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Simple, sensitive, and specific detection of human immunodeficiency virus type 1 subtype B DNA in dried blood samples
I A Beck1, K D Drennan, A J Melvin
1Departments of Pediatrics, University of Washington, Seattle, Washington, USA.
Insights
A new HIV-1 DNA PCR assay on filter paper simplifies infant diagnosis. This method offers a practical, economical, sensitive, and specific approach for detecting human immunodeficiency virus type 1 (HIV-1) infection in infants.
Area of Science:
- Virology
- Molecular Biology
- Public Health
Background:
- Maternal antibodies interfere with standard human immunodeficiency virus type 1 (HIV-1) testing in infants.
- Accurate and early HIV-1 diagnosis in infants is crucial for timely intervention.
- Existing diagnostic methods can be complex and require specialized laboratory infrastructure.
Purpose of the Study:
- To develop and evaluate a simplified HIV-1 DNA PCR assay for infant diagnosis.
- To assess the assay's performance using whole blood collected on filter paper.
- To determine the sensitivity, specificity, and practicality of the novel assay.
Main Methods:
- Whole blood collected on filter paper, eliminating cell lysis and DNA extraction.
- DNA remained bound to filter paper throughout the nested PCR amplification of the HIV-1 pol gene.
- Validated with copy number standards, various HIV-1 subtypes, and clinical specimens from infants and adults.
Main Results:
- Reproducible detection of as few as 5-10 copies of HIV-1 per 5 microl of whole blood.
- Assay sensitivity remained stable after 3 months of storage at room temperature or heat exposure.
- Achieved high sensitivity (98.4%-99%) and specificity (98.3%-100%) in diverse populations.
Conclusions:
- The filter paper-based HIV-1 DNA PCR assay is a practical, economical, sensitive, and specific method.
- This assay simplifies specimen collection and processing, making it suitable for resource-limited settings.
- It offers a reliable tool for diagnosing HIV-1 subtype B infection in infants.
Abstract:
The detection of virus is used to diagnose human immunodeficiency virus type 1 (HIV-1) infection in infants due to the persistence of maternal antibodies for a year or more. An HIV-1 DNA PCR assay with simple specimen collection and processing was developed and evaluated. Whole blood was collected on filter paper that lysed cells and bound the DNA, eliminating specimen centrifugation and extraction procedures. The DNA remained bound to the filter paper during PCR amplification. Assays of copy number standards showed reproducible detection of 5 to 10 copies of HIV-1 in 5 microl of whole blood. The sensitivity of the assay did not decrease after storage of the standards on filter paper for 3 months at room temperature or after incubation at 37 or 45 degrees C for 20 h. The primers used for nested PCR of the HIV-1 pol gene amplified templates from a reference panel of multiple HIV-1 subtypes but did not amplify a subtype A or a subtype C virus from children living in Seattle. The assay had a sensitivity of 98.4% and a specificity of 98.3% for testing of 122 specimens from 35 HIV-1-infected and 16 uninfected children and 43 seronegative adults living in Washington. The assay had a sensitivity of 99% and a specificity of 100% for testing of 102 HIV-1-positive (as determined by enzyme immunoassay) Peruvian women and 6 seropositive and 34 seronegative infants. This assay, with adsorption of whole blood to filter paper and no specimen processing, provides a practical, economical, sensitive, and specific method for the diagnosis of HIV-1 subtype B infection in infants.
