RESUMO
The major human immunodeficiency virus type 1 subtype circulating in Brazil is B, followed by F and C. We have genotyped 882 samples from Brazilian patients for whom highly active antiretroviral therapy failed, and we found subtype B and the unique recombinant B/F1 forms circulating. Due to codon usage variation, there is a significantly lower incidence of the substitutions L210W, Q151M, and F116Y in subtype F1 isolates than in the subtype B counterparts.
Assuntos
Terapia Antirretroviral de Alta Atividade , Códon/genética , Farmacorresistência Viral/genética , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Mutação , Brasil , Contagem de Linfócito CD4 , Feminino , Genótipo , Infecções por HIV/virologia , Protease de HIV/genética , Transcriptase Reversa do HIV/genética , Soropositividade para HIV/tratamento farmacológico , Soropositividade para HIV/virologia , HIV-1/classificação , HIV-1/genética , Humanos , Masculino , RNA Viral/sangue , Falha de TratamentoRESUMO
The I50V protease inhibitor (PI) resistance mutation was found in 87.4% of protease gene fragments sequenced from 199 nucleic acid isolates extracted using an NASBA virus load assay, performed between 1997 and 2001 in Brazil. This mutation is an amprenavir-related mutation, and at that particular time this PI was seldom used in Brazil. This mutation was found both in patients with and without therapeutic success. Q calibrators showed the PI resistance mutation I50V when directly amplified and sequenced from the 423-bp PCR product targeting protease gene. The majority of the patients' samples had a mixture of I50I and I50V; however, this artifact was nor seen when a 989-bp PCR product was used. These results show that RNA extracted using virus load kits need to be critically evaluated before being used in home-brew genotypic tests.
Assuntos
Reações Falso-Positivas , Inibidores da Protease de HIV/farmacologia , HIV-1/genética , Kit de Reagentes para Diagnóstico/normas , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/uso terapêutico , Farmacorresistência Viral/genética , Infecções por HIV/genética , Infecções por HIV/virologia , Protease de HIV/genética , Protease de HIV/metabolismo , Inibidores da Protease de HIV/uso terapêutico , HIV-1/efeitos dos fármacos , Humanos , Mutação , RNA Viral/análise , Kit de Reagentes para Diagnóstico/estatística & dados numéricos , Carga ViralRESUMO
We report an improved method for the detection and identification of mycobacteria using PCR and the heteroduplex mobility shift assay (HMA). The HMA for detection of mycobacteria was based on the microheterogeneity within the DNA coding sequences for 16S rRNA. A remarkable shift between single-stranded, heteroduplex and homoduplex bands in PAGE was observed among the Mycobacterium spp. tested. The Mycobacteria HMA (MHMA) of amplified PCR products from mycobacteria DNA coding for 16S rDNA derived from culture showed a specific heteroduplexes formed among different Mycobacterium species. Other bacterium species were distinguished from Mycobaterium due to slow migrating heteroduplexes mobility bands observed when M. bovis (BCG), M. avium, or M. fortuitum were used as a standard. The specific heteroduplexes were detected when as little as 1 etag of DNA template was used, although better results were obtained with 5 etag and when PCR products of sample test and mycobacterium standard were mixed at a ratio of 1.8. To correctly evaluate the feasibility of using MHMA to detect and identify mycobacteria, 15 clinical sample patients were tested. All MTB-positive clinical samples were identified by MHMA as well as the negative samples. In addition, MHMA will, in principle, be applicable to the detection and classification of any microorganism showing differences within the 16S rRNA as well as to the identification of new and unrecognized bacterial species.
