HIV Drug Resistance Testing by High-Multiplex "Wide" Sequencing on the Illumina MiSeq.

2015 | Antimicrob Agents Chemother

Program(s): Laboratory |

Abstract:

Limited access to HIV drug resistance testing in low- and middle-income countries impedes clinical decision-making at the individual patient level. An efficient protocol to address this issue must be established to minimize negative therapeutic outcomes for HIV-1 infected individuals in this setting. This is an observational study to ascertain the potential of newer genomic sequencing platforms, such as the Ilumina MiSeq instrument, to provide accurate HIV drug resistance genotypes for hundreds of samples simultaneously. Plasma samples were collected from Canadian patients during routine drug resistance testing (n=759) and from a Ugandan cohort study (n=349). Amplicons spanning HIV reverse transcriptase codons 90-234 were sequenced with both MiSeq and conventional Sanger methods. Sequences were evaluated for nucleotide concordance between methods using coverage and mixture parameters for quality control. Consensus sequences were also analyzed for disparities in identifying drug resistance mutations. Sequencing was successful in 881 (80%) and 892 (81%) samples by Sanger and MiSeq respectively with 832 samples having results by both methods. Most failures were in samples with viral load <3.0 log₁₀ HIV RNA copies/mL. Overall 99.3% nucleotide concordance was observed between methods. MiSeq achieved 97.4% sensitivity and 99.3% specificity in detecting resistance mutations identified by Sanger. Findings suggest that the Illumina MiSeq platform can obtain high-quality data using a high-multiplex "wide" sequencing approach. This strategy can be applied for multiple HIV subtypes, demonstrating the potential for widespread individual testing and annual population surveillance in resource limited settings.

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Lapointe H

BC Centre for Excellence in HIV/AIDS

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HIV Drug Resistance Testing by High-Multiplex "Wide" Sequencing on the Illumina MiSeq.

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