Holly Bazmi

More sensitive assays for human immunodeficiency virus type 1 (HIV-1) RNA are needed to detect, quantify, and characterize persistent viremia in patients who are receiving antiretroviral therapy and whose plasma HIV-1 RNA levels are suppressed to less than 50 to 75 copies/ml. We therefore developed an internally controlled real-time reverse transcriptase-initiated PCR assay that quantifies HIV-1 RNA concentrations down to 1 copy per ml of plasma. This assay with single-copy sensitivity (the single-copy assay) generates a reproducible linear regression plot of input copy number versus threshold cycle by using HIV-1 RNA transcripts at copy numbers ranging from 1 to 10(6) per reaction mixture. The single-copy assay was compared to the ultrasensitive AMPLICOR HIV-1 MONITOR assay and a more sensitive modification of the ultrasensitive assay by repeatedly testing a low-copy-number panel containing 200 to 0.781 copies of HIV-1 RNA per ml of plasma. This comparison showed that the single-copy assay had a greater sensitivity than the other assays and was the only assay that detected HIV-1 RNA at levels as low as 0.781 copies/ml. Testing of plasma samples from 15 patients who were receiving antiretroviral therapy and who had <75 HIV-1 RNA copies/ml revealed persistent viremia in all 15 patients, with HIV-1 RNA levels ranging from 1 to 32 copies/ml (median, 13 copies/ml). The greater sensitivity of the single-copy assay should allow better characterization of persistent viremia in patients who are receiving antiretroviral therapy and whose HIV-1 RNA levels are suppressed to below the detection limits of present assays.

To investigate the extent to which drug resistance mutations are missed by standard genotyping methods, we analyzed the same plasma samples from 26 patients with suspected multidrug-resistant human immunodeficiency virus type 1 by using a newly developed single-genome sequencing technique and compared it to standard genotype analysis. Plasma samples were obtained from patients with prior exposure to at least two antiretroviral drug classes and who were on a failing antiretroviral regimen. Standard genotypes were obtained by reverse transcriptase (RT)-PCR and sequencing of the bulk PCR product. For single-genome sequencing, cDNA derived from plasma RNA was serially diluted to 1 copy per reaction, and a region encompassing p6, protease, and a portion of RT was amplified and sequenced. Sequences from 15 to 46 single viral genomes were obtained from each plasma sample. Drug resistance mutations identified by single-genome sequencing were not detected by standard genotype analysis in 24 of the 26 patients studied. Mutations present in less than 10% of single genomes were almost never detected in standard genotypes (1 of 86). Similarly, mutations present in 10 to 35% of single genomes were detected only 25% of the time in standard genotypes. For example, in one patient, 10 mutations identified by single-genome sequencing and conferring resistance to protease inhibitors (PIs), nucleoside analog reverse transcriptase inhibitors, and nonnucleoside reverse transcriptase inhibitors (NNRTIs) were not detected by standard genotyping methods. Each of these mutations was present in 5 to 20% of the 20 genomes analyzed; 15% of the genomes in this sample contained linked PI mutations, none of which were present in the standard genotype. In another patient sample, 33% of genomes contained five linked NNRTI resistance mutations, none of which were detected by standard genotype analysis. These findings illustrate the inadequacy of the standard genotype for detecting low-frequency drug resistance mutations. In addition to having greater sensitivity, single-genome sequencing identifies linked mutations that confer high-level drug resistance. Such linkage cannot be detected by standard genotype analysis.

The (-) enantiomers of 2',3'-dideoxy-5-fluoro-3'-thiacytidine [(-)-FTC] and 2',3'-dideoxy-3'-thiacytidine [(-)-BCH-189] were recently shown to inhibit selectively human immunodeficiency viruses (HIV) and hepatitis B virus in vitro. In the current study, the potential for HIV type 1 (HIV-1) resistance to these compounds was evaluated by serial passage of the virus in human peripheral blood mononuclear cells and MT-2 cells in the presence of increasing drug concentrations. Highly drug-resistant HIV-1 variants dominated the replicating virus population after two or more cycles of infection. The resistant variants were cross-resistant to (-)-FTC, (-)-BCH-189, and their (+) congeners but remained susceptible to 2',3'-dideoxycytidine, 3'-azido-3'-deoxythymidine, 3'-fluoro-3'-deoxythymidine, 2',3'-dideoxyinosine, phosphonoformate, the TIBO compound R82150, and the bis(heteroaryl)piperazine derivative U-87201E. Reverse transcriptase derived from drug-resistant viral particles was 15- to 50-fold less susceptible to the 5'-triphosphates of FTC and BCH-189 compared with enzyme from parental drug-susceptible virus. DNA sequence analysis of the reverse transcriptase gene amplified from resistant viruses consistently identified mutations at codon 184 from Met (ATG) to Val (GTG or GTA) or Ile (ATA). Sequence analysis of amplified reverse transcriptase from a patient who had received (-)-BCH-189 therapy for 4 months demonstrated a mixture of the Met-184-to-Val (GTG) mutation and the parental genotype, indicating that the Met-184 mutation can occur in vivo.

The natural history of human immunodeficiency virus type 1 (HIV-1) viremia and its association with clinical outcomes after seroconversion was characterized in a cohort of homosexual men. HIV-1 RNA was measured by reverse-transcription polymerase chain reaction (RT-PCR) in stored longitudinal plasma samples from 269 seroconverters. Subjects were generally antiretroviral drug naive for the first 3 years after seroconversion. The decline in CD4 lymphocyte counts was strongly associated with initial HIV RNA measurements. Both initial HIV RNA levels and slopes were associated with AIDS-free times. Median slopes were +0.18, +0.09, and -0.01 log10 copies/mL, respectively, for subjects developing AIDS <3, 3-7, and>7 years after seroconversion. In contrast, HIV RNA slopes in the 3 years preceding AIDS and HIV RNA levels at AIDS diagnosis showed little variation according to total AIDS-free time. HIV RNA load at the first HIV-seropositive visit ( approximately 3 months after seroconversion) was highly predictive of AIDS, and subsequent HIV RNA measurements showed even better prognostic discrimination.

Human immunodeficiency virus type 1 (HIV-1) isolates resistant to (-)-beta-D-dioxolane-guanosine (DXG), a potent and selective nucleoside analog HIV-1 reverse transcriptase (RT) inhibitor, were selected by serial passage of HIV-1(LAI) in increasing drug concentrations (maximum concentration, 30 microM). Two independent selection experiments were performed. Viral isolates for which the DXG median effective concentrations (EC(50)s) increased 7.3- and 12.2-fold were isolated after 13 and 14 passages, respectively. Cloning and DNA sequencing of the RT region from the first resistant isolate identified a K65R mutation (AAA to AGA) in 10 of 10 clones. The role of this mutation in DXG resistance was confirmed by site-specific mutagenesis of HIV-1(LAI). The K65R mutation also conferred greater than threefold cross-resistance to 2',3'-dideoxycytidine, 2', 3'-dideoxyinosine, 2',3'-dideoxy-3'-thiacytidine, 9-(2-phosphonylmethoxyethyl)adenine, 2-amino-6-chloropurine dioxolane, dioxolanyl-5-fluorocytosine, and diaminopurine dioxolane but had only marginal effects on 3'-azido-3'-deoxthymidine (AZT) susceptibility. However, when introduced into a genetic background for AZT resistance (D67N, K70R, T215Y, T219Q), the K65R mutation reversed the AZT resistance. DNA sequencing of RT clones derived from the second resistant isolate identified the L74V mutation, previously reported to cause ddI resistance. The L74V mutation also decreased the AZT resistance when the mutation was introduced into a genetic background for AZT resistance (D67N, K70R, T215Y, T219Q) but to a lesser degree than the K65R mutation did. These findings indicate that DXG and certain 2',3'-dideoxy compounds (e.g., ddI) can select for the same resistance mutations and thus may not be optimal for use in combination. However, the combination of AZT with DXG or its orally bioavailable prodrug (-)-beta-D-2, 6-diaminopurine-dioxolane should be explored because of the suppressive effects of the K65R and L74V mutations on AZT resistance.