Sequence Variation in Amplification Target Genes and Standards Influences Interlaboratory Comparison of BK Virus DNA Load Measurement

Abstract

International guidelines define a BK virus (BKV) load of >4 log 10 copies/ml as presumptive of BKV-associated nephropathy (BKVN) and a cutoff for therapeutic intervention. To investigate whether BKV DNA loads (BKVL) are comparable between laboratories, 2 panels of 15 and 8 clinical specimens (urine, whole blood, and plasma) harboring different BKV genotypes were distributed to 20 and 27 French hospital centers in 2013 and 2014, respectively. Although 68% of the reported results fell within the acceptable range of the expected result ؎0.5 log 10 , the interlaboratory variation ranged from 1.32 to 5.55 log 10 . Polymorphisms specific to BKV genotypes II and IV, namely, the number and position of mutations in amplification target genes and/or deletion in standards, arose as major sources of interlaboratory disagreements. The diversity of DNA purification methods also contributed to the interlaboratory variability, in particular for urine samples. Our data strongly suggest that (i) commercial external quality controls for BKVL assessment should include all major BKV genotypes to allow a correct evaluation of BKV assays, and (ii) the BKV sequence of commercial standards should be provided to users to verify the absence of mismatches with the primers and probes of their BKV assays. Finally, the optimization of primer and probe design and standardization of DNA extraction methods may substantially decrease interlaboratory variability and allow interinstitutional studies to define a universal cutoff for presumptive BKVN and, ultimately, ensure adequate patient care.T he emergence of BK virus-associated nephropathy (BKVN) as a major cause of graft dysfunction and loss in kidney transplant recipients (KTR) arises from the use of highly potent immunosuppressive drugs (1-3). This is a growing medical problem as the population of transplant recipients continues to increase. In Europe and the United States, the number of kidney transplantations has increased up to 50% in the last 20 years (www.kidney .niddk.nih.gov and http://www.era-edta.org). BKV reactivation or reinfection occurs in 40 to 50% of KTR, followed by BKVN in 6.6% of KTR at 5 years posttransplant, ultimately leading to graft dysfunction and loss in up to 50% of cases (4). The diagnosis of BKVN is based on the documentation of viral cytopathic effects observed in tubular epithelial cells accompanied by inflammatory cell infiltration after renal biopsy (5, 6). Immunohistochemistry with SV40 staining is the gold standard for diagnosing definitive BKVN (7). Nevertheless, in the early stages of BKVN, kidney allograft biopsy results may be falsely negative at an estimated rate of 10 to 30% (8). Prospective studies showed that high BKV viruria usually precedes viremia by 4 to 12 weeks, with a sustained BKV viremia above the threshold of 4 log 10 copies/ml defined as presumptive of BKVN, with a positive predictive value of Ͼ80% (9, 10). These studies showed that BKVN can be effectively and safely prevented using a preemptive reduction in immunosuppression (11,12). There...

Neutralizing Antibody–Mediated Response and Risk of BK Virus–Associated Nephropathy

“…This cannot be explained by PCR assay performance because our PCR assay is able to detect and adequately quantify all BKV genotypes. 18,19 On the other hand, this result raises the hypothesis that BKV genotype II may be more easily cleared compared with genotype I and IV. Further investigations are needed to address this question.…”

Quantification of BK Virus Standards by Quantitative Real-Time PCR and Droplet Digital PCR Is Confounded by Multiple Virus Populations in the WHO BKV International Standard

“…An evaluation of year 2009 College of American Pathologists (CAP) proficiency test results showed a very large spread of results (3.84 log copies/mL) for 2 plasma samples, indicating that BK standardization may be needed (26 ). In addition, a multicenter French study found that only 68% of urine, whole blood, and plasma samples fell within an acceptable range of the expected result (Ϯ 0.5 log 10 ), and that interlaboratory variation ranged from 1.32-5.55 log 10 (27 ). Several studies have evaluated the performance of different PCR primer sets (1, 28 -30 ) and demonstrated the critical need to design primer sets that amplify equally with all 6 BK genotypes.…”

Calibration of BK Virus Nucleic Acid Amplification Testing to the 1st WHO International Standard for BK Virus

“…A multiinstitution evaluation of the first WHO international standard for CMV DNA demonstrated improved harmonization across assays, though clinically relevant variability remained (13). The factors previously cited to impact BKV NAAT quantification include the nucleic acid extraction method and the design of primers and probes that do not account for variation in BKV genotypes (7)(8)(9). The use of commercially available assays, such as Altona, may yield improved agreement between assays, as demonstrated in a study that found reliable and comparable performances of three commercial real-time BKV PCR assays (21).…”

“…Quantitative results reported by one assay may be vastly different from those reported by a different assay, making it difficult to compare results across assays. Such variability has been well described with negative impact on patient care, particularly when two laboratories report discrepant viral loads and management is initiated or delayed based on inaccurate or discordant quantitative results (6)(7)(8)(9)(10). Among the most cited factors contributing to this variability are the use of different standards for quantitation and primer and probe designs that do not account for BKV genotypic variation (7)(8)(9)11).…”