Workflow Optimization of the ViroSeqTM Human Immunodeficiency Virus-1 (HIV-1) Genotyping System

Background: Current therapy guidelines recommend resistance testing to guide therapy in antiretroviral (ARV) treatment naive patients as well as patients with suboptimal virologic responses or virologic failure. Objective: This study evaluated workflow optimization and automation improvements to the ViroSeq HIV-1 Genotyping System through the use of the semi-automated extraction platforms (Abbott Molecular Inc. m2000sp and bioMerieux Inc. NucliSens easyMAG) for sample extraction, NanoDrop 2000 Spectrophotometer (Thermo Scientific, CA) and Agilent Bioanalyzer 2100 (Agilent Technologies, Germany) for amplicon quantitation and Applied Biosystems Instruments (ABI) 3500 (Life Technologies, Ca) for increased sequencing throughput (Table 1). Methods: Clinical plasma samples were processed in parallel with Viroseq HIV-1 Genotyping System per manufacturer instructions along with above described automated options. Resistance associated mutations from the different approaches were evaluated and assessed for clinical impact. Results: All samples tested in this study gave valid results regardless of the procedure used. Samples extracted with m2000sp demonstrated resistance- associated mutations in 24/30 samples (80%) while complete drug resistance concordance was observed in 29/30 samples (97%). Ninety one percent (10/11) of the samples extracted with easyMAG had 98.7% concordance at the nucleotide level and 100% concordance for drug resistance mutations. No apparent difference was observed in sequence quality between manual and semi-automated extractions and comparable drug resistance profiles were obtained regardless of the extraction method. Conclusion: The ViroSeq HIV-1 Genotyping System is compatible with the semi-automated extraction systems, alternative amplicon quantitation devices as well as with high throughput sequencing platform such as the ABI 3500 sequencer. This study also indicated that the assay can accurately sequence HIV-1 virus below 1,000 copies/mL. Significant workflow and turnaround time advantages were observed in this study with the automation of the Viroseq HIV-1 Genotyping System.