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Detection of human immunodeficiency virus type 1 RNA in plasma samples from high-risk pediatric patients by using the
C E Bush1, R M Donovan, W R Peterson
1Department of Molecular Diagnostics, Baxter Diagnostics Inc., West Sacramento, California 95691.
Insights
A new method using self-sustained sequence replication (3SR) amplifies human immunodeficiency virus type 1 (HIV-1) RNA for sensitive detection in children. This rapid HIV-1 RNA detection shows promise for pediatric diagnosis and treatment monitoring.
Area of Science:
- Virology
- Molecular Biology
- Pediatric Infectious Diseases
Background:
- Accurate and rapid assessment of human immunodeficiency virus (HIV) infection in pediatric populations is critical.
- Existing methods for HIV-1 RNA detection in infants and children require improvement in speed and sensitivity.
Purpose of the Study:
- To evaluate the efficacy of self-sustained sequence replication (3SR) for direct amplification of HIV-1 RNA.
- To assess the sensitivity and performance of 3SR coupled with time-resolved fluorescence for pediatric HIV-1 detection.
Main Methods:
- Direct amplification of HIV-1 RNA using the 3SR reaction.
- Detection of amplified RNA via bead-based sandwich oligonucleotide capture hybridization.
- Quantification using rare earth metal chelate time-resolved fluorescence.
Main Results:
- The 3SR assay demonstrated high sensitivity, detecting less than 12 HIV-1 RNA copies with a 10(10)-fold amplification.
- Agreement between 3SR and viral culture was observed in 14 out of 19 pediatric plasma samples.
- Discrepancies in 5 samples highlighted the potential of 3SR to detect low-level viremia or early infection.
Conclusions:
- 3SR amplification combined with time-resolved fluorescence is a promising technology for sensitive HIV-1 RNA detection in pediatric patients.
- This method offers potential for improved HIV-1 diagnosis, monitoring disease progression, and evaluating therapeutic interventions in children.
- The technology aids in understanding HIV-1 pathogenesis and transmission dynamics in pediatric cohorts.
Abstract:
There is an urgent need for rapid and sensitive methods to assess human immunodeficiency virus (HIV) infection in infants and children. We evaluated an approach by using the self-sustained sequence replication reaction (3SR) to amplify HIV type 1 (HIV-1) RNA directly. The amplified RNA product was then detected by bead-based sandwich oligonucleotide capture hybridization and rare earth metal chelate time-resolved fluorescence. The sensitivity of this technology was determined to be less than 12 HIV-1 RNA copies with an amplification level of 10(10)-fold with purified HIV-1 RNA. Plasma samples from 19 high-risk pediatric patients younger than 5 years of age were examined, and results were compared with viral culture of patient plasma. Results from plasma culture and 3SR amplification agreed for 14 of these patients and disagreed for 5. Of the five samples which did not agree, four were positive by 3SR and negative by culture and one was positive by culture and negative by 3SR but became positive by 3SR at a subsequent testing. We conclude that 3SR amplification coupled with time-resolved fluorescence is a promising technology for investigating the relationship between the presence of HIV-1 RNA in plasma and progression of disease in HIV-infected pediatric patients. This technology should be important in the assessment of HIV-1 infection, in evaluating drug therapies, and in understanding the pathogenesis and transmission of the virus.