Background:  Zidovudine (ZDV) resistance increases progressively with the accumulation of thymidine analogue reverse transcriptase inhibitor (RTI) associated mutations (TAM) at positions 41, 67, 70, 210, 215, and 219. Two pathways of accumulation of TAM have been described: pathway 1 (M41L, L210W, T215Y) and pathway 2 (D67N, K70R, T215F, K219E/Q). Pathway 1 is predominant but the reasons for this are unclear. The availability of a large number of RT sequences linked to NRTI susceptibility data provides an opportunity to examine the clustering patterns and contribution to reduced susceptibility of the TAMs in both pathways.

Method:  ZDV and stavudine (d4T) susceptibility was analyzed in a dataset of 4907 clinical samples containing at least 1 nucleoside RTI-associated mutation (NAM, here defined as TAM plus E44D and V118I), but without Q151M, T69ins or M184V/I, and compared to 6200 samples without NAM. Combinations of NAM found in > 3% of samples were considered. Four cut-off values were used to categorize the ZDV fold-change (2.5, 4.5, 9.5, 50) and 3 for d4T (1.7, 2.5, 4.5) defining several ordinal susceptibility levels. A polytomous logistic regression model was used to compute odds ratio (OR) of the combination of one to 6 NAM relative to samples without NAM.

Results:  Among the 4907 samples, the proportion with 1, 2, 3, or 4 NAM was 19, 21, 22, and 17% respectively. M41L and T215Y were the most frequent mutations in the dataset. T215Y/F alone showed the greatest contribution to the reduction of ZDV and d4T susceptibility, while E44D and L210W had the lowest OR, among samples with a single NAM. Pathway 1 and 2 showed similar prevalence if <4 NAM were present (34% vs 37% for 3 NAM). The prevalence of pathway 2 (defined by the presence of T215F vs Y and K219E vs Q) declined markedly when 4 or more NAM were present:  10% of samples with 6 NAM were found in pathway 2 vs 73% in pathway 1. Most combinations in pathway 2 were associated with lower ZDV FC compared with pathway 1 even with a lower number of NAM. Numerous interactions between NAMs were found to be statistically significant in both ZDV and d4T models.

Conclusions:  Pathway 1 is preferred only in samples with >3 NAM. T215Y exhibits a greater effect on reduction of ZDV and d4T susceptibility than other NAM, giving a selective advantage to pathway 1. Negative interactions were revealed between additional NAM in pathway 1 increasing the ZDV and d4T resistance to a lesser degree.

Keywords: Mutation Pathway; TAMs; ZDV