Standard genotype assays may miss 75% of mutations when present in less than 35% of plasma sample
Simon Collins, HIV i-Base
Focusing on limitations of standard genotype analysis, Kearney and colleagues from NIH/NIAID also used single genome sequencing (SGS) to test the reliability for detecting minority populations.
They tested samples from 24 patients, either failing therapy or known to be infected with multi-drug resistant virus, by both standard and SGS and analysed with reference to the Stanford database.
All mutations present in composite genotype were detected by SGS. However, mutations present in <10% of single genomes, with one exception, were not detected by composite genotype. Mutations present in 10%-35% of single genomes were only detected in 25% of composite genotypes.
Several cases indicate the breadth of the missed mutations. Case one included 10 mutations conferring resistance to RTI, NNRTI and PI classes, but only present on 5-20% of the 20 genomes analysed. None of the PI mutations present in 15% of the genomes was detected by composite sequence including L10V, M46I, I84V and L90M.
A second sample missed five linked RT mutations present in 33% of genomes including K101E, Y181C, G190A and T215Y.
A third sample missed D67N in 30% of genomes and V118I present in 21%. All plasma samples contained at least one mutation that was identified by SGS but not by composite testing.
While these data appear alarming it is important to know that when a patient is on drugs and has truly developed resistance to these drugs with a documented rise in viral load the resistance mutations will generally be in excess of 50% of the total population and therefore will be picked up. However, once the selective pressure of the drug is removed their percentage in the viral population will fall quickly. Hence the rationale for obtaining the plasma sample for resistance testing while the patient is still taking the suspect drug.
Kearney M et al – Comparison of single-genome sequencing with standard genotype analysis for detection of HIV-1 drug-resistance mutations. XII International HIV Drug Resistance Workshop, Los Cabos, Mexico, 10-14 June 2003. Abstract 86. Published as part of Antiviral Therapy Volume 8 Issue 3.