Pre-screening HIV-1 reverse transcriptase resistance mutations in subtype B patients using a novel multiplex primer extension assay.
Antiretroviral therapy is standard treatment for HIV-infected patients. Such therapy has decreased mortality and morbidity, but treatment success is often jeopardized by the emergence of viral drug resistance. Moreover, in recent years there has been a reported rise in the incidence of transmitted drug resistance, highlighting the importance of pre-treatment resistance screening. In this report, we describe the development and utility of a sensitive multiplex approach for detecting mutations conferring drug resistance to HIV-1 reverse transcriptase inhibitors. This protocol, termed HIV-SNaPshot, utilizes a multiplex primer extension assay with capillary electrophoresis reporting altered nucleotides at nine important drug resistance mutation positions. Mutations were successfully detected to levels of 5% in viral quasispecies populations. Furthermore, although developed and optimised for HIV-1 subtype B, drug resistance mutations could also be detected in most non-B subtypes. Comparison of the HIV-SNaPshot with the commercial Viroseq genotyping system in 10 patients gave similar results, but importantly, additional resistance mutations were identified in several patients by the HIV-SNaPshot assay. Thus, the HIV-SNaPshot is a method capable to support standard genotyping for the determination of minority HIV-1 resistance mutations, with equivalent and perhaps greater sensitivity than Viroseq.
Jakobsen
Martin Roelsgaard
MR
Department of Infectious Diseases, Aarhus University Hospital, Skejby, Brendstrupgaardvej 100, DK-8200 Aarhus N, Denmark. mrj@svf.au.dk
Aggerholm
Anni
A
Jørgensen
Louise Bruun
LB
Laursen
Alex
A
Ostergaard
Lars
L
D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D024882:Drug Resistance, Viral; D019075:Electrophoresis, Capillary; D015658:HIV Infections / Q000821:virology*; D054303:HIV Reverse Transcriptase / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008403:Mass Screening / Q000379:methods*; D020125:Mutation, Missense; D012680:Sensitivity and Specificity
D017931:DNA Primers; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase