Development of Calibrated Viral Load Standards for Group M Subtypes of Human Immunodeficiency Virus Type 1 and Performance of an Improved AMPLICOR HIV-1 MONITOR Test with Isolates of Diverse Subtypes

Abstract

Accurate determination of plasma human immunodeficiency virus type 1 (HIV-1) RNA levels is critical for the effective management of HIV-1 disease. The AMPLICOR HIV-1 MONITOR Test, a reverse transcription-PCR-based test for quantification of HIV-1 RNA in plasma, was developed when little sequence information on HIV-1 isolates from outside North America was available. It has since become apparent that many non-subtype B isolates, particularly subtypes A and E, are detected inefficiently by the test. We describe here the AMPLICOR HIV-1 MONITOR Test, version 1.5, an upgraded test developed to minimize subtype-related variation. We also developed a panel of HIV-1 standards containing 30 HIV-1 isolates of subtypes A through G. The virus particle concentration of each cultured viral stock was standardized by electron microscopic virus particle counting. We used this panel to determine the performance of the original AMPLICOR HIV-1 MONITOR Test and version 1.5 of the test with HIV-1 subtypes A through G. The original test underestimated the concentration of HIV-1 subtype A, E, F, and G RNA by 10-fold or more, whereas version of the 1.5 test yielded equivalent quantification of HIV-1 RNA regardless of the subtype. In light of the increasing intermixing of HIV-1 subtypes worldwide, standardization of PCR-based tests against well-characterized viral isolates representing the full range of HIV-1 diversity will be essential for the continued utility of these important clinical management tools.

The Perils of Pathogen Discovery: Origin of a Novel Parvovirus-Like Hybrid Genome Traced to Nucleic Acid Extraction Spin Columns

“…(v) HIV-1-spiked plasma sample. HIV-1 isolate DJ263 (CRF02_AG) was cultured, purified as described previously (39), quantified by the Abbott RealTime HIV-1 assay (Abbott Molecular, Des Plaines, IL), and used at a copy number of 10 4 to spike BaseMatrix defibrinated human plasma (Seracare) negative for hepatitis B virus (HBV), HCV, and HIV. Viral NA was extracted using the EZ1 viral minikit (Qiagen), followed by a post-DNase step and cleanup using the RNeasy MinElute cleanup kit (Qiagen).…”

Guidelines for Laboratory Test Result Reporting of Human Immunodeficiency Virus Type 1 Ribonucleic Acid Determination: Recommendations from a CDC Working Group

“…Available test kits perform differently in detecting non-B subtypes (42)(43)(44)(45)(46)(47)(48). In the United States, the Roche Amplicor HIV-1 Monitor version 1.0 is the most popular test for determining viral load.…”

“…In the United States, the Roche Amplicor HIV-1 Monitor version 1.0 is the most popular test for determining viral load. However, it underdetects and underestimates non-B subtypes (e.g., A, E, F, and G) when quantifying viral load (42)(43)(44)(45)(46)(47). The Bayer HIV-1 RNA 3.0 Quantitative Assay (bDNA) probably quantifies RNA of different HIV-1 subtypes accurately as a result of redundancy of multiple probes (43)(44)(45).…”

“…However, it underdetects and underestimates non-B subtypes (e.g., A, E, F, and G) when quantifying viral load (42)(43)(44)(45)(46)(47). The Bayer HIV-1 RNA 3.0 Quantitative Assay (bDNA) probably quantifies RNA of different HIV-1 subtypes accurately as a result of redundancy of multiple probes (43)(44)(45). The Organon Teknika NucliSens HIV-1 QT assay also underdetects and underestimates divergent subtypes (e.g., A and E) (43)(44)(45).…”

Development of sensitive ddPCR assays to reliably quantify the proviral DNA reservoir in all common circulating HIV subtypes and recombinant forms

“…There are many available assays that have been reported and used to quantify HIV DNA, of which most assays target the ltr [16,17,[19][20][21][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58], gag [22,23,43,44,54,56,[59][60][61][62], or pol [19,25,58,60,[63][64][65][66][67] regions [68]. To the best of our knowledge, we queried the target sequence of the most prominent assays to all known subtype and CRF entries in the LANL HIV1 Complete Nucleotide Filtered web alignment (http://www.hiv.lanl.gov/).…”