Detection and molecular characterization of urinary tract HIV-1 populations

Abstract

Background: Identification of all possible HIV reservoirs is an important aspect in HIV eradication efforts. The urinary tract has however not been well studied as a potential HIV reservoir. In this pilot study we molecularly characterized HIV-1 viruses in urine and plasma samples to investigate HIV-1 replication, compartmentalization and persistence in the urinary tract.

Methods: Prospectively collected urine and blood samples collected over 12-36 months from 20 HIV-1 infected individuals were analysed including sampling points from prior to and after ART initiation. HIV-1 pol gene RNA and DNA from urine supernatant and urine pellets respectively were analysed and compared to plasma RNA viruses from the same individual.

Results: HIV-1 nucleic acid was detected in urine samples from at least one time point in 8/20 (40%) treatment-naïve subjects compared to 1/13 (7.7%) individuals on antiretroviral treatment (ART) during periods of plasma viral suppression and 1/7 (14.3%) individuals with virological failure. HIV-1 RNA was undetectable in urine samples after ART initiation but HIV-1 DNA was detectable in one patient more than 6 months after treatment initiation. There was co-clustering of urine-derived pol sequences but some urine-derived sequences were interspersed among the plasma-derived sequences.

Conclusions: Suppressive ART reduces HIV-1 replication in the urinary tract but HIV-1 DNA may persist in these cells despite treatment. A larger number of sequences would be required to confirm HIV compartmentalization in the urinary tract.

Fig. 1

CD4 T cell counts and viral loads over time in subjects showing detection of HIV nucleic acid in urine samples and plasma. Y-axis left: CD4 (cells/μl), black dotted line; Y-axis right: viral load (log₁₀ copies/ml), red line. Subject IDs: Nucleic acid was detected in urine supernatant and pellet samples in all the subjects except Subject 051. In Subject 100, nucleic acid was detected in urine pellet during treatment period including period of virological failure (month 30) and period of undetectable viremia in blood (month 24). BL baseline (enrolment) sample, UP urine pellet, US urine supernatant, P plasma. Positive (Pos) samples are highlighted in green, negative (neg) in grey, ND not determined. Grey-shaded graph regions denote period of antiretroviral therapy

Fig. 2

Phylogenetic tree of the sequenced pol gene from the urine and blood viruses. The evolutionary history was inferred using the Neighbour-Joining method. The bootstrap values (1000 replicates) are shown next to the branches. The evolutionary distances were computed using the Kimura 2-parameter method. Evolutionary analyses were conducted in MEGA6. The sequence clusters from the 8 subjects with HIV-1 nucleic acid detected in urine are indicated. Red triangles—plasma RNA, blue squares—urine supernatant variants, empty blue squares—urine pellet variants. Sequences are named using time points (e.g. 6 M = 6 months, BL baseline)