“Dynamic Range” of Inferred Phenotypic HIV Drug Resistance Values in Clinical Practice

Background: ‘Virtual ’ or inferred phenotypes (vPhenotypes) are commonly used to assess resistance to antiretroviral agents in patients failing therapy. In this study, we provide a clinical context for understanding vPhenotype values. Methods: All HIV-infected persons enrolled in the British Columbia Drug Treatment Program with a baseline plasma viral load (pVL) and follow-up genotypic resistance and pVL results were included up to October 29, 2008 (N = 5,277). Change from baseline pVL was determined as a function of Virco vPhenotype, and the ‘‘dynamic range’ ’ (defined here by the 10th and 90th percentiles for fold-change in IC50 amongst all patients) was estimated from the distribution of vPhenotye foldchanges across the cohort. Results: The distribution of vPhenotypes from a large cohort of HIV patients who have failed therapy are presented for all available antiretroviral agents. A maximum change in IC50 of at least 13-fold was observed for all drugs. The dideoxy drugs, tenofovir and most PIs exhibited small ‘‘dynamic ranges’ ’ with values of,4-fold change observed in.99 % of samples. In contrast, zidovudine, lamivudine, emtricitabine and the non-nucleoside reverse transcriptase inihibitors (excluding etravirine) had large dynamic ranges. Conclusion: We describe the populational distribution of vPhenotypes such that vPhenotype results can be interprete

N348I in the Connection Domain of HIV-1 Reverse Transcriptase Confers Zidovudine and Nevirapine Resistance

Analyzing HIV sequences from a Canadian cohort, Gilda Tachedjian and colleagues identify a common mutation in a little-studied domain of reverse transcriptase that confers resistance to two drug classes

Gene discovery in EST sequences from the wheat leaf rust fungus Puccinia triticina sexual spores, asexual spores and haustoria, compared to other rust and corn smut fungi

© 2011 Xu et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.DOI: 10.1186/1471-2164-12-161Background.Rust fungi are biotrophic basidiomycete plant pathogens that cause major diseases on plants and trees world-wide, affecting agriculture and forestry. Their biotrophic nature precludes many established molecular genetic manipulations and lines of research. The generation of genomic resources for these microbes is leading to novel insights into biology such as interactions with the hosts and guiding directions for breakthrough research in plant pathology. Results. To support gene discovery and gene model verification in the genome of the wheat leaf rust fungus, Puccinia triticina (Pt), we have generated Expressed Sequence Tags (ESTs) by sampling several life cycle stages. We focused on several spore stages and isolated haustorial structures from infected wheat, generating 17,684 ESTs. We produced sequences from both the sexual (pycniospores, aeciospores and teliospores) and asexual (germinated urediniospores) stages of the life cycle. From pycniospores and aeciospores, produced by infecting the alternate host, meadow rue (Thalictrum speciosissimum), 4,869 and 1,292 reads were generated, respectively. We generated 3,703 ESTs from teliospores produced on the senescent primary wheat host. Finally, we generated 6,817 reads from haustoria isolated from infected wheat as well as 1,003 sequences from germinated urediniospores. Along with 25,558 previously generated ESTs, we compiled a database of 13,328 non-redundant sequences (4,506 singlets and 8,822 contigs). Fungal genes were predicted using the EST version of the self-training GeneMarkS algorithm. To refine the EST database, we compared EST sequences by BLASTN to a set of 454 pyrosequencing-generated contigs and Sanger BAC-end sequences derived both from the Pt genome, and to ESTs and genome reads from wheat. A collection of 6,308 fungal genes was identified and compared to sequences of the cereal rusts, Puccinia graminis f. sp. tritici (Pgt) and stripe rust, P. striiformis f. sp. tritici (Pst), and poplar leaf rust Melampsora species, and the corn smut fungus, Ustilago maydis (Um). While extensive homologies were found, many genes appeared novel and species-specific; over 40% of genes did not match any known sequence in existing databases. Focusing on spore stages, direct comparison to Um identified potential functional homologs, possibly allowing heterologous functional analysis in that model fungus. Many potentially secreted protein genes were identified by similarity searches against genes and proteins of Pgt and Melampsora spp., revealing apparent orthologs. Conclusions. The current set of Pt unigenes contributes to gene discovery in this major cereal pathogen and will be invaluable for gene model verification in the genome sequence

A Mutation in the 3′ Region of the Human Immunodeficiency Virus Type 1 Reverse Transcriptase (Y318F) Associated with Nonnucleoside Reverse Transcriptase Inhibitor Resistance

The Y318F substitution in the 3′ region of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) has been linked to nonnucleoside RT inhibitor (NNRTI) resistance in vitro. A systematic search of a large phenotypic-genotypic database (Virco) linked the Y318F substitution with a >10-fold decrease in NNRTI susceptibility in >85% of clinically derived isolates. There was a significant association between Y318F and use of delavirdine (P = 10(−11)) and nevirapine (P = 10(−6)) but not efavirenz (P = 0.3). Site-directed HIV-1 Y318F mutants in an HXB2 background displayed 42-fold-decreased susceptibility to delavirdine but <3-fold-decreased susceptibility to nevirapine or efavirenz. Combinations of Y318F with K103N, Y181C, or both resulted in decreased efavirenz susceptibility of 43-, 3.3-, and 84-fold, respectively, as well as >100- and >60-fold decreases in delavirdine and nevirapine susceptibility, respectively. These results indicate the importance of the Y318F substitution in HIV-1 drug resistance

Technical and Regulatory Shortcomings of the TaqMan Version 1 HIV Viral Load Assay

<div><h3>Background</h3><p>The lower limit of detection of the original Roche Amplicor HIV plasma viral load (pVL) assay (50 copies/mL) has defined HIV treatment success. The Amplicor assay, however, has been replaced by the Roche TaqMan assay(s). Changes to the limits of detection and calibration have not been validated for clinical utility. Sudden increases in the number of patients with detectable pVL have been reported following the introduction of the TaqMan version 1 assay.</p> <h3>Methods</h3><p>Between October 2009 and April 2010 all routine pVL samples from British Columbia, Canada, with 40–250 copies/mL by TaqMan were re-tested by Amplicor (N = 1198). Subsequent short-term virological and resistance outcomes were followed in patients with unchanged therapy (N = 279; median 3.2 months follow-up).</p> <h3>Results</h3><p>TaqMan and Amplicor values correlated poorly at low pVL values. Low-level pVL by TaqMan was not associated with impending short-term virological failure; only 17% of patients with 40–250 copies/mL by TaqMan had detectable pVL by Amplicor at follow-up. During the follow-up period only 20% of patients had an increase in pVL by TaqMan (median [IQR]: 80 [36–283] copies/mL). In addition, in ∼2.4% of samples pVL was dramatically <em>underestimated</em> by TaqMan due to poor binding of the proprietary TaqMan primers.</p> <h3>Conclusions</h3><p>The replacement of Amplicor with the TaqMan assay has altered the previously accepted definition of HIV treatment failure without any evidence to support the clinical relevance of the new definition. Given the systematic differences in measurement in the low pVL range the British Columbia HIV treatment guidelines now use a threshold of >250 copies/mL by TaqMan to define treatment failure.</p> </div

Poor concordance between TaqMan v1 and Amplicor v1.5 at low plasma viral load levels (40

<p>–<b>250 HIV RNA copies/mL by TaqMan v1).</b> Between October 2009 and April 2010 plasma samples from British Columbia with low-level viremia (40–250 copies/mL) by the Roche TaqMan v1 assay (N = 1198) were systematically re-tested by the Amplicor v1.5 assay. Poor concordance was observed between TaqMan v1 and Amplicor v1.5 results, with 82% of values falling below the line of identity (red solid line). Points above the blue dashed line are samples in which TaqMan v1 <i>underestimated</i> pVL by ≥0.5 log₁₀ copies/mL relative to the Amplicor v1.5 assay.</p

Plasma viral load testing and reporting protocol in British Columbia between October 2009 and April 2010.

<p>All plasma samples were initially tested with the Roche COBAS AmpliPrep/COBAS TaqMan v1 HIV-1 Test (“TaqMan v1”) assay. TaqMan v1 pVL results <40 or ≥250 copies/mL were reported to physicians. Samples with TaqMan v1 pVL ≥40 and <250 copies/mL were re-tested by the Roche COBAS AmpliPrep/COBAS AMPLICOR HIV-1 MONITOR UltraSensitive Test, version 1.5 (“Amplicor v1.5”). The Amplicor v1.5 test results were reported to physicians.</p