Real time BKV PCR testinin analitik performansı

Abstract

Amaç: BKV, çocukluk çağında genellikle asemptomatik olarak geçirilen ve özellikle böbreklerde olmak üzere perifik kan ve beyinde latent olarak kalabilen bir virustür. BVK’nın aktivasyonu immünsuprasyonu olan hastalarda hemorajik veya hemorajik olmayan sistit, ürostenoz ve nefropatiye neden olabilir. Özellikle transplant alıcılarında nefropati sıklığı %5’lere kadar ulaşabilmekte ve transplante edilen organın %30-60 erken kaybı ve kötü bir klinik seyirle sonuçlanabilmektedir. BKV viral yükünün serum ve idrar örneklerinde polimeraz zincir reaksiyonu PZR ile kantitasyonu, erken tanı ve tedavinin yönetiminde önemli rol oynamaktadır. Real time PZR; klasik PZR’ye göre daha duyarlı, kantitasyon yapabilen, kontaminasyon riski az ve sonuçlanma süresinin kısa olması nedeniyle son yıllarda yaygın olarak kullanılmaktadır. Bu çalışmamızda BKV tanısı için laboratuvarımızda tasarlanan kantitatif real time BKV PZR testinin analitik performansının değerlendirilmesi amaçlanmıştır. Yöntem: BKV plazmidinden ATCC 45025 standardlar hazırlanmıştır. 15 x 107 kopya/ml’den 3 x 101 kopya/ml’ye kadar seri dilüsyonlar içeren BKV plazmidinin spektrofotometrik ölçümü yapılmıştır. Amplifikasyon reaksiyonları için BKV VP1 gen bölgesine uygun, 5’ ucu 6-karboksifloreseinle FAM , 3’ ucu 6-karboksitetrametilrodamin TAMRA ile işaretli primerler daha önce belirtildiği gibi kullanılmıştırBulgular: Çalışmada testin analitik performansının değerlendirilmesi için analitik duyarlılık, özgüllük, doğrusallık, doğruluk ve kesinlik parametreleri belirlenmiştir. Testin analitik sensitivitesi 15 x 102 kopya/ml ve saptama limiti 5 x 102 kopya/ml olarak belirlenmiştir. 15 x 107 kopya/ml ile 15 x 101 kopya/ ml konsantrasyonlar arasında çalışılan dilüsyonların standard deviasyonu SD 0,02 ile 0,644 arasında, varyasyon katsayısı CV ise %0,79 ile %11,47 arasında değişmiştir. 19 dış kalite kontrol örneğinin sonuçları ilgili programla %100 uyumludur. Testimiz 15 x 102 kopya/ml ile 15 x 108 kopya/ml arasında lineer bir seyir göstermiştir. Özgüllüğü ise %100 olarak belirlenmiştir. Ayrıca dış kalite kontrol programı ile kalite kontrol örneklerinin sonuçları çok yakın uygunluktadır. Sonuç: Elde edilen sonuçlarımız doğrultusunda laboratuvar tasarımlı real time PCR protokolümüz; duyarlı, özgül, kesin ve geniş dinamik aralıklı tekraralanabilirliği olan bir test olarak bulunmuştur.

Keywords

• Analitik performans, Real Time BKV PCR, Validasyon deneyleri

The analytical performance of a real time BKV PCR assay

Abstract

Objective: BKV is a virus usually undergone asymptomatically in the childhood and can remain latent in the peripheral blood, brain and especially in kidneys. Reactivation of BKV under immunosuppression can cause diseases like interstitial nephritis, haemorrhagic or non- haemorrhagic cystitis, ureterostenosis and nephropathy. Especially in transplant recipients nephropathy frequency can reach 5% and can be the cause of premature loss 30-60% of transplanted organs and poor outcome. Quantification of BKV viral load in urine and serum with real time polymerase chain reaction PCR plays important role for early diagnosis and management of the therapy. Since the Real time PCR assay is more sensitive than classical PCR, can do quantification and have a less risk of contamination and short turn-around time. The aim of our study was to evaluate the analytical performance of a real time quantitative BKV PCR assay which was developed in our laboratory. Method: Standards were prepared from BKV plasmid ATCC 45025 . BKV plasmid that contained 15 x 107 copies/ ml to 3 x 101 copies/ml serial dilutions was measured by spectrofotometery. Primers for BKV VP1 gene and dual labelled probe at the 5’ end with 6-carboxyfluoresceine FAM and the 3’ end with 6-carboxytetramethylrhodamine TAMRA as described previously were used for the amplification reactions. Results: To evaluate the analytical performance of the assay; analytical sensitivity, specificity, linearity, accuracy and precision was determined. The analytical sensitivity and the limit of detection of the assay were found 15 x 102 copies/ml and 5 x 102 copies/ ml, respectively. Standard deviation SD of dilutions varied from 0.02 to 0.644 and CV varied from 0.79% to 11.47% between 15 x 107 to 15 x 101 copies/ml concentrations. Nineteen proficiency samples results of quality control program were in close agreement 100% . The assay demonstrated a linear range from 15 x 102 to 15 x 108 copies/ml. Specificity of the assay was found 100%. In addition proficiency samples results of the external quality control program were in close agreement. Conclusion: According to our results the real time PCR protocol of BKV developed in our laboratory was found sensitive, specific, precise and reproducible with a broad dynamic range

Keywords

• Analytical performance, Real Time BKV PCR, validation experiments

References

1. 1. Major EO, Ryschkewitsch C, Valsamakis A, Hou J. Human polyomaviruses. In: Murray PR, Baron EJ, Jorgensen JH, Landry ML, Pfaller MA, eds. Manual of clinical microbiology. 9th ed. Washington DC: ASM Pres, 2007: 1612-21.
2. 2. Van Aalderen MC, Heutinck KM, Huisman C, Berge. IJM. BK virus infection in transplant recipients: Clinical manifestations, treatment options and the immune response. Neth J Med, 2012; 70: 172-83.
3. 3. Mischitelli M, Fioriti D, Anzivino E, Bellizzi A, Ferretti G, Gussman N et al. BKV QPCR detection and infection monitoring in renal transplant recipients. New Microbiol, 2007; 30: 271-4.
4. 4. Pollara CP, Corbellini S, Chiappini S, Sandrini S, Tomasi DD, Bonfanti C et al. Quantitative viral load measurement for BKV infection in renal transplant recipients as a predictive tool for BKVAN. New Microbiol, 2011; 34: 165-71.
5. 5. Anziviro E, Bellizzi A, Mitterhofer AP, Tinti F, Barile M, Colosimo MT et al. Early monitoring of the human polyomavirus BK replication and sequencing analysis in a cohort of adult kidney transplant patients treated with basiliximab. Virol J, 2011; 8: 407.
6. 6. Bechert CJ, Schnadig VJ, Payne DA, Dong J. Monitoring of BK Viral Load in Renal Allograft Recipients by Real-Time PCR Assays. Am J Clin Pathol, 2010; 133: 242-50.
7. 7. Abdelsalam NF, Hashad DI , Salem MA, El-Wakil HS, Adam AG. Occurrence of the Polyomavirus among Kidney Transplant Recipients: A Single-Center Study. Saudi J Kidney Dis Transpl, 2014; 25(2): 285-93.
8. 8. Kwak EJ, Vilchez RA, Randhawa P, Shapiro R, Butel JS, Kusne S. Pathogenesis and Management of Polyomavirus Infection in Transplant Recipients. Clin Infect Dis, 2002; 35: 1081–7.

9. 9. M, Passweg J, Hirsch HH, Knowles W, Dickhenmann Klimkait T, Mihatsch M, Steiger J. Prospective study of polyomavirus type BK replication and nephropathy in renal-transplant recipients. N Engl J Med, 2002; 347: 488-96.
10. 10. Vats A, Shapiro R, Singh Randhawa P, Scantlebury V, Tuzuner A, Saxena M, et al. Quantitative viral load monitoring and cidofovir therapy for the management of BK virus-associated nephropathy in children and adults. Transplant, 2003; 75: 105-12.
11. 11. Hirsch HH, Randhawac P, and the AST Infectious Diseases Community of Practice. BK Polyomavirus in Solid Organ Transplantation. Am J Transplant, 2013; 13: 179–88.
12. 12. Randhawa P, Ho A, Shapiro R, Vats A, Swalsky P, Finkelstein S, et al. Correlates of Quantitative Measurement of BK Polyomavirus (BKV) DNA with Clinical Course of BKV Infection in Renal Transplant Patients. J Clin Microbiol, 2004; 3: 1176–80.
13. 13. Sung H, Choi BH, Pyo YJ, Kim MN, Han DJ. Quantitation of BK Virus DNA for Diagnosis of BK Virus-Associated Nephropathy in Renal Transplant Recipients. J Korean Med Sci, 2008; 23: 814-8.
14. 14. Marinelli K, Bagnarelli P, Gaffi G, Trappolini S, Leoni P, Paggi AM, et al. PCR real time assays for the early detection of BKV-DNA in immunocompromised patients. New Microbiologica, 2007; 30: 275-8.
15. 15. Holman CJ, Van Burik JAH, Hinrichs SH, Balfour Jr HH. Specific Detection of Human BKPolyomavirus in Urine Samples of Immunocompromised Patients. Clin Diagn Lab Immunol, 2003; 10: 66–9.
16. 16. Hoorfar J, Wolffs P, Rådstro P. Diagnostic PCR: validation and sample preparation are two sides of the same coin. APMIS, 2004; 112: 808–14.
17. 17. Hayden RT, Yan X, Wick MT, Rodriguez AB, Xiong X, Ginocchio CC, et al. Caliendog for the College of American Pathologists Microbiology Resource Committee. Factors Contributing to Variability of Quantitative Viral PCR Results in Proficiency Testing Samples: a Multivariate Analysis. J Clin Microbiol, 2012; 50: 337–45.
18. 18. Green RL, Roinestad IC, Boland C, Hennessy LK. Developmental Validation of the QuantifilerTM Real-Time PCR Kits for the Quantification of Human Nuclear DNA Samples. J Forensic Sci, 2005; 50: 1-17.
19. 19. Moret H, Brodard V, Barranger C, Jovenin N, Joannes M, Andre Oletti L. New Commercially Available PCR and Microplate Hybridization Assay for Detection and Differentiation of Human Polyomaviruses JC and BK in Cerebrospinal Fluid, Serum, and Urine Samples. J Clin Microbiol, 2006; 44: 1305–9.
20. 20. Chevaliez S, Bouvier-Alias M, Brillet R, Pawlotsky JM. Overestimation and Underestimation of Hepatitis C Virus RNA Levels in a Widely Used RealTime Polymerase Chain Reaction–Based Method. Hepatology, 2007; 46: 22-30.
21. 21. Hoffman NG, Cook L, Atienza EE, Limaye AP, Jerome KR. Marked Variability of BK Virus Load Measurement Using Quantitative Real-Time PCR among Commonly Used Assays. J Clin Microbiol, 2008; 46: 2671–80.
22. 22. Zhang CW, Chen XQ, Bai YH, Pan XD, Wang SL, Cai Y et al. Establishment of a real-time PCR assay for simultaneously detecting human BKV and CMV DNA and itsapplication in renal transplantation recipients. Bing Du Xue Bao, 2013; 29(4): 410-4.
23. 23. Wu CZ, Chen XQ, Wang ZY, Pan XD, Bai YH, Yang YR et al. J Virol Methods, 2014; 210: 40-4.
24. 24. Mitui M, Leos NK, Lacey D, Doern C, Rogers BB, Park JY. Development and validation of a quantitative real time PCR assay for BK virus. Mol Cell Probes, 2013; 27: 230–6.
25. 25. Bergallo M, Astegiano S, Sidoti F, Mantovani S, Segoloni GP, Cavallo R et al. Real-time RT-PCR assay for the quantitation of polyomavirus BK VP1 mRNA levels in urine. Mol Biotechnol, 2010; 45: 82-6.