Drug Resistance Program at the NIH (abstract 37) developed a high-throughput real-time RT-PCR assay that quantifies virus resistant to non-nucleoside reverse transcriptase inhibitors (NNRTIs) at frequencies of 0.1% or lower. Super-sensitive detection of resistance to NNRTIs is of importance as single mutations can confer high-level resistance to this class of antiviral.
Plasma samples were obtained from patients before and during NNRTI therapy and after its cessation. They found three patterns of the emergence of NNRTI resistance. In the first pattern resistance simply persisted after discontinuing the NNRTI efavirenz (EFV; Sustiva). The variant containing a change at RT amino acid 103 (K103N) persisted at a frequency of 7-15% in patients, and for at least 2.5 years without further NNRTI therapy.
A second pattern of "codon switching" was seen. The K103N amino acid mutation is encoded by a change of DNA code to either AAT or AAC. The AAC and AAT switch of K-to-N-at-103 were present in different proportions during EFV exposure, and these proportions changed after EFV was stopped. However, as resistance is encoded by either AAT or AAC, it persisted off EFV.
In the third pattern seen in some patients, K103N resistance disappeared. However, as the authors called this "apparent" disappearance, the implication made was that drug resistance was still there at a very low level but undetectable by this super-sensitive assay.
Standard Genotype Test Did Not Detect Resistance
For the same group, Mellors (abstract 39) presented the logical extension of these findings. In an older ACTG study 398, 212 NNRTI-experienced and 269 NNRTI-naïve patients were randomized to therapy combination that did or did not include EFV. Genotypes prior to this therapy were obtained in 452 of the 481 patients by a standard method, or the super-sensitive single genome RT-PCR and sequencing system. Failure of EFV-containing therapy was associated with NNRTI experience (p <0.001), baseline NNRTI mutations (p <0.001), and development of EFV resistance (p <0.001). Standard genotype did not identify NNRTI mutations in baseline samples from 48 (22%) NNRTI-experienced patients. Virologic response in this group with low-level NNRTI mutations detected at baseline was no better than in the experienced group with baseline NNRTI mutations, suggesting that standard genotype was insensitive for NNRTI-resistant variants and that this low level of NNRTI resistance matters.
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