Background: The DHHS HIV treatment guideline from 10-29-04 states, "Drug resistance testing is not advised for persons with viral loads <1,000 copies/mL, since amplification of virus is unreliable." However, the ability to have reliable genotypic resistance results for HIV patients with persistent low levels of viremia may prove to be valuable for guiding therapeutic decisions. We did this study to validate resistance testing on viral loads <1,000 c/mL by comparing high to low HIV RNA samples.
Methods: Twenty consecutive patient samples were selected with multiple resistance mutations leading to complex resistance profiles. The samples were diluted to ~100 c/mL; median 136, range <75-254 c/mL (bDNA, Bayer). Resistance patterns were obtained using standard commercial methods: manual extraction of 140-uL plasma with the QiaAmp® Viral RNA kit (Qiagen) and amplification with the TruGene® HIV-1 Genotyping Kit (Bayer). Seven dilutions did not amplify the first time. The diluted virus was then concentrated by ultracentrifugation at 23,500g, 75 min, 4°C and reassayed. All 99 Protease (20 PI resistance) codons and 208 Reverse
Transcriptase (22 NRTI/NtRTI & 14 NNRTI resistance) codons were sequenced. Three techs processed and analyzed specimens in a blinded manner.
Results: There was high concordance between the 20 "
original vs. diluted" paired samples (Table). All antiretroviral drug resistance interpretations were identical for 16 pairs. The remaining 4 pairs collectively had 2 PI, 5 NRTI/NtRTI, and 3NNRTI resistance interpretation discordances based on codon mismatches at PR 84 and 90 and RT 74, 108, and 210.
Conclusion: The TruGene® assay can reliably amplify circulating HIV <1,000 c/mL to produce clinically acceptable drug resistance interpretations.
Summary of HIV-1 Genotype Percent Concordances for 20 Original vs. Diluted Clinical Samples| Resistance Interpretationsa (n) | Codon Mutations (n) | Base Mutations (n) | All Codons (n) | All Bases (n) |
| ..NRTI/NtRTI 96.4% (140) | 94.5% (91) | 95.5% (112) | 98.9% (440) | 99.6% (1,320) |
| ...........NNRTI 95.0% (60) | 93.9% (33) | 91.7% (36) | 99.3% (280) | 99.6% (840) |
| ....................PI 98.6% (140) | 95.9% (123) | 94.7% (133) | 98.8% (400) | 99.4% (1,200) |
| NRTI/NtRTI=nucleoside/nucleotide reverse transcriptase inhibitors | | All Codons (n) | All Bases (n) |
NNRTI=non-nucleoside reverse transcriptase inhibitor
PI=protease inhibitor | Reverse
Transcriptase | 99.5% (4,160) | 99.5% (12,480) |
| aGuideLines® 9.0 Rules (Bayer) | Protease | 99.0% (1,980) | 99.3% (5,940) |
