Comparison of ultra-deep versus Sanger sequencing detection of minority mutations on the HIV-1 drug resistance interpretations after virological failure.
OBJECTIVE
Drug-resistance mutations are routinely detected using standard Sanger sequencing, which does not detect minor variants with a frequency below 20%. The impact of detecting minor variants generated by ultra-deep sequencing (UDS) on HIV drug-resistance interpretations has not yet been studied.


METHODS
Fifty HIV-1 patients who experienced virological failure were included in this retrospective study.


METHODS
The HIV-1 UDS protocol allowed the detection and quantification of HIV-1 protease and reverse transcriptase variants related to genotypes A, B, C, F and G. DeepChek-HIV simplified drug-resistance interpretation software was used to compare Sanger sequencing and UDS.


RESULTS
The total time required for the UDS protocol was found to be approximately three times longer than Sanger sequencing with equivalent reagent costs. UDS detected all of the mutations found by population sequencing and identified additional resistance variants in all patients. An analysis of drug resistance revealed a total of 643 and 224 clinically relevant mutations by UDS and Sanger sequencing, respectively. Three resistance mutations with more than 20% prevalence were detected solely by UDS: A98S (23%), E138A (21%) and V179I (25%). A significant difference in the drug-resistance interpretations for 19 antiretroviral drugs was observed between the UDS and Sanger sequencing methods. Y181C and T215Y were the most frequent mutations associated with interpretation differences.


CONCLUSIONS
A combination of UDS and DeepChek software for the interpretation of drug resistance results would help clinicians provide suitable treatments. A cut-off of 1% allowed a better characterization of the viral population by identifying additional resistance mutations and improving the drug-resistance interpretation.
aLaboratoire Alphabio, Marseille, France bAdvanced Biological Laboratories (ABL), Luxembourg, Luxembourg cHôpital Européen, Marseille, France dLaboratoire d'Immunologie des Tumeurs et Centre INSERM de Recherche en Cancérologie, Institut Paoli Calmettes, Marseille, France.
Sofiane
S
Mohamed
Guillaume
G
Penaranda
Dimitri
D
Gonzalez
Claire
C
Camus
Hacène
H
Khiri
Ronan
R
Boulmé
Chalom
C
Sayada
Patrick
P
Philibert
Daniel
D
Olive
Philippe
P
Halfon
D019380:Anti-HIV Agents / Q000627:therapeutic use*; D024882:Drug Resistance, Viral; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology*; D015497:HIV-1 / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D020125:Mutation, Missense; D012189:Retrospective Studies; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D017211:Treatment Failure
D019380:Anti-HIV Agents