Background: We described a patient with PHI with an MDR virus, who without receiving  therapy precipitously lost all evidence of resistance in conjunction with an increase in viral load. Initial sequence analysis revealed many changes between resistant (strain 1) and susceptible (strain 2) variants raising the possibility of superinfection. We have used phylogenetic analysis and estimates of in vivo and in vitro fitness to address this issue.

Methods: Direct consensus PCR-sequencing of pol was performed on serial samples. Replication capacity (RC) was measured using a single-cycle assay based on the same amplicon. Motifscan was used to screen for potential CTL epitopes.

Results: Plasma viral load (pVL) was initially 5.5 log₁₀, declining to 3.0 log₁₀ by day 30. It remained stable until day 110, then increased to 4.5 log₁₀ by day 173, when  the switch in phenotype occurred. Phylogenetic analyses on all 1200 nucleotides, or on third base positions alone, confirmed the 2 viral strains cluster separately among strains from other PHI patients, indicating co-infection with 2 independent virus strains. In vitro RC assays revealed strain 1 had a 4-fold higher fitness in vitro than strain 2. This fitness advantage is consistent with the success of strain 1 immediately after infection, but outgrowth of strain 2 after day 110 is inconsistent with the RC data. From its rate of increase in frequency, we estimate the fitness of strain 2 in vivo is at least 5% higher than strain 1 at day 110, implying a significant difference between strains in a fitness component not represented in the in vitro RC assay. We hypothesized that this outgrowth represented escape from immune control. Class I HLA typing (A3,24; B35,40) permitted screening of sequences from strains 1 and 2 for HLA-matched CTL epitopes. 18 potential A3-restricted epitopes were identified, 2 of which were present in the initial sample and missing from the later sequence and could represent the escape mutations. 

Conclusions: We propose that the patient was co-infected with a drug-susceptible strain and a more fit MDR strain, an effective immune response was elicited but the susceptible strain subsequently suffered a mutation in a critical CTL epitope, permitting outgrowth after day 110.