Thursday, 31 August 2017 16:03

Comparing molecular methods to detect HIV drug susceptibility status

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Published in mBiosphere

Human immunodeficiency virus (HIV) is famous for its ability to gain resistance to antiretroviral therapy (ART); the poor fidelity of the reverse transcriptase when converting the RNA genome into DNA results in errors averaging 1 of every 17,000 incorrect nucleotides. These mutations can give progeny virions a selective advantage if the mutation facilitates antiviral avoidance, and some areas of the HIV genome are well characterized for their ability to be mutated and confer resistance.

Identifying potential drug resistance is important when diagnosing an HIV infection. In resource-rich countries, Sanger sequencing HIV genomic regions associated with resistance helps guide ART therapy choices. Many resource-constrained areas instead use amplification-refractory mutations system (ARMS)-PCR, a process where differential amplification based on primer mismatch can evaluate specific point mutations by agarose gel electrophoresis. A recent Journal of Clinical Microbiology report compares these two methods with new next-generation sequencing (NGS) methods to sequence the entire HIV genome.

JClinMicro: Multimethod longitudinal HIV drug resistance analysis in antiretroviral-therapy-naïve patients

To compare these methods, a large collaborative research team headed by first author Aubin Nanfack and senior scientist Ralf Duerr looked at a cohort of ART-naïve HIV-infected patients in Cameroon. The team used the three resistance-testing methods for 5 known mutation sites within the HIV pol gene. Mutations in this gene can confer resistance to NRTI and NNRTI class antiretrovirals.

Patients are often infected with one HIV variant, but due to high mutation rates, viral genomes with slightly different sequences called quasispecies begin to accumulate within the patient. Because NGS (in this case, miSeq Illumina technology) can sequence multiple sequences simultaneously, it was able to detect the different quasispecies infections within patients. For detecting resistance-associated mutations, Sanger sequencing and NGS worked equally well, most of the time. In two cases, NGS detected resistant mutations in quasispecies consisting of less than 20% of the total sequences within a patient; in these cases, the largest proportion remained sensitive and weren’t detected by Sanger sequencing. 63 of 66 patients showed the same ARMS-PCR results as the Sanger sequencing results, with three false-positive results, and importantly, no false-negatives.

HIV mol testing 2HIV Env protein from one patient, 42 mo. apart. Mutant reverted to WT sequences, leading to a susceptible variant protein. Source.

21 of the 66 patients provided samples over a 4-year period, allowing the researchers to investigate the resistance profiles of their infections over time. During this time, 5 patients had changes in the HIV pol gene sequence affecting antiviral drug efficacy, and 2 of these were resistance mutations that appeared despite the patients not being on a selective antiviral drug, though the researchers speculate that patients may have received drugs from HIV-positive family or friends on an intermittent basis. Resistance mutations reverted to sensitivity was detected, due to either superinfection (a second infection with a sensitive HIV strain) or intrinsic conversion.

HIV mol testing 1ARMS-PCR testing for one mutation. The presence of 2 bands is indicative of mutant virus presence, while only one band indicates no mutation. Source.

From the comparative study, the scientists concluded that ARMS-PCR compares well to Sanger sequencing to identify specific ART resistance mutations. ARMS-PCR is a labor-intensive process, since each individual mutation requires its own PCR interrogation (see example from the study, right), but its sensitivity presently provides the most cost-effective measure of ART susceptibility in drug-naïve patients. As sequencing costs continue to drop and become available to resource-scarce areas, new technologies may continue to improve diagnostic capabilities in some of the areas that need it most. 

Last modified on Thursday, 31 August 2017 16:33
Julie Wolf

Julie Wolf is the ASM Science Communications Specialist. She contributes to the ASM social media and blog network and hosts the Meet the Microbiologist podcast. She also runs workshops at ASM conferences to help scientists improve their own communication skills. Follow Julie on Twitter for more ASM and microbiology highlights at @JulieMarieWolf.

Julie earned her Ph.D. from the University of Minnesota, focusing on medical mycology and infectious disease. Outside of her work at ASM, she maintains a strong commitment to scientific education and teaches molecular biology at the community biolab, Genspace. She lives in beautiful New York City.