In vivo emergence of drug-resistant mutations at less than 50 HIV-1 RNA copies/mL that are maintained at viral rebound in longitudinal plasma samples from human immunodeficiency virus type-1-infected patients on highly active antiretroviral therapy.

OBJECTIVE: Emergence of human immunodeficiency virus type-1 (HIV-1) genotypic drug resistance is generally attributed to noncompliance, poorly absorbed drugs, or drug-to-drug interaction. Attempts to determine emerging genotypic drug resistance from study subjects on highly active antiretroviral therapy (HAART) relied on insensitive polymerase chain reaction (PCR) techniques, revealing wild type HIV-1 or precursor resistant genotypes from few plasma samples successfully amplified with <50 copies/mL. STUDY DESIGN/METHODS: In this analysis, using Applied Biosystems' ViroSeq HIV-1 Genotyping Systems, Version 2.0 (Applied Biosystems, Foster City, CA, USA) and the supplemental, for research use only, nested PCR primers, genotypic drug resistance was determined in longitudinal plasma samples from 11 study subjects on HAART. RESULTS: In 4 of 11 study subjects, newly emerging genotypic primary resistant mutations were detected in plasma samples with <50 copies/mL. Most of these primary drug-resistant mutations were detected in subsequent longitudinal samples with detectable viral load (viral breakthrough). CONCLUSIONS: This analysis suggests sufficient viral replication <50 copies/mL to generate genotypic drug resistance in study subjects on suppressive HAART.

Quantification of cytomegalovirus DNA in peripheral blood leukocytes by a branched-DNA signal amplification assay.

Quantification of cytomegalovirus (CMV) DNA in blood may aid in the identification of patients at highest risk for developing CMV disease, the evaluation of new therapeutics, and the prompt recognition of drug-resistant CMV strains. A branched-DNA (bDNA) assay was developed for the reliable quantification of CMV DNA in peripheral blood leukocytes. The bDNA assay allowed for the highly specific and reproducible quantification of CMV DNA in clinical specimens. Furthermore, the bDNA assay was at least as sensitive as culture techniques and displayed a nearly 3 log10 dynamic range in quantification. Changes in CMV DNA levels measured by the bDNA assay in a human immunodeficiency virus-positive patient undergoing therapy were consistent with CMV culture, antigen, and genotype results and correlated with disease progression and resistance markers. The bDNA assay for the quantification of CMV DNA may provide a useful tool that can be used to aid physicians in monitoring disease progression, evaluating therapeutic regimens, and recognizing viral resistance and drug failure.

Quantitation of HBV DNA in human serum using a branched DNA (bDNA) signal amplification assay.

The aim of this study was to establish the performance characteristics of a nonradioisotopic branched DNA (bDNA) signal amplification assay for quantitation of hepatitis B virus (HBV) DNA in human serum. Quantitation was determined from a standard curve and expressed as HBV DNA equivalents/mL (Eq/mL; 285,000 Eq = 1 pg of double stranded HBV DNA). The bDNA assay exhibited a nearly four log dynamic range of quantitation and an analytical detection limit of approximately 100,000 Eq/mL. To ensure a specificity of 99.7%, the quantitation limit was set at 700,000 Eq/mL. The interassay percent coefficient of variance for quantification values ranged from 10% to 15% when performed by novice users with different sets of reagents. Using the bDNA assay, HBV DNA was detected in 94% to 100% of hepatitis B e antigen-positive specimens and 27% to 31% of hepatitis B e antigen-negative specimens from chronic HBV-infected patients. The bDNA assay may be useful as a prognostic and therapy monitoring tool for the management of HBV-infected patients undergoing antiviral treatment.

Quantitation of HIV-1 RNA in plasma predicts outcome after seroconversion.

OBJECTIVE: To investigate the relation between the quantity of human immunodeficiency virus type 1 (HIV-1) RNA in plasma and the risk for the acquired immunodeficiency syndrome (AIDS) or a decline in the CD4+ T-cell count after seroconversion. DESIGN: Prospective study. PATIENTS: 62 homosexual men with documented HIV-1 seroconversion. SETTING: University outpatient setting. MEASUREMENTS: Clinical status, CD4+ T-cell counts, and plasma and serum samples were obtained every 6 months. Human immunodeficiency virus RNA in plasma was quantitated with a branched-DNA (bDNA) assay. Serum samples were assayed for neopterin, beta 2-microglobulin, and immune complex dissociated HIV-1 p24 antigen. RESULTS: 18 of 62 (29%) men developed AIDS; 21 (34%) had a significant decline in the CD4+ T-cell count without AIDS; and 23 (37%) had a stable CD4+ T-cell count. For each participant, HIV-1 RNA results were categorized into one of four groups: 1) detection of HIV-1 RNA (> 1 x 10(4) genome equivalents/mL [Eq/mL]) in all samples; 2) detection in most samples (> or = 50%); 3) detection in fewer than 50% of samples; and 4) detection in none of the samples. Detection of HIV-1 RNA in all or most samples was strongly associated with AIDS (16 of 18 patients) and a decline in the CD4+ T-cell count (13 of 21 patients) compared with a stable CD4+ T-cell count (4 of 23 patients; P < 0.001). Conversely, the absence of HIV-1 RNA (< 1 x 10(4) Eq/mL) in all or most samples was associated with stable CD4+ T-cell counts (19 of 23 patients) and a lower risk for AIDS or decline in the CD4+ T-cell count (10 of 39 patients; P < 0.001). In multivariate analysis of all laboratory values at the seroconversion visit, a plasma HIV-1 RNA level greater than 1 x 10(5) Eq/mL was the most powerful predictor of AIDS (odds ratio, 10.8; P = 0.01). CONCLUSIONS: Plasma HIV-1 RNA is a strong, CD4+ T-cell-independent predictor of a rapid progression to AIDS after HIV-1 seroconversion.