Performance of the TruGene Human Immunodeficiency Virus Type 1 Genotyping Kit and OpenGene DNA Sequencing System on Clinical Samples Diluted to Approximately 100 Copies per Milliliter

Abstract

The TruGene human immunodeficiency virus type 1 (HIV-1) genotyping kit/OpenGene DNA sequencing system (Bayer HealthCare, Tarrytown, NY) reliably produced clinically acceptable resistance profiles for reverse transcriptase and protease inhibitors on patient samples diluted to ϳ100 copies/ml following extraction with the QIAamp viral RNA minikit (QIAGEN Inc., Valencia, CA). One modification of the standard protocol was made to guarantee PCR amplification: a centrifugation step to concentrate virus was added before RNA extraction. For genotypic antiretroviral resistance testing, no significant differences in the identification and sensitivity of detection for codon mutations, base mutations, and multibase sites were found between the original and diluted samples.Genotypic antiretroviral resistance testing (GART) is a routine tool in the management of human immunodeficiency virus (HIV) patients on highly active antiretroviral therapy (2). Commercial assays require Ն1,000 copies/ml, but noncommercial, modified commercial, and proprietary GART assays have successfully detected resistance at much lower levels (1,3,4,(6)(7)(8). Since 2001, the Infectious Diseases Laboratory at the Veterans Affairs Medical Center in Washington, D.C., has performed genotypic testing using the TruGene HIV type 1 (HIV-1) genotyping kit/OpenGene DNA sequencing system (Bayer HealthCare LLC, Tarrytown, NY). We observed that this commercial assay was consistently able to genotype clinical samples well below 1,000 copies/ml. We designed this study to validate genotypic testing at very low viral loads because an earlier recognition of resistance to antiretroviral agents may well enhance the clinician's opportunity to alter therapy, prevent the emergence of added resistance, and better preserve future treatment options.(Results of this study were presented in part at the 45th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, D.C., 16 to 19 December 2005.) MATERIALS AND METHODSPeripheral blood was drawn by venipuncture into EDTA tubes, kept at room temperature, and centrifuged within 4 h of drawing at 1,000 ϫ g for 15 min. Plasma was stored at Ϫ80°C and thawed in a 10 to 15°C water bath for 15 min before use. Twenty consecutive clinical GART specimens were chosen for the presence of HIV RNA with multiple mutations leading to complex resistance profiles and with Ͼ1,000 copies/ml (median, 13,933 copies/ml; range, 1,032 to 345,560 copies/ml; Versant HIV-1 RNA 3.0 assay [bDNA]; Bayer HealthCare). The samples were diluted in human serum-based dilution matrix (AcroMetrix Corporation, Benecia, CA) or HIV-negative plasma to ϳ100 copies/ml (median, 136 copies/ml; range, Ͻ75 to 254 copies/ml) ( Table 1). The diluted samples were stored and thawed in the same manner as the original samples before processing.Viruses in the diluted sample were only when PCR amplification failed concentrated: one milliliter was centrifuged at 23,500 ϫ g and 4°C for 75 min in a 2-ml microtube (PN 72.693.005; Sarstedt Inc., Newton, NC), and 800 l of sup...

Improved Viral Suppression After Treatment Optimization in HIV-Infected Patients With Persistent Low-Level Viremia

“…To address this issue, several assays have recently been developed for genotyping samples with viral load below 1000 copies per milliliter. [3][4][5] A high prevalence of resistance mutations among this patient subset has been reported, supporting the practice of HIV genotyping in patients with low viral loads. [6][7][8][9] However, these studies have not addressed the utility of optimizing antiretroviral therapy based on genotyping data obtained from this patient population.…”

“…5,11 Because most commercially available genotyping tests are validated for viral loads .1000 copies per milliliter, obtaining genotyping data for these patients can be difficult. [3][4][5] In the present study, we show that an in-house genotyping assay is able to obtain reliable data from samples with as few as 8 copies per milliliter. Similar to previous studies reporting genotyping data from this patient population, we show that these patients harbor viruses with high frequencies of resistanceassociated mutations.…”

Sensitive Oligonucleotide Ligation Assay for Low-Level Detection of Nevirapine Resistance Mutations in Human Immunodeficiency Virus Type 1 Quasispecies

“…Direct DNA sequencing of cDNA from patient plasma or from peripheral blood mononuclear cell (PBMC) extracts, such as with the Trugene system (Bayer Diagnostics) or the ViroSeq system (Applied Biosystems), may be the most common method; but it is also the least sensitive method used to detect SNPs as minority base changes (Ͼ20 to 30%) (10,20,28,44). Subcloning and sequencing of HIV-1 clones (Ͼ100) can counter this low sensitivity of bulk sequencing, but they are very costly.…”

Description of Two Commercially Available Assays for Genotyping of HIV-1

“…Nowadays, clinical routine is quite different, and hence the commercial systems needed adaptations. Therefore, both assays have been tested intensely in terms of performance characteristics on different blood collection systems [33], including dried blood spot specimens [34,35,36], different extraction procedures [33,37,38,39,40] and modified PCR reactions [41]. Using dried blot samples as a source for genotyping is of major interest in the resource-limited setting.…”