HIV-1 diagnosis of perinatally exposed children is usually performed by molecular biology-based methods, allowing the direct detection of the virus. Thus, HIV-1 genomic variability within and across strains plays a major role in relation to the sensitivity of these tests, often leading to misdiagnosis. We describe the performance of an in-house multiplex nested PCR (nPCR) for early detection of HIV-1 infection in perinatally exposed children born in Argentina, where the percentage of diverse BF recombinants is as high as 80%. After evaluation of 1316 HIV-1 perinatally exposed children collected over a 7-year period, the specificity and sensitivity of the diagnostic nPCR was of 100% and 99.2% respectively, with only two false negative cases indicating a good performance of the diagnostic nPCR in the Argentine pediatric cohort. In search of unusual HIV-1 subtypes among 22 HIV-1 infected cases presenting partial or complete HIV-1 gene amplification failure, we performed phylogenetic and recombination analysis of a vpu-env fragment in addition to gag and env Heteroduplex Mobility Assay screening. The most unusual findings included two subtypes A and a novel BC recombinant, while the majority of the strains were a variety of different BF recombinants. These results indicate the presence of novel and heterogeneous genotypes in our country and the need of continuous viral surveillance not only for diagnostic test optimization but also for the eventual implementation of a successful vaccine.

El diagnóstico temprano de infección por HIV-1 en niños expuestos perinatalmente al virus se realiza con técnicas de biología molecular, detectando el virus en sangre. Por ello, la variabilidad genómica intra e inter subtipo del HIV-1 juega un rol importante en relación a la sensibilidad de estos tests. Describimos aquí la performance de una PCR multiplex anidada artesanal (nPCR), rutinariamente usada para el diagnóstico temprano de la infección por HIV-1 en niños expuestos por vía perinatal en Argentina, donde cerca del 80% de las cepas circulantes son diversas recombinantes BF. La nPCR se utilizó para el diagnóstico de infección en 1316 niños en un período de 7 años, obteniéndose una especificidad de 100% y una sensibilidad de 99.2%, con sólo 2 falsos negativos. En busca de subtipos inusuales de HIV-1 entre los 22 casos que presentaron falla completa o parcial en la amplificación por nPCR, se subtipificaron fragmentos de gag y env mediante el Ensayo de Movilidad de Heteroduplex, y de los genes vpu-env por análisis filogenético y de recombinación. Se encontraron un subtipo A en 2 hermanos, y una recombinante BC, mientras que el resto de las cepas de HIV-1 pertenecieron a diversas recombinantes BF. Los resultados indican que en nuestro país circula una variedad de genotipos de HIV-1 con influencia sobre el diagnóstico. El continuo monitoreo de las cepas circulantes es necesario tanto para la optimización de las técnicas de diagnóstico como para la implementación de una vacuna efectiva en el futuro.

This study aimed at implementing a Nested-polymerase chain reaction (Nested-PCR) for the molecular diagnosis of human T-cell lymphotropic virus type I/II (HTLV-I and HTLV-II) infections in peripheral blood mononuclear cells of infected subjects in Argentina. The sensitivity and specificity of the assay for the detection of regional strains were assessed by comparing them with the molecular assay of reference PCR-hybridization. The Nested-PCR detected 1 MT-2 cell (³ 8 proviral copies)/1x106 non-infected cells showing high sensitivity for provirus detection. While both molecular assays showed high specificity (100%) for HTLV-I and HTLV-II detection, the sensitivity values differed: 100% for Nested-PCR and 67% for PCR-hybridization assay. Moreover, this technique showed less sensitivity for the detection of DNA sequences of HTLV-II (33%) than for the detection of DNA sequences of HTLV-I (75%). The high sensitivity and specificity of the Nested-PCR for regional strains and its low costs indicate that this assay could replace the PCR-hybridization assay for the molecular diagnosis of HTLV-I/II infections. It will be interesting to assess the usefulness of this assay as a tool for the molecular diagnosis of HTLV-I/II infections in other developing countries. Other studies that include a greater number of samples should be conducted.