World-wide variation in HIV-1 phenotypic susceptibility in untreated individuals: biologically relevant values for resistance testing.

OBJECTIVES: To examine the natural phenotypic variability in drug susceptibility among recombinant HIV-1 isolates from a large number of untreated HIV-positive individuals from wide-ranging geographic locations, and to use this information to establish biologically relevant cut-off values for phenotypic antiretroviral susceptibility testing. METHODS: Phenotypic susceptibility to 14 antiretroviral agents was determined for HIV-1 samples from > 1000 treatment-naive individuals in seven clinical trials. Samples were from the USA (n = 351), Germany (n = 306), Canada (n = 265), and South Africa (n = 358). Geometric mean fold-resistance and confidence intervals were determined relative to a standard laboratory wild-type virus. RESULTS: Baseline fold-resistance was approximately log-normally distributed for all antiretroviral agents examined. There was no evidence of large geographical differences in average antiviral susceptibility. Geometric mean fold-resistance for each of 14 antiviral agents was similar (+/- 0.5-fold) for samples derived from the USA, Canada, Germany, or South Africa. The non-nucleoside reverse transcriptase inhibitors (NNRTI) exhibited the broadest distribution of susceptibility; approximately 97.5% of all isolates had < 2.5-4.0, < 3.0-4.5, and < 5-10 fold-decrease in susceptibility to five protease inhibitors, six nucleoside analogues, and three NNRTI, respectively. No consistent geographic pattern or clade effect (B versus C) in either the mean or the distribution of baseline antiretroviral susceptibility was observed. CONCLUSIONS: Phenotypic drug susceptibility of HIV-1 in untreated individuals varies markedly from drug to drug, with broadly similar patterns world-wide. These results have important implications in defining the 'normal range' of phenotypic susceptibility to antiretroviral agents and establish biologically relevant cut-off values for this phenotypic drug susceptibility test.

Clinical implications of identifying non-B subtypes of human immunodeficiency virus type 1 infection.

Although human immunodeficiency virus type 1 (HIV-1) infection in the United States has predominantly involved subtype B, increasing global travel is leading to wider dissemination of genetically heterogeneous subtypes. While physicians depend on HIV-1 viral load measurements to guide antiretroviral therapy, commonly used molecular assays may underestimate the viral load of patients with non-B subtypes. Nine patients with non-B subtypes of HIV-1 were identified by physicians who suspected a non-B subtype on the basis of a low or undetectable HIV-1 viral load, by the Amplicor HIV-1 Monitor test, version 1.0, in conjunction with either a declining CD4 cell count or history of travel outside the United States. Use of version 1.5 of the Amplicor HIV-1 Monitor test detected a median HIV-1 viral load that was 2.0 log(10) RNA copies/mL higher than was determined with version 1.0. Clinical management was altered in all cases after diagnosis of a non-B-subtype infection. These cases demonstrate that it is critical for physicians to suspect and diagnose non-B subtypes of HIV-1 so that an assay with reliable subtype performance can be used to guide antiretroviral therapy.

Prevalence of genotypic and phenotypic resistance to anti-retroviral drugs in a cohort of therapy-naïve HIV-1 infected US military personnel.

OBJECTIVE: While transmission of drug-resistant HIV-1 has been reported, estimates of prevalence of resistance in drug-naïve populations are incomplete. We investigated the prevalence of genotypic mutations and phenotypic antiretroviral resistance in a cohort of HIV-1 infected U.S. military personnel prior to the institution of antiretroviral therapy. DESIGN: Cross-sectional cohort study. METHODS: Plasma was obtained from 114 recently HIV-1 infected subjects enrolled in an epidemiological study. Genotypic resistance was determined by consensus sequencing of a PCR product from the HIV-1 pol gene. Sequences were interpreted by a phenotypic-genotypic correlative database. Resistance phenotypes were determined by a recombinant virus cell culture assay. RESULTS: Genotypic mutations and phenotypic resistance were found at a higher than expected frequency. Resistance to non-nucleoside reverse transcriptase inhibitors was most common, with a prevalence of 15% of 95 subjects by genotype and 26% of 91 subjects by phenotype. Genotypic and phenotypic resistance respectively were found in 4% and 8% of subjects for nucleoside reverse transcriptase inhibitors and in 10% and 1% for protease inhibitors. One subject harbored virus with resistance to all three drug classes. CONCLUSIONS: A substantial frequency of resistance to antiretroviral drugs was identified in a therapy-naïve U.S. cohort. In most cases, the genotypic and phenotypic assays yielded similar results, although the genotypic assay could detect some protease inhibitor resistance-associated mutations in the absence of phenotypic resistance. These data suggest the need for optimization of treatment guidelines based on current estimates of the prevalence of drug resistance in HIV-1 seroconverters.

Performance of the Affymetrix GeneChip HIV PRT 440 platform for antiretroviral drug resistance genotyping of human immunodeficiency virus type 1 clades and viral isolates with length polymorphisms.

The performance of a silica chip-based resequencing method, the Affymetrix HIV PRT 440 assay (hereafter referred to as the Affymetrix assay), was evaluated on a panel of well-characterized nonclade B viral isolates and on isolates exhibiting length polymorphisms. Sequencing of human immunodeficiency virus type 1 (HIV-1) pol cDNAs from clades A, C, D, E, and F resulted in clade-specific regions of base-calling ambiguities in regions not known to be associated with resistance polymorphisms, as well as a small number of spurious resistance polymorphisms. The Affymetrix assay failed to detect the presence of additional serine codons distal to reverse transcriptase (RT) codon 68 that are associated with multinucleoside RT inhibitor resistance. The increasing prevalence of non-clade B HIV-1 strains in the United States and Europe and the identification of clinically relevant pol gene length polymorphisms will impact the generalizability of the Affymetrix assay, emphasizing the need to accommodate this expanding pool of pol genotypes in future assay versions.

Genomic organization and functional characterization of the chemokine receptor CXCR4, a major entry co-receptor for human immunodeficiency virus type 1.

CXCR4 is both a chemokine receptor and entry co-receptor for T-cell line-adapted human immunodeficiency virus type 1. The genomic organization and promoter function for the entire transcription unit of CXCR4 were determined. The gene contains 2 exons of 103 and 1563 base pairs (bp) interrupted by a 2132-bp intron precisely between codons 5 and 6 of the coding sequences. A transcription start site was identified 88 bp upstream of the initiation codon, and a polyadenylate addition site was identified 22 bp 3' to a polyadenylation signal. Transient expression assays defined a minimal promoter at positions -114 to +43 relative to the transcription start site. This region contains a TATA box, a nuclear respiratory factor-1 (NRF-1) site, and two GC boxes. Specific factor binding to the NRF-1 site and GC boxes were demonstrated by gel mobility shifts and DNase I footprinting. Site-directed mutagenesis showed that the NRF-1 site is crucial for promoter activity providing the first evidence for the regulation of a signal transduction gene by NRF-1. Sequences between -691 and -191 repress CXCR4 promoter activity. Further study of these regulatory elements will be important to understanding how CXCR4 functions as both a chemokine receptor and human immunodeficiency virus type 1 entry co-receptor.

Treatment of chronic graft-versus-host disease with clofazimine.

Clofazimine (Lamprene) is an antimycobacterial drug that has antiinflammatory activity in a number of chronic autoimmune skin disorders. We report 22 patients treated with clofazimine for chronic graft-versus-host disease (cGVHD). The initial dose was 300 mg orally in a single daily dose for 90 days. After 90 days, the dose was lowered to 100 mg orally each day and the medication continued indefinitely as tolerated. Treatment courses lasted 7 to 835 days and were generally well tolerated. Gastrointestinal side effects occurred in eight of 22 patients (36%) and hyperpigmentation was noted in 12 of 22 patients (55%), which resolved upon decrease or discontinuation of the drug. Over 50% of patients with skin involvement, flexion contractures, or oral manifestations achieved complete or partial responses. Seven of 22 patients (32%) were able to reduce other immunosuppressive medications. Thus, clofazimine is safe and has encouraging efficacy in cGVHD, particularly if sclerodermatous skin, joint contractures, or oral manifestations are present. The mechanism by which clofazimine induces a response is unknown, but might be secondary to suppression of alloreactive T-cell function in cGVHD target organs. Clofazimine deserves further study for the treatment of cGVHD.

Sampling lymph node content of human immunodeficiency virus type 1 nucleic acids and p24 antigen by fine-needle aspiration in early-stage patients.

The potential of lymph node fine-needle aspiration (LNFNA) for sampling viral load was evaluated in excised, peripheral lymph nodes from five patients with early-stage human immunodeficiency virus type 1 HIV-1 disease (asymptomatic, CD4 cells > 300/mm3). The preponderance (> 80%) of viral RNA was within follicular germinal centers as noted by in situ hybridization on lymph node frozen sections (LNFSs) as well as within cohesive groups of 10-20 lymphoid cells (microfragments) in LNFNA preparations. Quantification of cells expressing HIV-1 RNA by in situ hybridization, quantification of HIV-1 gag RNA and gag DNA per 10(5) cells by polymerase chain reaction, and measurement of p24 antigen per 10(5) cells yielded similar values for LNFNA, lymph node mononuclear cells (LNMCs) from tissue homogenates by Ficoll-Hypaque separation, and LNFS. Sampling of lymph node viral load by LNFNA appears to capture viral components associated with both individual expressing cells and follicular germinal centers. Due to the advantages in terms of patients' morbidity, repeatability, and cost, assessment of lymphoid tissue viral load by LNFNA warrants an in vivo feasibility trial as an alternative to lymph node biopsy.