ABSTRACT
We assessed the intralaboratory reproducibility of a system for sequencing human immunodeficiency virus type 1 (HIV-1) protease (PR) and reverse transcriptase (RT) by using replicate subanalyses of 46 plasma samples collected from HIV-1-infected, antiretroviral-experienced patients in order to determine the relative contributions of the different procedural steps to final sequence variability. Complete sequence concordance between duplicates of each sample was 99.4%. Complete and partial mismatches occurred scattered throughout the PR-RT genome segment at >300 positions. Approximately 75% of the discordances involved mixtures, some of which appeared at key resistance sites. Most differences were the result of the first-round RT-PCR procedure. Inter-rater concordance for sequence analysis and assembly was >99.9%. There was no observed correlation between the number or frequency of mismatches and plasma viral loads. A separate longitudinal analysis of a single routine control sample sequenced 103 times over 9 months consistently gave highly reproducible sequences (median percentage of nucleotide discordances, 0.04%; range, 0 to 0.2%). Finally, sequence data from 168 sequential samples collected from 22 patients with long-term, predominantly wild type HIV showed that intrapatient nucleotide concordance with individual index sequences ranged from 96.5 to 100%. Together, these results confirm that sequence-based genotyping can be a precise and reliable tool for monitoring HIV drug resistance, and they suggest that efforts to reduce variability should focus on the first RT-PCR step. Consequently, the data suggest that the composition of external quality assessment panels should be based on clinical HIV isolates rather than DNA clones.
Subject(s)
Drug Resistance, Viral/genetics , HIV Protease/genetics , HIV Reverse Transcriptase/genetics , HIV-1/drug effects , Laboratories/standards , Sequence Analysis, DNA/standards , Amino Acid Sequence , Antiretroviral Therapy, Highly Active , Genotype , HIV Infections/drug therapy , HIV Infections/virology , HIV-1/classification , HIV-1/genetics , Humans , Microbial Sensitivity Tests/methods , Microbial Sensitivity Tests/standards , Mutation , RNA, Viral/blood , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/standards , Sequence Analysis, DNA/methodsABSTRACT
We investigated the performance of a double-antigen sandwich recombinant enzyme immunoassay (EIA; Abbott Laboratories, North Chicago, Ill.) and compared it with that of a synthetic-peptide-based EIA (Biochem Immunosystems, Montreal, Quebec, Canada) for the detection of human immunodeficiency type 1 (HIV-1) and HIV-2 antibodies in 2,321 clinical serum samples. The results of both EIA methods and Western blot (immunoblot) were in agreement for 1,046 HIV-1 and 10 HIV-2 specimens from a panel of known positives. From a prospective panel of 1,085 specimens, 38 proved to be positive by both EIAs and Western blot, 3 were positive by the recombinant EIA only, and 9 were positive by the peptide EIA only, for calculated specificities of 99.71 and 99.04%, respectively. Of 180 specimens from a seroconversion panel collected from 77 patients, the results for 170 were in agreement by all antibody testing methods and 10 were found to be repeat reactive for HIV antibodies by the recombinant EIA only. All 10 were initial specimens of seroconverting patients; 7 were also reactive for HIV p24 antigen. An examination of four of these sera by radioimmunoprecipitation assay showed gp120 and gp160 bands in each. Analysis of the anti-Env antibody class in three of these samples showed that one consisted of immunoglobulin M (IgM) only and two contained both IgG and IgM antibodies. Although both EIA procedures were sensitive and specific in the detection of antibodies to HIV-1 and HIV-2 and both were capable of detecting early antibodies, the recombinant assay was more sensitive for antibody detection during early seroconversion.
Subject(s)
HIV Antibodies/blood , HIV Seropositivity/immunology , HIV-1/immunology , HIV-2/immunology , Immunoenzyme Techniques , Evaluation Studies as Topic , Gene Products, env/blood , HIV Core Protein p24/blood , HIV Envelope Protein gp120/blood , HIV Envelope Protein gp160 , HIV Seropositivity/virology , HIV-1/isolation & purification , HIV-2/isolation & purification , Humans , Immunoenzyme Techniques/statistics & numerical data , Immunoglobulin G/blood , Immunoglobulin M/blood , Protein Precursors/blood , Sensitivity and Specificity , Time FactorsABSTRACT
The purpose of this study is to evaluate the fascial stapler (a new technique) in the plication of the musculoaponeurotic fascia abdominoplasty in comparison to conventional (sutured) techniques. Thirty-eight patients underwent abdominoplasty with rectus sheath plication. Patients were randomized into staple and suture groups. Similar degrees of plicationing were performed in both groups (range, 12-20 cm). Fascial repairs were evaluated postoperatively at approximately 1 month and 6 months. A small, but comparable, subclinical fascial separation was demonstrated immediately in both the stapled and sutured groups. No progression of fascial separation was observed in either group at 1 month and 4 months postoperatively. No complications attributable to the fascial closure were noted in either group. Operative time was considerably less with the stapled technique. The results, although early, suggest that the use of fascial staples for plicationing of the musculoaponeurotic fascia during abdominoplasty is comparable to conventional (sutured) techniques regarding complication rate and disruption rate, but appreciably decreases operative time.
