Table 1.
The major strengths and challenges of current technologies for genotypic antiretroviral resistance testing.
| Method | Strengths | Challenges |
|---|---|---|
| Dye terminator ‘consensus’ or ‘population’ sequencing | Mutations correlated with reference to clinical outcomes | Limited potential for automation |
| Cost savings can be incurred by the use of in-house methods, shorter fragment sequencing and the use of qualitative polymerase chain reaction (PCR) for screening of patients with possible failure without performing viral load testing [75] | Not suited for parallela testing | |
| Cannot detect variants that occur at a low frequency | ||
| Point mutation assays (e.g. allele-specific PCR, oligonucleotide ligation assays and multiplex allele-specific assays) | High sensitivity for minor variants | No commercial assays available |
| Relatively economical | Multiplexing allows for detection of a set of common mutations only | |
| Next generation sequencing (various platforms including: 454, Illumina and Ion torrent sequencers) | Parallel testing: ability to pool multiple labelled specimens is cost-saving | Complex workflow is labour intensive |
| High sensitivity for minor variants (ultradeep sequencing) | Requirement for specialised facilities | |
| Long turn-around times PCR errors can lead to overestimating resistance Possible read problems dependent on template compositionb |
Parallel testing refers to the ability to test multiple HIV templates in a viral population. In addition, many patients can be tested at once through the use of ‘barcoding’ or ‘indexing’ the sequences. Although a single next generation sequencing reaction is costly this may allow for pooled testing of multiple samples. Allele-specific assays are more affordable than dye terminator sequencing or next generation sequencing, and can suffice if the requirement is to look for a few specific mutations; however, when required to detect more mutations, this approach become more costly.
Assays that are dependent on pyrosequencing (e.g. 454 and Iontorrent) may be inaccurate in determining the sequence in regions with homopolymers, whereas sequencing by synthesis methods (e.g. Illumina) may be prone to unequal sequencing coverage depending to the CG : AT composition of the genome.