Rastreamento molecular por pirosequenciamento de mutações relacionadas à resistência a Lamivudina em subpopulações de HBV e HIV isoladas de pacientes coinfectados

Approximately 10% of individuals infected with human immunodeficiency virus (HIV) are chronic carriers of hepatitis B virus (HBV). The monitoring of these patients deserves special attention, as they progress more rapidly to cirrhosis and death from liver disease than HBV monoinfected. For this reason, the antiviral therapy against the two viruses is a priority in most cases. Some drugs act in reverse transcriptase (RT), an important enzyme in the replication of both viruses and, therefore, can be effective in the treatment of both diseases. However, resistance mutations may be selected during therapy, leading to reduced efficacy of the used drugs. The aim of this study was to detect and quantify lamivudine (3TC) resistance mutations in isolated populations from HBV/HIV coinfected patients by pyrosequencing technique. Forty -five serum samples from HBV/HIV coinfected patients were analyzed for primary mutation rtM204V/I and compensatory mutations rtV173L and rtL180M inHBV and M184V/I mutation in HIV–1 genomes. All samples were subjected to a nested-PCR assay to amplify a fragment of HBV viral polymerase and an RT - PCR amplification of the HIV-1 RT region. Twenty-five samples were successfully amplified for HBV and subjected to pyrosequencing of HBV resistance mutations. The HIV RNA was amplified in thirteen samples, which were submitted to analysis of the M184V/I mutation. The Sanger sequencing of the HBV S genomic region was performed on 15 samples and the HIV YMDD motif was performed on 2 samples for comparison with the results of pyrosequencing. The analysis of the M204V/I mutation was successfully performed on 23 samples. Most samples (69.6%, 16 /23) showed a mixture of wild populations (M204) and mutants (M204V/I). Only M204V mutant population was found in 21.7 % (5 /23) and 8.7 % (2/23 ) of samples studied were composed only of wild populations. The V173L mutation was investigated in 20 samples. Only wild populations were found in 90 % (18/20) of the samples, and only mutant populations were found in 10% (2 /20) of the samples. In the samples subjected to Sanger sequencing it was found the same results for both methods, except for samples with mixed population in pyrosequencing, which the standard sequencing detected only the majority populations. The L180M mutation have been investigated only in 15 samples and only wild strains were found by pyrosequencing method , however , this result was disregarded due to a likely failure of the primer used for this mutation, since the Sanger sequencing detected this mutation in 4 samples. The M184V / I mutation of HIV was investigated in 9 samples, and only wild populations were found by pyrosequencing. The same result was confirmed by standard sequencing. The pyrosequencing is a sensitive technique that can be useful in the detection and quantification of subpopulations of resistant viruses that may not be detected by other methods. However, some adjustments must also be made for the detection of mutations rtL180M, which was found only in the samples analyzed by the Sanger technique. The high sensitivity of pyrosequencing enables one to evaluate the best treatment for a patient, because it is possible to detect the ratio of wild type and mutant strains infecting an individual, allowing early intervention during treatment.