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By Liã Bárbara Arruda, Laura I. Weber, Marisa dos Santos, Edson M. Kawakubo and Ana Maria B. Martínez


<p id="para1">The method used by YAGYU et al. for the subtype-specific polymerase chain reaction (PCR) amplification of the gp41 transmembrane region of the human immunodeficiency virus type-1 (HIV-1) env gene, was tested. HIV-1 proviral DNA from 100 infected individuals in Itaja&#237;, South Brazil was used to analyze this method. Seventy individuals were determined according to this method as having PCR products at the expected size for subtypes B, C, D and F. Of these individuals, 26 (37.1%) were observed as having the expected amplification for subtype C, and 42 (60%) were observed as having the expected products for subtypes B and D. Of the subtype B and D amplicons, 16 (22.9%) were classified as subtype D, and 26 (37.1%) were classified as subtype B. Two individuals (2.9%) had amplicons that were observed after subtype F-specific amplification was performed. Sequencing and comparing the patient sequences to reference sequences confirmed the classification of sequences of subtypes C and B. However, sequences that were falsely determined as being D and F in the PCR assay were determined as being subtypes C and B, respectively, by sequence analysis. For those individuals from whom no amplified products were obtained, a low viral load that was indicated in their patient history may explain the difficulty in subtyping by PCR methods. This issue was demonstrated by the results of ANOVA when testing the effect of viral load on the success of PCR amplification. The alignment of the obtained sequences with HIV-1 reference sequences demonstrated that there is high intra-subtype diversity. This indicates that the subtype-specific primer binding sites were not conserved or representative of the subtypes that are observed in the Brazilian populations, and that they did not allow the correct classification of HIV-1 subtypes. Therefore, the proposed method by YAGYU et al. is not applicable for the classification of Brazilian HIV-1 subtypes.<br><p id="para2">A metodologia para amplifica&#231;&#227;o subtipo-espec&#237;fica por PCR da regi&#227;o transmembrana do gene env (gp41) do HIV-1, descrita por Yagyu e colaboradores, foi testada a partir de DNA proviral de 100 pacientes infectados pelo HIV-1 de Itaja&#237;, Sul do Brasil. Setenta indiv&#237;duos apresentaram produtos amplificados e correspondentes aos subtipos B, C, D e F de acordo com a metodologia escolhida. Destes indiv&#237;duos, 26 (37,1%) apresentaram a amplifica&#231;&#227;o esperada para o subtipo C de acordo com a metodologia; 42 (60%) apresentaram os produtos esperados para os subtipos B e D, sendo que na etapa seguinte de diferencia&#231;&#227;o destes subtipos, 16 (22,9%) corresponderam ao subtipo D e 26 (37,1%) ao subtipo B. Dois indiv&#237;duos (2,9%) mostraram produtos amplificados ap&#243;s a amplifica&#231;&#227;o espec&#237;fica para o subtipo F. O sequenciamento e a compara&#231;&#227;o com sequ&#234;ncias refer&#234;ncias confirmou a subtipagem de HIV-1 C e B obtida pela metodologia. No entanto, indiv&#237;duos subtipados erroneamente como HIV-1 D e F pela metodologia, foram classificados pela compara&#231;&#227;o com sequ&#234;ncias refer&#234;ncias como subtipos C e B, respectivamente. Em rela&#231;&#227;o aos indiv&#237;duos que n&#227;o mostraram produtos amplificados, a baixa carga viral observada no hist&#243;rico destes pacientes seria em parte respons&#225;vel pela dificuldade na subtipagem pela metodologia de PCR, como demonstrado pelo resultado significativo no ANOVA ao testar o efeito da carga viral no sucesso da amplifica&#231;&#227;o. O alinhamento das sequ&#234;ncias obtidas com sequ&#234;ncias refer&#234;ncias de HIV-1 correspondentes &#224; regi&#227;o da gp41 demonstrou que h&#225; uma alta diversidade intra-subtipo e que as regi&#245;es a partir das quais foram desenhados os oligonucleot&#237;deos iniciadores HIV-1 subtipo-espec&#237;ficos n&#227;o s&#227;o conservadas nem suficientemente representativas dos subtipos observados nas popula&#231;&#245;es brasileiras para permitir sua correta identifica&#231;&#227;o. Portanto, esta metodologia n&#227;o &#233; aplic&#225;vel para popula&#231;&#245;es virais brasileiras

Topics: HIV-1, gp41, Viral load, Subtypes, PCR, South Brazil, Arctic medicine. Tropical medicine, RC955-962
Publisher: Universidade de São Paulo
Year: 2013
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