Viral resistance to the antiretroviral drugs used for treatment of human immunodeficiency virus type 1 (HIV-1) infection has been an important cause of treatment failure and limits options for alternative antiretroviral regimens. The utility of monitoring HIV-1 for drug resistance in the clinical setting has not been fully defined. Several studies have suggested that evaluating HIV-1 drug resistance increased the likelihood of suppressing viral replication with subsequent treatment (2
; C. Cohen, S. Hunt, M. Sension, C. Farthing, M. Conant, S. Jacobson, J. Nadler, W. Verbiest, K. Hertogs, M. Ames, and A. Rinehart, Abstr. 7th Conf. Retrovir. Opportunistic Infect., abstr. 237, 2000), whereas other studies found that testing for drug resistance did not improve outcome (R. Haubrich, P. Keiser, C. Kemper, M. Witt, J. Leedom, D. Forthal, M. Leibowitz, J. Hwang, E. Seefried, J. A. McCutchan, N. Hellmann, D. Richman, et al., Antivir. Ther., abstr. 80, 2001; J. L. Meynard, M. Vray, L. Morand-Joubert, et al., Antivir. Ther., abstr. 85, 2000). In addition, transmission of HIV-1 drug-resistant variants has been reported (1
; S. J. Little, S. Holte, J. P. Routy, E. S. Daar, M. Markowitz, A. C. Collier, R. A. Coup, B. Conway, E. Connick, M. S. Saag, A. Mwatha, L. Corey, P. H. Keiser, M. Kilby, K. Dawson, J. M. Whitcomb, N. S. Hellmann, and D. D. Richman, Antivir. Ther., abstr. 25, 2001) with an increasing incidence, which is apparently related to the widespread use of antiretrovirals. Early reports suggest that highly active antiretroviral therapy was less effective in suppressing viral replication in individuals infected with drug-resistant virus, and when suppression of replication occurred, it was less durable (Little et al., Antivir. Ther.; K. Van Vaerenbergh, L. Debaisieux, I. Derdelinckx, N. De Cabooter, K. De Smet, K. Fransen, D. Marissens, K. Miller, G. Muyldermans, S. Sprecher, D. Vaira, C. Verhofstede, G. Zissis, M. Van Ranst, E. De Clercq, and A. M. Vandamme, Antivir. Ther., abstr. 130, 2001). In this context, HIV-1 drug resistance screening may prove useful in guiding the choice of initial therapeutic regimens by identifying drugs that are unlikely to suppress viral replication.
Oligonucleotide ligation assays (OLAs) are rapid, specific, and sensitive reactions for the detection of known point mutations (15
). Ligation assays are based on the covalent joining of two adjacent oligonucleotide probes by a DNA ligase when they are hybridized to a cDNA template, usually a PCR product. The specificity of the ligation is regulated by three factors: (i) the hybridization of the oligonucleotides to complementary sequences within the template, (ii) the need for these primers to anneal directly adjacent to one another in a 5′ to 3′ orientation on the target, and (iii) the requirement that the oligonucleotides have two bases complementary with the target in each direction at the site of the junction (15
). These characteristics allow nonstringent ligation conditions, which can be used to type multiple nucleotide substitutions in a single assay yet require specificity at the site of interest.
Our laboratory has developed an OLA for the detection of mutations in the HIV-1 pol
gene associated with resistance to zidovudine, dideoxyinosine, and lamivudine (9
) to nonnucleoside reverse transcriptase inhibitors (unpublished data) and, in collaboration with others, to mutations associated with multinucleoside drug resistance (29
). This high-throughput ligation-based system uses differentially modified oligonucleotides specific for wild-type or mutant sequences, allowing sensitive detection of both genotypes in a single well of a microtiter plate. In this report, we describe an OLA for the detection of primary mutations in HIV-1 pol
associated with high-level resistance to protease inhibitors currently in clinical use: nelfinavir, saquinavir, ritonavir, amprenavir, indinavir, and lopinavir.
While sequencing HIV-1 nucleic acids is commonly used to evaluate the presence of drug resistance mutations and provides the opportunity to comprehensively evaluate multiple mutations associated with HIV-1 antiretroviral resistance and novel mutations, the OLA has several advantages over consensus sequencing. First, the assay has a high throughput that makes it ideal for epidemiologic studies or clinical trials that evaluate a large number of specimens for specific mutations. Second, the OLA is highly sensitive in the detection of small populations of mutant genotypes among wild-type viral sequences (9
). Third, the results of the OLA are simple to interpret either visually or by a spectrophotometer. These attributes, together with its low cost, make this assay suitable for genotypic evaluation of HIV-1 drug resistance in laboratories where the costly equipment, software, and technical expertise needed for sequencing analysis may not be available.