Human immunodeficiency virus bDNA assay for pediatric cases

Techniques to quantify plasma HIV-1 RNA viral load (VL) are commercially available, and they are adequate for monitoring adults infected by HIV and treated with antiretroviral drugs. Little experience on HIV VL has been reported in pediatric cases. In Argentina, the evaluation of several assays for VL in pediatrics are now being considered. To evaluate the pediatric protocol for bDNA assay in HIV-infected children, 25 samples from HIV-infected children (according to CDC criteria for pediatric AIDS) were analyzed by using Quantiplex HIV RNA 2.0 Assay (Chiron Corporation) following the manufacturer's recommendations in a protocol that uses 50 microliters of patient's plasma (sensitivity: 10,000 copies/ml). When HIV-RNA was not detected, samples were run with the 1 ml standard bDNA protocol (sensitivity: 500 HIV-RNA c/ml). Nine samples belonged to infants under 12 months of age (group A) and 16 were over 12 months (group B). All infants under one year of age had high HIV-RNA copies in plasma. VL ranged from 30,800 to 2,560,000 RNA copies/ml (median = 362,000 c/ml) for group A and < 10,000 to 554,600 c/ml (median = < 10,000) for group B. Only 25 of children in group B had detectable HIV-RNA. By using the standard test of quantification, none of the patients had non detectable HIV-RNA, ranging between 950 and 226,200 c/ml for group B (median = 23,300 RNA c/ml). The suggested pediatric protocol could be useful in children under 12 months of age, but 1 ml standard protocol must be used for older children. Samples with undetectable results from children under one year of age should be repeated using the standard protocol.

Evaluación de la inmunofluorescencia indirecta como prueba suplementaria para el diagnóstico de la infección por HIV-1 / Evaluation of indirect inmunofluorescence as a confirmatory test for the diagnosis of HIV-1 infection

Se comparó la eficiencia diagnóstica de la inmunofluorescencia indirecta (IFI) como método confirmatorio de la infección por HIV-1 en muestras de suero de 362 personas con conductas de alto y bajo riesgo. El panel compuesto por 220 positivos, 122 negativos y 20 indeterminados por Western blot (WB) fue ensayado por una técnica de IFI desarrollada en nuestro laboratorio. La sensibilidad calculada fue 98,63 por ciento y la espicificidad 98,36 por ciento, indicando que la IFI es un método alternativo para la confirmación de la presencia de anticuerpos contra el HIV-1. Dado que su costo es menor que el 10 por ciento comparado con el del WB, se justifica su introducción en el algoritmo de diagnóstico serológico de HIV-1. Se observó también una relación directa entre la reactividad de las proteínas del WB y los resultados de IFI. En 15 muestras con resultado indeterminado por WB e inespecífico por IFI, las bandas más observadas fueron la p24 seguida de la gp160; por otro lado los anticuerpos contra las glicoproteínas virales son los que presentan mayor frecuencia en las muestras positivas débiles, demostrando su alto valor predictivo

Detección de la carga viral y su uso como marcador virológico en el seguimiento de pacientes HIV-1 positivos / Viral load detection and its role as virological marker for the monitoring of HIV-1 positive patients

Se realizaron los primeros estudios de carga viral en pacientes HIV-1 positivos provenientes de diferentes instituciones asistenciales de la Ciudad de Buenos Aires. Se evaluó la carga viral como marcador virológico y su correlación con la clínica y el recuento de los linfocitos CD4+ para 216 pacientes HIV-1 positivos. La técnica utilizada fue bDNA (Quantiplex HIV RNA 2.0 assay, Chiron Corporation). Se observó una tendencia al aumento de la carga viral en los pacientes con menor cantidad de linfocitos CD4+ y en los estadíos clínicos con sintomatología. En pacientes que no recibieron ninguna terapia antirretroviral se encontraron valores desde < 10000 copias de ARN viral/ml de plasma hasta 48995 c/ml. En aquéllos que recibieron terapia antirretroviral se observó mayor variación en los valores de la carga viral como lo mostró un rango de < 10000 c/ml hasta 96605 c/ml. Se obtuvieron muestras consecutivas en 25 pacientes y se observaron diferencias entre ambas muestras que permitieron corroborar la utilidad de la técnica en el seguimiento de los pacientes infectados con HIV

HIV-1 viral load: comparative evaluation of three commercially available assays in Argentina

Viral load (HIV-RNA copies per milliliter of plasma) has good correlation to prognosis considering progression to AIDS. The evaluation of commercial kits to measure viral load has become a need to find the most specific, sensitive and reproducible procedure to follow up HIV-infected patients. Hereby, a comparative analysis was done by using three different assays available in Argentina for quantitation of HIV-RNA in plasma. A plasma panel: 20 from HIV-1 infected individuals (9 asymptomatic and 11 symptomatic) and 9 from HIV-1 seronegative individuals was studied. Samples were run by Amplicor HIV-1 Monitor (Roche Diagnostic System, USA) Quantiplex HIV-1 RNA 2.0 Assay (Chiron Corporation, USA) and NASBA HIV-1 RNA QT (Organon Teknika, Holland). RNA was extracted from 0.2 ml of plasma for Amplicor, 0.1 ml and 1 ml of plasma for NASBA and, duplicates of 1 ml of plasma was centrifuged and pellet was used for bDNA assay no RNA extraction step. For a given specimen, a log difference of <0.5 between assays was considered as concordant result. All seronegative samples were bellow the detection limit of all assays (Amplicor 200 c/ml, NASBA 400 c/ml and Quantiplex (bDNA) 500 c/ml). Two samples from asymptomatic patients were not detectable by NASBA (Sensitivity: 90 per cent) Sensitivity was increased to 100 per cent by using 1 ml of plasma. All samples were detectable by the other assays (sensitivity: 100 per cent). For NASBA-bDNA, 74 per cent samples were concordant, 35 per cent for Amplicor-bDNA and 53 per cent for NASBA-Amplicor. By using 1 ml of plasma from asymptomatic patients, concordance was 65 per cent for NASBA-bDNA and 60 per cent for NASBA Amplicor. Comparing samples from asymptomatic patients, only 22 per cent was concordant in both cases. Reproducibility of NASBA was low (33 per cent, with differences lower than 0.5 Log) when 0.1 and 1 ml were used. Due to the levels of concordance of these results, it would be suggested to use always the same technique to follow up HIV-1 infection. The reproducibility of the assays should be tested by every laboratory and for every technician in charge of the assay in order to have confidence in the results specially to follow up HIV-infected patients or to monitor anti-viral therapies.

Different integrated and unintegrated HIV-1 DNA after superinfection and cell to cell transmission

Efficient superinfection of H9HTLVIIIB cell line (persistently infected with HIVHXB2 strain) with HIVMN strain is reported. The superinfecting viral DNA was found in the chromosomic and extrachromosomic fractions at early stages, but at 48 hours post superinfection, it remained mainly unintegrated. Interestingly, superinfected cells only produced HIVHXB2 in the supernatant and no increase of viral yield of this persistent virus was observed. Remarkably, virions of both strains, HIVHXB2 and HIVMN, were recovered after cocultivating superinfected cells with MT2 cell line. In the extrachromosomic fractions of seven different superinfected subclons of H9HTLVIIIB, viral DNA of the superinfecting HIVMN strain predominated while in the chromosomic fraction, the proportion of superinfecting viral DNA differed. The study of the presence of different integrated and unintegrated genomes in a single cell could be crucial in the understanding of HIV biology.

Factores de riesgo de infección por el virus de la inmunodeficiencia humana tipo 1 en un grupo de mujeres de Buenos Aires / Risk factors of infection by human inmunodeficiency virus type 1 in a women group of Buenos Aires city

Se estudiaron 204 mujeres de la ciudad de Buenos Aires, con el objeto de determinar los factores de riesgo de infección por el virus de la inmunodeficiencia humana tipo 1. Se recogieron datos epidemiológicos sobre factores de riesgo y se realizó un estudio serológico en muestras de sangre tomadas en el momento de la admisión. Las mujeres adictas a drogas por vía endovenosa tuvieron una tasa de infección del 65,85 por ciento, 8 veces superior al de las no adictas; esta tasa se elevó al 90,62 por ciento en aquellas mujeres que además tenían contactos sexuales con hombres seropositivos para el virus de la inmunodeficiencia humana tipo 1. En 7 casos registrados de transfusión sanguínea como único factor de riesgo reconocido, uno solo fue seropositivo. En este estudio, de las 64 pacientes con serología positiva el 84,37 por ciento de ellas eran drogadictas endovenosas, confirmando nuevamente que la drogadicción endovenosa fue el factor de mayor riesgo de infección por el virus de la inmunodeficiencia humana tipo 1

Análisis molecular del principal epítope de neutralización ([quot ]V3 loop[quot ]) del virus de la inmunodeficiencia humana tipo 1 de la Argentina / [Molecular analysis of the principal neutralization epitope (V3 loop) of human immunodeficiency virus type 1 in Argentina]

The main goal of the present paper was to analyze the molecular diversity of the principal neutralizing domain (V3 loop) of the HIV 1 gp120 in samples from patients of Argentina. The study was carried out on a total of 30 HIV 1 positive blood samples, obtained during 1991-1992, belonging to 15 intravenous drug users (group A), 5 homosexual men (group B), 8 children born to HIV 1 positive mothers (group C) and 2 AIDS patients (group D). By using extracted DNA from peripheral blood lymphocytes and from infected cells of the viral isolates in the case of the 2 AIDS patients, the V3 loop region was amplified by means of polymerase chain reaction. Direct sequencing by Sanger methodology was then performed on DNA fragments and nucleotide sequences obtained were translated into the correspondent amino acids. Consensus sequences for each group and a general consensus sequence were established (Table 1). Its alignment with V3 loop amino acid sequences of the major HIV 1 strains isolated worldwide is showed in table 2. Homology analysis between each sequence of the study population and sequence of different HIV 1 isolates showed that most of these samples share high homology with SF2 and BH10 strains. In contrast a low homology was found with JH3 and MN isolated (table 3). The presence of highly conserved amino acid residues as substitutions and insertions was determined in the Argentinian V3 loop sequences giving them a local pattern. The present paper is of great importance for our country, considering that the V3 loop is the main neutralizing domain becoming a major target in the development of HIV 1 vaccine. To our knowledge, this is the first report on the sequencing of the principal neutralizing domain of the Human Immunodeficiency Virus type 1 in Latin America.

Análisis molecular del principal epítope de neutralización ([quot ]V3 loop[quot ]) del virus de la inmunodeficiencia humana tipo 1 de la Argentina / [Molecular analysis of the principal neutralization epitope (V3 loop) of human immunodeficiency virus type 1 in Argentina]

The main goal of the present paper was to analyze the molecular diversity of the principal neutralizing domain (V3 loop) of the HIV 1 gp120 in samples from patients of Argentina. The study was carried out on a total of 30 HIV 1 positive blood samples, obtained during 1991-1992, belonging to 15 intravenous drug users (group A), 5 homosexual men (group B), 8 children born to HIV 1 positive mothers (group C) and 2 AIDS patients (group D). By using extracted DNA from peripheral blood lymphocytes and from infected cells of the viral isolates in the case of the 2 AIDS patients, the V3 loop region was amplified by means of polymerase chain reaction. Direct sequencing by Sanger methodology was then performed on DNA fragments and nucleotide sequences obtained were translated into the correspondent amino acids. Consensus sequences for each group and a general consensus sequence were established (Table 1). Its alignment with V3 loop amino acid sequences of the major HIV 1 strains isolated worldwide is showed in table 2. Homology analysis between each sequence of the study population and sequence of different HIV 1 isolates showed that most of these samples share high homology with SF2 and BH10 strains. In contrast a low homology was found with JH3 and MN isolated (table 3). The presence of highly conserved amino acid residues as substitutions and insertions was determined in the Argentinian V3 loop sequences giving them a local pattern. The present paper is of great importance for our country, considering that the V3 loop is the main neutralizing domain becoming a major target in the development of HIV 1 vaccine. To our knowledge, this is the first report on the sequencing of the principal neutralizing domain of the Human Immunodeficiency Virus type 1 in Latin America.