Current Status of Point-of-Care Testing for Human Immunodeficiency Virus Drug Resistance

Abstract

Healthcare delivery has advanced due to the implementation of point-of-care testing, which is often performed within minutes to hours in minimally equipped laboratories or at home. Technologic advances are leading to point-of-care kits that incorporate nucleic acid-based assays, including polymerase chain reaction, isothermal amplification, ligation, and hybridization reactions. As a limited number of single-nucleotide polymorphisms are associated with clinically significant human immunodeficiency virus (HIV) drug resistance, assays to detect these mutations have been developed. Early versions of these assays have been used in research. This review summarizes the principles underlying each assay and discusses strategic needs for their incorporation into the management of HIV infection.

Keywords: HIV; drug resistance; point-of-care tests.

Figure 1.

Point-of-care test to detect human immunodeficiency virus drug resistance. A, Simplified kit with single-use reagents to test one specimen for drug resistance mutations prior to nonnucleoside reverse transcriptase inhibitor–based antiretroviral therapy. Kit detects mutant codons predictive of virologic failure [17]. The kit amplifies DNA using premade dried polymerase chain reaction (PCR) mixture. The product is added to a dried ligation mix and subsequently detected in a paper-strip test cartridge. B, Targets are PCR amplified, then the multiplex allele-specific (MAS) assay uses allele-specific primer extension (ASPE) with specific primers mixed together in one reaction tube containing reaction reagent mixture and a template. When the primer complementary to the 3ʹ-terminal nucleotide of the target, primer extension occurs and biotinylated deoxycytodine triphosphates (dCTPs) are incorporated into the extended products. ASPE products are hybridized to microspheres through the specificity of “TAG”/”Anti-TAG” recognition and read with the suspension array system. C, Premixed pan-degenerate amplification and adaptation (PANDAA) with quantitative PCR (qPCR) enzymes, buffer, primers, and probes labeled with 3 distinct fluorophores to detect 2 drug resistance mutations and quantify total viral nucleic acid. Viral RNA from plasma, DNA from whole blood, or PCR amplicon previously generated for Sanger sequencing can be used as the input template. PANDAA is a one-step reaction that does not require a first-round cDNA synthesis or PCR step prior to qPCR. PANDAA can be run on any qPCR machine that can distinguish the FAM, VIC, and NED fluorophores (or equivalent fluorophores with a similar emission spectra). Automated data analysis allows the relative abundance of each drug resistance mutation to be quantified with additional data handling by the user.

Figure 2.

Overview of the simple method to amplify RNA targets (SMART). A, The RNA sequences, isolated from a clinical sample, are put in a solution containing 2 probes that bind to specific sequences within the target RNA, allowing testing of RNA mutations. The first probe (capture probe) is attached to a magnetic bead, which hybridizes RNA via a general consensus sequence. The 5ʹ end of the biotinylated capture probe readily binds to a streptavidin-coated magnetic bead. Simultaneously a mutation-specific probe molecule (~25 nucleotides, SMART or amplification probe) that hybridizes with the RNA. The center sequence of the SMART probe molecule is the reverse complement (RC) of the target strain sequence. The sequence of the 2 flanking ends (called “hybrid seq RC” and “primer 2”) of the SMART probe can be adjusted by the user to optimize amplification reaction kinetics. At the conclusion of (A), a chain-linked molecule complex of streptavidin-coated magnetic beads—biotinylated oligo–RNA—SMART probe is centered about the target region of the RNA. B, The magnetic bead bound complex is microfluidically separated from the unbound SMART probes and/or other molecules in reservoir W1 to reservoir W2. C, Amplification of the SMART probe is performed via an isothermal scheme that utilizes the designed primer sequences for optimal reaction kinetics. Here, various enzymes (AMV-RT, RNase, and T7 polymerase) are used for transcription and amplification. Subsequently, molecular beacons or other fluorescent molecules can be used for detection of amplified SMART probes. The SMART scheme employs an isothermal and exponential amplification of SMART probes, which is suitable for point-of-care testing.