Proviral burden quantitation in peripheral blood mononuclear cells from HIV-1 infected patients by the method of PCR-enzyme-linked-capture-hydroxyapatite-assay (ELCHA) as subrogated marker for AIDS prediction.

Important note: Information in this article was accurate in 1993. The state of the art may have changed since the publication date.

Abstr Gen Meet Am Soc Microbiol. 1993;93:433 (abstract no. T-9). Unique Identifier : AIDSLINE ASM93/93291809
Garcia-Cabrera L; Ruiz-Palacios G; Instituto Nacional de la Nutricion Salvador Zubiran, Mexico,; D.F.

Abstract: CD4+ lymphocytes are the only recognized standard marker to measure the level of immunodeficiency and progression to AIDS in HIV-1 infected patients. It is important to find other markers for AIDS prediction. In this study we developed an enzymatic-colorimetric assay to quantify proviral burden in peripheral blood mononuclear cells (PBMC) amplified by PCR and we studied its correlation with CD4+ levels. PCR conditions were established using two primers to amplify specific DNA fragments of gag and pol genes. PCR products were analysed by Southern blot and ELCHA, the quantitative developed method, which uses biotinylated oligonucleotides coupled with hydroxyapatite capture and colorimetric reaction. DNA standard curve of O.D. was constructed with known quantities of HIV-1 proviral DNA per micrograms of cellular DNA. Study population: 15 AIDS patients, 30 asymptomatic HIV-1 patients with different CD4+ levels and 10 Healthy individuals. Specificity and sensitivity of ELCHA compared with Southern blot, were 100% and 100%, respectively. The mean of proviral DNA copies/micrograms of cellular DNA for AIDS patients was 425 +/-249; for asymptomatic patients CD4+ < 500 74 +/-21.5, and asymptomatic patients CD4+ > 500 65.11 +/-34.7, R = -0.917, p < 0.0001. ELCHA has the advantage over Southern blot of reducing the processing time by 2 days, avoiding the use of radioisotopes. Our data suggest that an increase in the proviral burden is coupled to a decrease of CD4+ and to the appearance of symptoms. Even though our results show only one expression of HIV-1, proviral burden could be used as a predictor marker of AIDS.

Keywords: Acquired Immunodeficiency Syndrome/*BLOOD/MICROBIOLOGY Blotting, Southern Colorimetry/METHODS DNA, Viral/ANALYSIS/GENETICS Genes, gag Genes, pol Human Hydroxyapatites HIV-1/GENETICS/*ISOLATION & PURIF Monocytes/*MICROBIOLOGY Polymerase Chain Reaction/METHODS Proviruses/GENETICS/*ISOLATION & PURIF Virology/METHODS ABSTRACT
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M9391162

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