HIV drug resistance testing by high-multiplex "wide" sequencing on the MiSeq instrument.
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 such settings. This is an observational study to ascertain the potential of newer genomic sequencing platforms, such as the Illumina 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 study cohort (n = 349). Amplicons spanning HIV reverse transcriptase codons 90 to 234 were sequenced with both MiSeq sequencing and conventional Sanger sequencing 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 the identification of drug resistance mutations. Sanger and MiSeq sequencing was successful for 881 samples (80%) and 892 samples (81%), respectively, with 832 samples having results from both methods. Most failures were for samples with viral loads of <3.0 log10 HIV RNA copies/ml. Overall, 99.3% nucleotide concordance between methods was observed. MiSeq sequencing achieved 97.4% sensitivity and 99.3% specificity in detecting resistance mutations identified by Sanger sequencing. Findings suggest that the Illumina MiSeq platform can yield high-quality data with a high-multiplex "wide" sequencing approach. This strategy can be used for multiple HIV subtypes, demonstrating the potential for widespread individual testing and annual population surveillance in resource-limited settings.
British Columbia Centre for Excellence in HIV/AIDS, Vancouver, Canada.
H R
HR
Lapointe
British Columbia Centre for Excellence in HIV/AIDS, Vancouver, Canada.
W
W
Dong
British Columbia Centre for Excellence in HIV/AIDS, Vancouver, Canada.
G Q
GQ
Lee
Harvard School of Public Health, Boston, Massachusetts, USA Massachusetts General Hospital, Boston, Massachusetts, USA Mbarara University of Science of Technology, Mbarara, Uganda.
D R
DR
Bangsberg
University of California, San Francisco, San Francisco, California, USA.
J N
JN
Martin
University of California, San Francisco, San Francisco, California, USA.
A R
AR
Mocello
Mbarara University of Science of Technology, Mbarara, Uganda.
Y
Y
Boum
British Columbia Centre for Excellence in HIV/AIDS, Vancouver, Canada.
A
A
Karakas
British Columbia Centre for Excellence in HIV/AIDS, Vancouver, Canada.
D
D
Kirkby
British Columbia Centre for Excellence in HIV/AIDS, Vancouver, Canada University of British Columbia, Vancouver, Canada.
A F Y
AF
Poon
British Columbia Centre for Excellence in HIV/AIDS, Vancouver, Canada University of British Columbia, Vancouver, Canada prharrigan@cfenet.ubc.ca cbrumme@cfenet.ubc.ca.
P R
PR
Harrigan
British Columbia Centre for Excellence in HIV/AIDS, Vancouver, Canada prharrigan@cfenet.ubc.ca cbrumme@cfenet.ubc.ca.
C J
CJ
Brumme
D002170:Canada; D024882:Drug Resistance, Viral / Q000235:genetics*; D060005:Genotyping Techniques; D054303:HIV Reverse Transcriptase / Q000235:genetics; D015497:HIV-1 / Q000187:drug effects / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D019562:Viral Load
D054303:HIV Reverse Transcriptase