Analysis of cycle threshold values in SARS-CoV-2-PCR in a long-term study.

BACKGROUND: Cycle threshold (Ct) values can be used in an attempt to semiquantify results in the qualitative real-time polymerase-chain-reaction (PCR) for the new coronavirus SARS-CoV-2. The significance of Ct values in epidemiological studies and large cohorts is still unclear. OBJECTIVE: To monitor Ct values in a long-term study and compare the results with demographic data of patients who tested positive for SARS-CoV-2 by real-time PCR. STUDY DESIGN: S gene SARS-CoV-2 Ct values were analyzed retrospectively from consecutive patients between March 15th to September 15th 2020 with special regard to age, gender, and in- or outpatient status. RESULTS: In total, 65,878 patients were tested, 1103 (1.7 %) of whom were positive for SARS-CoV-2. Twenty-six positive patients were excluded, because the respective PCR runs did not meet the stability requirements (Ct value of the positive controls between 26 and 29). Of the remaining 1077 patients, females (n = 566; 53 %) were significantly older than males (n = 511; 47 %) (50.9 versus 45.1 years; p = 0.006) and had slightly higher mean Ct values than males (25.4 vs. 24.8; p = 0.04). Patients in the age groups >80 years had significantly higher Ct values than the remaining age groups (p < 0.001). Children (0-19 years) showed Ct values in the range of those found in adults (25.2 vs. 25.1, p = 0.9). There were no statistically different Ct values between in- and outpatients (p = 0.1), however, SARS-CoV-2 positive inpatients were significantly older than outpatients (p < 0.0001). CONCLUSIONS: CT values are suitable for more detailed monitoring of the SARS-CoV-2 pandemic. Age is an important cofactor in SARS-CoV-2 positive patients and may have influence on Ct values in SARS-CoV-2-PCR.

A rolling circle amplification screen for polyomaviruses other than BKPyV in renal transplant recipients confirms high prevalence of urinary JCPyV shedding.

OBJECTIVES: Multiple novel human polyomaviruses (HPyVs) have been discovered in the last few years. These or other, unknown, nephrotropic HPyVs may potentially be shed in urine. METHODS: To search for known and unknown HPyVs we investigated BKPyV-negative urine samples from 105 renal transplant recipients (RTR) by rolling circle amplification (RCA) analysis and quantitative JCPyV PCR. Clinical data was analysed to identify risk factors for urinary polyomavirus shedding. RESULTS: In 10% (11/105) of the urine samples RCA with subsequent sequencing revealed JCPyV, but no other HPyV sequences. Using quantitative JCPyV PCR, 24% (25/105) of the samples tested positive. Overall sensitivities of RCA of 44% (11/25) in detecting JCPyV in JCPyV DNA-positive urine and 67% (10/15) for samples with JCPyV loads >10,000 copies/ml can be assumed. Despite frequent detectable urinary shedding of JCPyV in our cohort, this could not be correlated with clinical risk factors. CONCLUSION: Routine urinary JCPyV monitoring in BKPyV-negative RTR without suspected polyomavirus-associated nephropathy might be of limited diagnostic value. As RCA works in a sequence-independent manner, detection of novel and known polyomaviruses shed in sufficient quantities is feasible. High-level shedding of HPyVs other than BKPyV or JCPyV in the urine of RTR is unlikely to occur.

Donor origin of BKV replication after kidney transplantation.

BACKGROUND: BK virus associated nephropathy (BKVN) leads to renal allograft dysfunction and loss. However, it is still unclear whether BKV replication in the transplant recipient is a result of reactivation in the recipient's native kidneys or whether BKV originates from the donor kidney. STUDY DESIGN: Urine of 249 donor/recipient pairs was investigated for the presence of BKV-DNA by qPCR before living transplantation (Tx) and consecutively after Tx. In BKV positive samples, the VP1 typing region (TR) and, in case of the presence of sufficient amount of DNA, the complete VP1 gene, the NCCR and a fragment of the Large T-antigen were sequenced and compared between donors and corresponding recipients before and after Tx. RESULTS: In 20 pairs, sequencing of the BKV TR succeeded in donors and corresponding recipients after Tx. The derived sequences were completely identical in donor and post-Tx recipient samples. For comparison, identical TR sequences were detected in only 24% of 1068 randomly assembled pairs. This difference was statistically highly significant (p<0.0001, Fisher's exact test). Furthermore, all VP1, Large T-antigen and NCCR BKV sequences were also identical between donors and corresponding post-Tx recipients. In two of the 20 donor/recipient pairs, VP1 TR sequencing was also successful from the recipient before Tx. In both cases the sequence differed from the sequence detected in donor and recipient after Tx giving further evidence that recipient BKV was replaced by donor BKV after Tx. CONCLUSIONS: Our study for the first time provides evidence of BKV donor origin in renal transplant recipients.

Factors influencing viral clearing and renal function during polyomavirus BK-associated nephropathy after renal transplantation.

BACKGROUND: The course of BK virus nephropathy (BKVN) is difficult to predict. METHODS: Between 2008 and 2010, we diagnosed BKVN in 46 (5.5%) of 859 patients with transplant biopsies by simian virus 40 (SV40) staining and routine serum polymerase chain reaction. We measured the influence of different variables on glomerular filtration rate (ΔGFR increasing or decreasing) and the time for viral polymerase chain reaction reduction by 1 log (≤13 or >13 weeks). At diagnosis, we either reduced calcineurin inhibitor (CNI) and mycophenolate mofetil by 30% to 50% (n=23), or we switched from CNI to mammalian target of rapamycin (mTOR) inhibitor (n=7) or from CNI to mTOR inhibitor as a second step in patients with protracted viral reduction (n=16). Results are the following: GFR stabilized or increased in 61% of patients and decreased in 39% (graft failure, 15%). Viral reduction by 1 log was rapid in 54% (≤13 weeks) and slow in 46% (>13 weeks). Rapid viral reduction was associated with stable or increasing GFR (84%), compared with slow viral reduction (33%; P=0.0004). High peak viral load, tacrolimus treatment, and late diagnosis (biopsy for cause vs. protocol biopsy) had a negative influence on GFR and viral reduction time. Defining 1-log viral load reduction as an event, tacrolimus compared with cyclosporine was associated with slow viral reduction (P=0.0043). In 88% of patients with slow viral reduction, the secondary switch from CNI to mTOR inhibitor favored viral load decrease. CONCLUSIONS: We conclude that peak viral load, tacrolimus treatment, delayed diagnosis, and viral reduction time influence outcomes in patients with BKVN.