Phenotypic Assays and Sequencing Are Less Sensitive Than Point Mutation Assays for Detection of Resistance in Mixed HIV-1 Genotypic Populations

Abstract

The sensitivity and discriminatory power of the 151 and 215 amplification refractory mutation system (ARMS) were evaluated, and their performance for the detection of drug resistance in mixed genotypic populations of the reverse transcription (RT) gene of HIV-1 were compared with T7 sequencing, cycle sequencing, the line probe assay (LiPA) HIV-1 RT test, and the recombinant virus assay (RVA). ARMS and the LiPA HIV-1 RT test were shown to be able to detect minor variants that in particular cases comprised only 1%. T7 sequencing on an ALF semiautomated sequencer could correctly score mixtures only when variants were present at 50%. Cycle sequencing on an ABI PRISM 310 improved the sensitivity for mixtures to about 25%. Using RVA, it was shown that at least 50% of the virus population needed to carry the resistance mutation at codon 184 to afford phenotypic resistance against lamivudine. The two point mutation assays therefore proved to be more sensitive methods than sequencing and RVA to reliably determine a gradual shift in HIV-1 drug resistance mutations in follow-up of patients infected with HIV-1. In 4 of 5 treated patients who were followed by ARMS, a gradual shift in resistant genotypic populations was observed during a period of 6 to 19 months. For 1 patient, a shift from wild to mutant type at position 151 occurred within 2 months, without mixed genotypic intermediate type's being detected.

Sequencing-Based Detection of Low-Frequency Human Immunodeficiency Virus Type 1 Drug-Resistant Mutants by an RNA/DNA Heteroduplex Generator-Tracking Assay

“…I13V, K20R, D30N, L33IL, E35D, M36I, I54V, L63P, A71T, N88D, L90M 71 a Differences between linkage groups are underlined. Non-sequencing-based methods are generally highly sensitive for detecting minority drug resistance mutations (65,76,77), but they suffer from several limitations. The exact sequence context of any mutation cannot be determined without sequencing the entire gene.…”

“…Direct PCR sequencing is primarily used in the clinical setting, but one of its major limitations is its inability to consistently detect minority variants present at frequencies below 10 to 25% (47,49,64,76). The presence of mixed bases in clinical samples is also largely responsible for discordant results when the same samples are analyzed in different laboratories or using different nonsequencing methods (20,28,36,38,66).…”

Feasibility of Detecting Human Immunodeficiency Virus Type 1 Drug Resistance in DNA Extracted from Whole Blood or Dried Blood Spots

“…While plasma RNA represents the population of short-lived actively replicating virus, viral DNA from infected cells is composed of a heterogeneous mix of DNA from acutely infected, actively virus-producing cells as well as quiescent cells that form the viral reservoir (13,27,40). Due to the limited capacities of current population-based sequencing genotyping methods, minor variants can remain undetectable (38). The virus replicating in plasma in the early stage of resistance development most probably derives from a small number of cells in the reservoir, and therefore, detection of resistant virus in plasma will precede detection in the cells.…”

A Combination of Poor Adherence and a Low Baseline Susceptibility Score is Highly Predictive for HAART Failure

“…The nested ARMS-151 PCR was performed as described previously (Van Laethem et al, 1999), starting from the same outer product as used for LiPA. Starting from control HIV-1 RNA the wild-type and the mutant primers have a detection limit of 100 copies/ml on RNA WT and MT template, respectively.…”