RESUMEN
AIM: To determine the relative effect of sample matrix on the quantitation of HIV RNA in plasma. METHOD: Two HIV-positive specimens were diluted into five and 10 different HIV-negative plasma samples, respectively. Branched DNA signal amplification technology and reverse-transcriptase polymerase chain reaction were used to measure the viral load. RESULTS: In one sample the viral load by polymerase chain reaction ranged from undetectable to 1.9 x 10(5) copies/ml, and the branched DNA results ranged from 2.6 x 10(4) to 4.2 x 10(4) HIV RNA equivalent/ml. In the other sample the corresponding figures were 6.3 x 10(4) to 5.5 x 10(5) copies/ml and 5.7 x 10(4) to 7.5 x 10(4) HIV RNA equivalents/ml. CONCLUSION: In contrast to reverse-transcriptase polymerase chain reaction the branched DNA signal amplification assay does not require a separate extraction step or enzymatic amplification of the target. Therefore this measurement is less affected by the sample matrix and the signal generated is directly proportional to the viral load.
Asunto(s)
Infecciones por VIH/microbiología , VIH-1/genética , VIH-1/aislamiento & purificación , ARN Viral/sangre , ARN Viral/genética , Virología/métodos , ADN Viral/genética , Amplificación de Genes , Infecciones por VIH/sangre , Humanos , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa/estadística & datos numéricos , Sensibilidad y Especificidad , Viremia/sangre , Viremia/microbiología , Virología/estadística & datos numéricosRESUMEN
The level of human immunodeficiency virus type 1 (HIV-1) RNA in human plasma has been quantitated directly with use of a solid-phase nucleic acid hybridization assay, based on branched DNA (bDNA) signal amplification technology with chemiluminescent detection. Signal amplification is accomplished by the incorporation of sites for 1,755 alkaline phosphatase-labeled probes per genome of HIV-1, after successive hybridization of target-specific oligonucleotides and bDNA amplifier molecules. The assay is performed in microwells, much like an immunoassay, and is amenable to routine laboratory use. Reproducibility and specificity studies indicated that the bDNA method was precise and showed no reactivity with seronegative donors. HIV-1 RNA levels were quantitated for 348 seropositive specimens, with a detection rate of 83% for those specimens from patients with < 500 CD4+ T-cell counts. Plasma RNA levels were found to change with disease stage, and in response to antiviral therapy. Quantitation of HIV-1 RNA in the plasma of HIV-1-infected patients, with use of the bDNA assay, may be a useful method for monitoring HIV-1 disease progression and therapeutic response.
Asunto(s)
Seropositividad para VIH/virología , VIH-1/genética , ARN Viral/sangre , Aciclovir/uso terapéutico , Recuento de Linfocito CD4 , ADN de Cadena Simple , Didanosina/uso terapéutico , Progresión de la Enfermedad , Quimioterapia Combinada , Amplificación de Genes , Genes pol , Seropositividad para VIH/diagnóstico , Seropositividad para VIH/tratamiento farmacológico , VIH-1/aislamiento & purificación , Humanos , Estudios Longitudinales , Hibridación de Ácido Nucleico , Sondas de Oligonucleótidos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Zidovudina/uso terapéuticoRESUMEN
The goal of this study was to determine the relationship between plasma human immunodeficiency virus (HIV) load and cytokine expression. HIV-RNA plasma levels were determined in 34 HIV-seropositive (HIV+) asymptomatic subjects [range: 0.5 to 211 kiloequivalents (kEq)/ml HIV-RNAJ, by a modified branched-DNA (bDNA) assay. Plasma HIV-RNA levels were positively correlated with increased plasma levels of TNF-alpha, soluble TNF receptor type II, soluble IL-2 receptor, beta 2-microglobulin, and neopterin, but not with plasma IL-6 levels. In contrast, increased viral load and diminished CD4 counts correlated weakly. TNF-alpha mRNA levels, as determined by bDNA technology, were not significantly increased in peripheral blood mononuclear cells (PBMC) isolated from HIV-infected subjects, compared to HIV-seronegative (HIV-) subjects, and were not correlated with plasma levels of HIV-RNA, cytokines, or activation markers. These results are consistent with the hypothesis that a self-reinforcing mechanism exists between TNF-alpha production and generalized immune activation on one hand with HIV replication on the other.
Asunto(s)
Infecciones por VIH/genética , Infecciones por VIH/inmunología , VIH/genética , ARN Viral/sangre , Factor de Necrosis Tumoral alfa/metabolismo , Biomarcadores/sangre , Estudios de Cohortes , Citocinas/sangre , VIH/inmunología , Infecciones por VIH/sangre , Humanos , Activación de Linfocitos , Masculino , ARN Mensajero/sangre , Receptores del Factor de Necrosis Tumoral/sangreRESUMEN
We have developed small-volume (50 or 250 microl)-format branched-DNA assays for human immunodeficiency virus type 1 (HIV-1) RNA for use with specimens in which the volume is limited and/or a high viral load is anticipated. These formats exhibited good correlation with the standard 1-ml format; high specificity, reproducibility, and linearity; and no significant difference in the quantification of HIV-1 subtypes.
Asunto(s)
Infecciones por VIH/virología , VIH-1/fisiología , ARN Viral/sangre , Adolescente , Fármacos Anti-VIH/uso terapéutico , Niño , Preescolar , ADN Viral , Progresión de la Enfermedad , Infecciones por VIH/tratamiento farmacológico , Humanos , Lactante , Recién Nacido , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Carga ViralRESUMEN
Highly sensitive assays that quantitate human immunodeficiency virus type 1 (HIV-1) RNA may be valuable for clinical research and the treatment of HIV-1-infected patients. In this study we evaluated the reproducibility and accuracy of the first-generation branched DNA (bDNA-1.0) signal amplification assay under conditions that are relevant to routine use in a clinical context. We show that the bDNA-1.0 assay was able to discern two- to three-fold changes in plasma HIV-1 RNA levels as significant. Reverse transcription coupled to polymerase chain reaction (RT-PCR) was less reproducible and required a 3.7- to 5.8-fold change in plasma HIV-1 RNA levels to be statistically significant. The accuracy of the bDNA-1.0 assay in RNA quantitation was not affected by HIV-1 genotypic variation or by the presence of hemoglobin, bilirubin, lipemia, or any of a dozen therapeutic drugs. Using the bDNA-1.0 assay, we show that HIV-1 RNA levels in plasma specimens were stable when stored at -80 degrees C and were able to withstand at least three freeze-thaw cycles without significant loss. We also examined the performance of an ultrasensitive bDNA assay with improvements to the signal amplification technology. The ultrasensitive bDNA assay displayed a quantitation limit of approximately 500 RNA Eq/ml, yet maintained a dynamic quantitation range up to 1.6 x 10(6) RNA Eq/ml. Like the bDNA-1.0 assay, the ultrasensitive bDNA assay was not affected by HIV-1 genotype variability.
Asunto(s)
VIH-1/genética , ARN Viral/sangre , Anticoagulantes/normas , Antivirales/farmacología , Bilirrubina/sangre , Conservación de la Sangre , ADN Viral/análisis , Amplificación de Genes , Variación Genética , Genotipo , Hemólisis , Humanos , Lípidos/sangre , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Manejo de Especímenes , Transcripción GenéticaRESUMEN
The quantification of human immunodeficiency virus type 1 (HIV-1) RNA has facilitated clinical research and expedited the development of antiretroviral drugs. The branched-DNA (bDNA) assay provides a reliable method for the quantification of HIV-1 RNA in human plasma and is considered one of the most reproducible assays ready for use in clinical trials. A series of oligonucleotide probe design and solution changes have been developed to enhance the sensitivity of the bDNA assay while maintaining its performance characteristics. Among the changes incorporated into the enhanced-sensitivity bDNA (ES bDNA) assay to reduce the background level and enhance the signal are the use of shorter overhang sequences of target probes for capture, the cruciform design of target probes for amplification, and the addition of preamplifier molecules. The ES bDNA assay is at least 20-fold more sensitive than the first-generation bDNA assay, yet it maintains a high level of accuracy, linearity, and reproducibility. Further, quantification values obtained with the ES bDNA assay and the first-generation bDNA assay are highly correlated, thus allowing for meaningful comparisons of HIV-1 RNA levels in specimens tested with either assay. The ES bDNA assay may be useful in determining the prognostic value of HIV-1 RNA levels of below 10,000 copies per ml and in assessing the clinical benefit of antiretroviral therapy-induced decreases in plasma HIV-1 RNA sustained at levels of below 10,000 copies per ml.