The Application Of Pyrosequencing In Detection Of Minor HIV-1Drug Resistant Strains In Chinese Treatment-naive HIV/AIDS Patients Experiencing Virological Failure

[Objective]:To establish a pyrosequencing method in detection&quantification of multiple loci responsible to NRTIs or NNRTIs-related drug resistance in HIV-1and reveal the contribution of minor HTV-1drug resistant strains to virological failure in treatment-naive HIV/AIDS patients of a cART cohort of China.[Methods]:Sets of carefully designed oligo-nucleotides were used to determine the linearity, sensitivity&stability of pyrosequencing. We designed both PCR and sequencing primers of several important loci of RT region in HIV-1genome responsible for NRTIs and NNRTIs-drug resistance. From a massive cART cohort, we selected40treatment-naive HIV/AIDS patients who suffer from virological failure. HIV RNA were extracted from plasma samples of these patients both before initiation of cART and after viral load rebounce, and the RT gene were amplified by RT-PCR and nested-PCR. Then we performed pyrosequencing to quantify mutations of loci described above. To determine the contribution of minor HIV-1drug resistant mutations to virological failure, we also summarized and analyzed correlated clinical data (including CD4+T lymphocyte count, HIV viral load, adherence, TDM data and in-house sequencing data) of these patients.[Results]:1. By using standard oligo-nucleotide and synthesis wild type&mutant sequence of codon103on RT gene, we determined the lower limit of sensitivity in pyrosequencing to be1%. We also do the linear regression between data yielded from pyrosequencing and theoretical outcomes, and fund the R2=0.9978.2. We designed PCR primers, sequencing primers and dispensation order targeting8NNRTIs or NRTIs related drug resistant loci of HIV-1, including103,181,184,67,70,210,215and219. We tested all8sets of protocol on serum specimen bearing HIV of subtype B and subtype AE.3. From the HIV/AIDS therapy cohort of the National11th Five-year Plan, we selected40patients with negative baseline DR screen results and no previous cART experience. Using the pyrosequencing methods demonstrated above, we found19minor drug resistant related loci in18patients, with K103N and Y181C to be the most common loci.4. We found5cases with baseline pyrosequencing results in consistence with conventional sequencing data after VL rebounce. We also gathered serum specimen of different time from patients and successfully demonstrated the correlation between minor drug resistant strain and prognosis as well as timely evolution of HIV-1drug resistant strain under pressure of cART.[Conclusions]:We designed and successfully performed pyrosequencing protocol to determine the minor drug resistant strain of HIV-1for the first in China. We selected40treatment-naive patients exercising virological failure from a large cohort of HIV/AIDS patients and conducted pyrosequencing on baseline specimen as well as specimen after VL rebounce. Combining data from pyrosequencing, conventional sequencing and other clinical data, our results support the idea of early screen of minor drug resistant mutations in HIV/AIDS patients.