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Background:  Incompatibility between K65R and thymidine analogue mutations (TAM) or L74V mutation on the same virus has been described in previous studies. At the contrary, other studies have suggested a link between K65R and reverse transcriptase (RT)-mutations as S68G or V75I. In clinical practice, some patients can harbour a genotypic resistance test with a combination of these different mutations. In most of these cases, the K65R and/or the others associated mutations are present as mixtures with wild type. We wanted to know precisely the RT genotype of individual molecular clones derived from these samples and show whether these all mutations are really linked with K65R on the same strain.

Methods:  We analysed 5 samples harbouring plasma genotype with K65R associated with TAM, L74V, S68G, and/or V75I in 3, 1, 3, and 2 cases, respectively. In all of them, K65R emergence was observed after a tenofovir regimen failure. For the clonal analyses, the RT fragments were cloned into pCR2.1-TOPO vector, before sequencing (automated population-based-full sequence analysis).

Results:  Clonal analyses of 2 patients showed that K65R can be linked with TAM as M41L, D67N, L210W, and K219E in several clones, and more rarely with the T215Y mutation in a sole clone. At the contrary, analysis of another patient showed that K65R was never linked with the L74V mutation. The S68G was always or never or sometimes linked with the K65R, depending on the case. The V75I was not linked with K65R in one patient, while it was sometimes in the other case with the additional Q151M mutation.

Conclusions:  The co-existence of the both L74V and K65R mutations seems produce too strong a replicative impairment to be observed in vivo. However, if these 2 mutations are incompatible on the same virus, they can be selected on different strains in plasma of the same patient. The incompatibility between K65R and TAM are more relative and perhaps more likely depends on the presence of antiretroviral compounds as zidovudine in the treatment. The V75I mutation is not necessarily linked with K65R. The same observation is done about the S68G substitution that is not necessarily a compensatory mutation of the K65R like previous studies have suggested.

Keywords: K65R; TAMs; L74V