ABSTRACT
The detection of HIV-1 proviral DNA in children born to seropositive mothers was studied using the polymerase chain reaction with either a radioactive electrophoretic method or a noval procedure that employs colorimetric microwell visualization. Peripheral blood mononuclear cell lysates from 18 HIV-1 infected children and 28 uninfected subjects were assayed for a 142 bp fragment of DNA from the gag region of HIV-1 using the primer pair SK145-431. Detection of amplified DNA was carried out by hybridization with a radiolabeled SK102 probe, or with a tagged SK102 probe permitting colorimetric detection. The radioactive detection procedure demonstrated 100% specificity and correlated with the serological results. The assay was more sensitive than the p24 antigen test, but two false negative results were obtained. One was from a sample taken at 2 weeks, an age at which undetectable provirus levels were reported in almost all HIV-1 infected newborns. The second was probably due to a low copy number of proviral DNA, as positive results were obtained in all other (6) samples from this child. Comparative analysis in a limited number of specimens of radioactive and colorimetric detection following PCR revealed 100% specificity and comparable sensitivity with 4 discordant results. The results show that PCR is the best method for early diagnosis of HIV-1 infection in pediatric subjects. The study also demonstrated the value of a colorimetric detection method for PCR products. This colorimetric microwell plate procedure may prove a useful technique in routine diagnosis of HIV-1 infection in children.
Subject(s)
Acquired Immunodeficiency Syndrome/diagnosis , DNA, Viral/analysis , HIV-1/genetics , Polymerase Chain Reaction , Child , Child, Preschool , Colorimetry , Female , HIV Antibodies/blood , Humans , Infant , Infant, Newborn , MaleABSTRACT
OBJECTIVE: To evaluate an acid pretreatment method designed to dissociate HIV p24 antigen from immune complexes in serum. DESIGN: Patient sera and sera containing experimental immune complexes were quantified for p24 antigen before and after immune complex dissociation (ICD). The clinical application of ICD was assessed in 1328 serum and plasma samples collected from HIV-infected patients. METHODS: Immune complexes were created artificially by mixing purified p24 antigen with antibody-positive sera or a standardized concentration of human antibody to p24. ICD was achieved by incubation of samples with an equal volume of Glycine HCl for 90 min at 37 degrees C followed by neutralization with Tris NaOH. Samples were quantified for p24 antigen using a commercial enzyme-linked immunosorbent assay (ELISA) kit. RESULTS: ICD resulted in significant release of purified antigen from simulated immune complexes in antibody-positive sera. Variation in antigen sequestration and dissociation was related to anti-gag antibody titers. ICD resulted in complete recovery of 500 pg of antigen complexed with human anti-p24 antibody at concentrations up to 2.5 U/ml. In seropositive patients, the mean level of serum antigen was 3.5-fold higher after ICD, and an additional 21% were antigen-positive. CONCLUSIONS: Pretreatment greatly improved antigen detection in HIV-antibody-positive sera by effectively dissociating immune complexes without compromising reactivity of the antigen itself. The treatment also facilitated routine monitoring of patients by revealing fluctuations in serum antigen that were indistinguishable or poorly defined in untreated sera.
