title: Optimized protocol for detection of HIV-1 drug mutations in patients with low viral load.
abstract: The TRUGENE HIV-1 Genotyping Kit for HIV-1 drug resistance testing is limited by the need for samples with HIV-1 RNA loads of at least 1000 copies/mL. In order to enable sequencing of clinical samples with viral loads under 1000 copies/mL, an optimized automated sample preparation protocol on the VERSANT kPCR Sample Preparation (SP) Module was evaluated. In order to prove the concept of successful sequencing of low-titer clinical samples with the optimized protocol, a dilution series of a routine clinical sample was analyzed. Furthermore, 57 routine clinical samples with viral loads below 1000 HIV-1 RNA copies/mL were tested. Finally, samples obtained from two patients with low viral loads were tested retrospectively for HIV-1 drug resistances and results were compared with those of the preceding and the subsequent sample. The dilution containing 92 HIV-1 RNA copies/mL was the lowest yielding an analyzable sequence with the optimized protocol. When routine clinical samples with viral loads between 100 and 1000 HIV-1 RNA copies/mL were tested, a sequence was obtained in 90.5%. Samples with low viral load of two patients that could not be analyzed with the routine protocol showed identical drug mutations in both, the low viral-load and the subsequent samples. Together with the optimized automated sample preparation protocol, the TRUGENE HIV-1 Genotyping Kit allows successful sequencing of the majority of samples with HIV-1 RNA loads between 100 and 1000 copies/mL. Detection of resistance mutations in low viral-load samples may lead to an earlier optimized antiretroviral therapy.
authors.affiliation: Research Unit Molecular Diagnostics, Center of Applied Biomedicine, Medical University of Graz, Universitaetsplatz 4, 8010 Graz, Austria. evelyn.stelzl@medunigraz.at
authors.forename: Evelyn
authors.initials: E
authors.lastname: Stelzl
authors.forename: Katharina T
authors.initials: KT
authors.lastname: Troppan
authors.forename: Michaela
authors.initials: M
authors.lastname: Winkler
authors.forename: Klaus
authors.initials: K
authors.lastname: Korn
authors.forename: Harald H
authors.initials: HH
authors.lastname: Kessler
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology*; D001331:Automation / Q000379:methods; D024882:Drug Resistance, Viral; D005838:Genotype; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D020125:Mutation, Missense; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D019562:Viral Load; D014773:Virology / Q000379:methods*
chemical_list: D019380:Anti-HIV Agents; D012367:RNA, Viral