Reference sequences from the panel of HIV isolates that we PCR
Reference sequences from the panel of HIV isolates that we PCR amplified with our universal primer set (Additional file 4). As shown in Additional file 4, all samples from this cohort cluster with subtype B viruses, including the HXB2 control samples that we sequenced. In addition, the two time points from each sample clustered with each other and not with other samples, indicating that there was no cross-contamination resulting in the changes in drug resistance mutations found between the two time points from each patient sample.Drug resistance mutations detected in specimens from ALIVE*Boldface mutations are major drug resistance mutations, non-boldface mutations are accessory mutations.further upstream in the protease region, however, primers design is constrained when trying to keep the primers universal across multiple HIV clades. As mentioned above,To detect HIV drug resistance mutations in virus from the 29 ALIVE participants failing treatment after release from prison/jail, we analyzed the pol gene sequencing reads from two time points. First, the ALIVE participants successfully suppressed HIV before incarceration by using antiretroviral therapy. The first time point we sampled was at the time point just prior to this viral suppression. Some patients were PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27107493 drug na e at this time point, but others were previously drug experienced (as outlined in Additional file 3) and either changed regimens or began taking ARTs after a break in treatment to reach viral suppression at the subsequent time point. The second time point represents the virus after treatment failure,Dudley et al. Retrovirology (Page 7 ofas detected at an ALIVE study visit, after release from jail or prison. We obtained sequence data from both time points from 26/29 specimens. Relative to the first time point sequenced (baseline), new drug resistance mutations emerged in 54 (14/26) of the patients exhibiting treatment failure that were not detected at AZD3759 biological activity baseline (Figure 3A and Table 3). One additional sample (patient 27) contained major drug resistance mutations at both time points tested that may have contributed to treatment failure at the second time point (Table 4). These mutations, I54V, L90M, K103N and M184V, were present at frequencies >85 at both time points with most being present at >99 of sequences. The remaining samples without emerging drug resistance mutations contained primarily accessory mutations at baseline or at the second time point or very low level major drug resistance mutations that disappeared by the second time point and therefore likely do not contribute to treatment failure. Therefore, these patients may be failing treatment for reasons other than drug resistance mutations in the pol gene product. For example, some patients may fail due to lack of adherence or presence of mutations in Gag or the cytoplasmic tail of Env, which have been recently reported to affect drug resistance to protease inhibitors [34-36]. In the future it will be possible to expand our technique to include these regions outside of PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/26780312 the pol gene, however, it would require amplification of more than one amplicon prior to fragmentation and sequencing.Association of major drug resistance mutations with treatment in ALIVEA 23 n=6 46 n=12 19 n=5 Accessory mutations only Major mutations only Major and accessory mutations No mutations B 31 n=9 12 n=41 n=As a way to assess our drug resistance genotyping assay, we compared the major drug resistance mutations identified by our.
