Early monitoring of the human polyomavirus BK replication and sequencing analysis in a cohort of adult kidney transplant patients treated with basiliximab

Abstract

Background: Nowadays, better immunosuppressors have decreased the rates of acute rejection in kidney transplantation, but have also led to the emergence of BKV-associated nephropathy (BKVAN). Therefore, we prospectively investigated BKV load in plasma and urine samples in a cohort of kidney transplants, receiving basiliximab combined with a mycophenolate mofetil-based triple immunotherapy, to evaluate the difference between BKV replication during the first 3 months post-transplantation, characterized by the non-depleting action of basiliximab, versus the second 3 months, in which the maintenance therapy acts alone. We also performed sequencing analysis to assess whether a particular BKV subtype/subgroup or transcriptional control region (TCR) variants were present.

Methods: We monitored BK viruria and viremia by quantitative polymerase chain reaction (Q-PCR) at 12 hours (Tx), 1 (T1), 3 (T2) and 6 (T3) months post-transplantation among 60 kidney transplant patients. Sequencing analysis was performed by nested-PCR with specific primers for TCR and VP1 regions. Data were statistically analyzed using χ² test and Student's t-test.

Results: BKV was detected at Tx in 4/60 urine and in 16/60 plasma, with median viral loads of 3.70 log GEq/mL and 3.79 log GEq/mL, respectively, followed by a significant increase of both BKV-positive transplants (32/60) and median values of viruria (5.78 log GEq/mL) and viremia (4.52 log GEq/mL) at T2. Conversely, a significantly decrease of patients with viruria and viremia (17/60) was observed at T3, together with a reduction of the median urinary and plasma viral loads (4.09 log GEq/mL and 4.00 log GEq/mL, respectively). BKV TCR sequence analysis always showed the presence of archetypal sequences, with a few single-nucleotide substitutions and one nucleotide insertion that, interestingly, were all representative of the particular subtypes/subgroups we identified by VP1 sequencing analysis: I/b-2 and IV/c-2.

Conclusions: Our results confirm previous studies indicating that BKV replication may occur during the early hours after kidney transplantation, reaches the highest incidence in the third post-transplantation month and then decreases within the sixth month, maybe due to induction therapy. Moreover, it might become clinically useful whether specific BKV subtypes or rearrangements could be linked to a particular disease state in order to detect them before BKVAN onset.

Figure 1

Schematic representation of the BKV TCR variants detected in urine and in blood specimens. The four blocks (P, Q, R and S) commonly used to denote archetypal TCRs [11] are indicated by rectangles. Above each colored rectangle is a letter confirming the name of the block, followed by a pair of numbers to indicate the range of nucleotides present. The S block of the WWLA2 archetype has one nucleotide insertion represented by a T below the line between nucleotide positions 49 and 50.

Figure 2

Alignment of the WWLA1 and WWLA2 TCR sequences with BKV archetype WW strain TCR sequence. The WW TCR is shown at the top of the figure while the nucleotide sequences of the WWLA1 and WWLA2 variants, determined directly from urine and blood samples of our study group, are shown below in relation to the WW TCR (GenBank accession no. AB211371), with similar nucleotides indicates by hyphens. Blocks (P, Q, R and S) commonly used to denote archetypal TCRs [11] are indicated under the TCR sequences, while proven and putative binding sites for transcription factors are illustrated above the WW TCR sequence.