Comparison of a commercial and a laboratory developed BKV polymerase chain reaction tests for measurement of BKV viral load in transplant patients

BKV infections are common all over the world and it's seroprevalence reachs up to 90% of the general population. Typically, BKV remains latent in the urogenital tract after primary infection. BKV is known to be associated with a variety of complications in immunocompromised hosts, including: BK virus-associated nephritis (BKVAN) and hemorrhagic cystitis (BKV-HC). BKV viral load monitoring has a very important role in the clinical control of BKVAN and allograft damage in transplant recipients. In this study, it was aimed to compare and evaluate BKV DNA measurement values performed with a commercially available Artus BKV PCR assay and laboratory developed BKV PCR assay in plasma samples of transplant recipients. Between May 2021 and June 2022, 199 plasma samples were collected from 124 adult and pediatric patients who had kidney transplant and hematopoietic stem cell transplant in Akdeniz University Medical Faculty Hospital. Viral nucleic acid extraction was performed with automatized systems for laboratory developed BKV PCR assay and commercially available Artus BKV PCR assay (EZ1&2ᵀᵐ Virüs Mini Kit (Qiagen, Germany) and QIAsymphony DSP Virus/Pathogen Kit (Qiagen, Germany); respectively). Rt-PCR procedure was carried out laboratory developed test using QuantiNova® Pathogen +IC, an amplification kit with a primer and probe sequences for the VP1 gene region and commercially available Artus BK Virus RG PCR Kit containing the primer and probe sequences for the VP2 and VP3 gene region using Rotor Gene (Qiagen, Germany). In both of the assays, 125 (62.8%) of the samples were found as positive, 36 (18.1%) of samples were found as negative and a moderate agreement was found between qualitative results of two real-time PCR methods (kappa= 0.505, p< 0.001). There were 38 qualitatively discrepant samples. Twenty nine samples were Artus BKV PCR positive and laboratory developed BKV PCR negative, and three of them were detected less than the Artus assay's lower limit of quantitation. Median viral load values were detected 7.329 copies/ml (136-2.731.125) by Artus BKV Rt-PCR and 35.543 copies/ml (314 -17.905.381) by laboratory developed BKV PCR for positive samples. Bland-Altman analysis yielded a mean difference of 0.61 log10 (standard deviation: 0.98) copies/mL. Supporting this result, according to the correlation analysis of quantitative results, moderate (r:0.616, p< 0.001) correlation was found. In addition, out of 116 samples, which were the results in the dynamic measurement range of both tests; a measurement difference of more than 0.5 log10 was detected in 108 (93.1%) and a measurement difference of more than 1 log10 was detected in 61 (52.5%) samples. The median viral load value of the samples with a measurement difference of more than 1 log10 in the results of both assays was found to be 1.27 log10 copies/mL (1.02-2.95). Only 6.9% of the reported results fell within the acceptable range of the expected result ±0.5 log10 for quantitative Rt-PCR assays. Artus BKV PCR measurements were lower than laboratory developed BKV PCR measurements. In conclusion, a significant difference in measurement was detected between the results of two BKV PCR tests, one laboratory developed and the other commercially available. This may have serious consequences in the management of patients. Updating the WHO's international quantitation standard and calibrating the tests with this standard can reduce the significant measurement difference between the tests. Keywords: BK Virus, PCR, BKV, Transplantation, Laboratory Developed, Standardization