Historical genotype resistance testing and HGRT-adjusted ART
Any plasma genotype resistance result recorded before CSF collection was sum up and included in the cumulative HGRT for each participant. A subgroup of patients had resistance testing performed also in CSF; CSF results were described when available but not included in multivariable modelling due to the limited sample size and the likely selection bias.
HGRT was performed with direct full-population sequencing of plasma/CSF samples on the ABI PRISM 3130xl genetic Analyzer (Applied Biosystem, CA, USA) using the ViroSeq HIV-1 genotyping system (Abbott, IL, US) modified in that a nested PCR step was introduced to enhance the sensitivity for samples with HIV-1 RNA <1000 copies/mL27. After nucleic acid extraction from 1 mL of plasma/CSF, sequences of HIV-1 RT and PT regions were constructed for each sample with seven different primers targeting the majority of HIV-1 PT and RT genes, then analyzed by ViroSeq HIV-1 genotyping software vs2.8 (Celera Diagnostics, CA, USA).
Stanford Drug resistance database (http://hivdb.stanford.edu) and the International AIDS Society RAM mutation list (https://www.iasusa.org/resources/hiv-drug-resistance-mutations/) were used to evaluate the impact of RAMs on ART regimen at the time of LP.
The effectiveness of ART at the time of LP was estimated for each participant by adjusting the CNS penetration and effectiveness (CPE) score (proxy of effectiveness and pharmacokinetics of ARVs within CNS) for HGRT, as previously suggested12,28. Briefly, genotypic susceptibility scores were calculated using GRT results and assigning a score of 0 for resistant, of 0.5 for intermediate resistance, and of 1 for susceptible profile to each drug; HGRT-adjusted CPE scores were thereafter calculated by multiplying the CPE value by the genotypic susceptibility score for each drug regimen and summing the scores.