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

Abstract

BackgroundNowadays, 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.MethodsWe 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.ResultsBKV 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.ConclusionsOur 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.

Possible antiviral effect of ciprofloxacin treatment on polyomavirus BK replication and analysis of non-coding control region sequences

“…BKV variants with rearranged NCCR have been identified in various studies including kidney transplant recipients [31]. Alignment of all NCCR sequences, detected from each BKV-positive clinical sample of our patient, with the prototypic NCCR sequence proposed by Yogo and colleagues [32], revealed that all specimens were defined by the presence of two nucleotide substitutions and a single nucleotide deletion occurring at nucleotide positions 52, 65 and 254, respectively (Figure 3).…”

Archetype and Rearranged Non-coding Control Regions in Urothelial Bladder Carcinoma of Immunocompetent Individuals

“…Regarding BKPyV, the 327 bp VP1 gene sequence was amplified in three urine, one plasma and two low-grade urothelial carcinoma samples. The amplified PCR products were then sequenced in order to classify each BKPyV strain into the corresponding subtype/subgroup, analysing the SNPs within the amplified VP1 region and aligning our 327 bp typing isolates with the consensus sequences generated for each BKPyV subtype/subgroup (33). Based on the SNPs and on the consensus sequences, subtype I/subgroup b-2 was detected in two out of three urine samples (66.7%) and in one out of two bioptical samples (50%), while subtype II was detected in urine, in plasma and in bioptical specimens belonged to the same patient.…”

Significance of polyomavirus detection in urine cytology: an update

“…12 Screening must be started early: a prospective study using real-time PCR assessment of viraemia and viruria found that viral replication started within hours of engraftment, reached a peak at the third month, and had a drop off in incidence after the sixth month; this pattern of highest risk in the first year has lead to guidelines recommending regular screening in the first months after transplant followed by decreasing frequency of testing after the allograft has been in place for more than two years. 9,14,17,22 While the method of screening (cytology versus viral load by PCR) is left up to clinician choice in many guidelines, 9,14 urine cytology is an available method of screening in laboratories without a BK virus molecular assay. 7,23 Regardless of method used, viruria is not specific for BKVAN.…”

“…So far in a research setting BK subgroups have been identified but as of yet there are no data regarding different virulence patterns or differing prognosis or treatments. 22 There are multiple choices of PCR assays available as described in Bechert et al's recent review. 34 In our laboratory we use a real-time PCR methodology with a labelled primer and quantitation by a fluorescence resonance energy transfer hybridization methodology, whereby a fluorophore lights to signal each new copy.…”