M.A. De Lorenzis, C. Pultrone, S. Zonca, M. Gilardi, D. Rigamonti, I. Merli, M.L. Incandela, L. Spinelli
Polyomaviruses are ubiquitous, species specific, members of the Polyomaviridae family Human Polyomavirus 1 (BKV) and Human Polyomavirus 2 (JCV) are the most commonly known viruses that can cause mild pathologies in childhood, and more serious disease states if reactivated or acquired by immunocompromised patients1 2 3 4. While BKV is mainly associated with nephropathies, JCV can cause progressive multifocal leukoencephalopathy. Both viruses are diagnosed by multiplex Real-Time PCR assays.
The aim of this work was to evaluate the analytical performances of STAT-NAT® SN 200 BKV and STAT-NAT® SN 200 JCV (Sentinel Diagnostics) kits for the detection and quantification, respectively, of BKV and JCV on different human matrices, using the fully automated system SENTiNAT® 200 (Sentinel Diagnostics).
Human plasma and urine samples were analyzed to evaluate the performances of STAT-NAT® SN 200 BKV kit and human plasma and blood samples were analyzed to evaluate the performances of STAT-NAT® SN 200 JVC kit to determine the analytical sensitivity (LoD, LoQ), linearity, precision and specificity on 22 pathogens, following the CLSI 5 (Clinical and Laboratory Standards Institute) guidelines.
DNA from samples was extracted using SENTiNAT® X 48 Pathomag Extraction kit (Sentinel Diagnostics). The extraction, PCR set up and amplification reaction were performed with SENTiNAT® 200 a fully automated, sample-to-result platform.
For the STAT-NAT® SN 200 BKV and STAT-NAT® SN 200 JCV kits, the analytical sensitivity was evaluated by calculating the Limit of Detection (LoD) and the Limit of Quantitation (LoQ) using different concentrations of EDX (Exact Diagnostics, Bio Rad Laboratories).
Results showing a 95% probability of having a positive LoD and LoQ result were calculated on multiple sample replicates (Table A).
Linearity of the STAT-NAT® SN 200 BKV kit was investigated using an 8 level panel of EDX BKV virus particles (Exact Diagnostics) The BKV assay presents a linear trend from a concentration of 1 x 102 to 1 x 108 IU/mL (Figure 1). As for BKV, also the linearity of the STAT-NAT® SN 200 JCV kit was investigated using an 8 level panel of EDX JCV (Exact Diagnostics) and the assay shows a linear trend from 1 x 102 to 1 x 108 IU/mL (Figure 2).
Figures 1 and 2. Linearity plots of BKV and JCV in plasma matrix. Linearity of STAT-NAT ® SN 200 BKV and STAT-NAT® SN 200 JCV kits was investigated in the range from 1 x 10 2 to 1 x 108 IU/ mL.
|Herpes Simplex Virus 1||NO||NO|
|Herpes Simplex Virus 2||NO||NO|
|Human herpes virus 8||NO||NO|
|Human immunodeficiency virus 1||NO||NO|
|Human immunodeficiency virus 2||NO||NO|
|Hepatitis B virus||NO||NO|
|Human betaherpesvirus 7||NO||NO|
|Human herpes virus 6||NO||NO|
Table B. Evaluation of in vitro cross reactivity. A panel of 22 pathogens was used for STAT-NAT® SN 200 BKV and STAT-NAT® SN 200 JCV kits.
|Interfering substances||Tested concentrations|
|Mycophenolate mofetil||40 µg/mL|
|Conjugated bilirubin||0,25 g/L|
|Unconjugated bilirubin||0,25 g/L|
|Human genome||2 mg/L|
Table C. Exogenous and endogenous interferents tested. The results obtained show an irrelevant interfering effect of the endogenous or exogenous molecules on the analytical sensitivity of the kits.
The evaluated STAT-NAT® kits, used in combination with the SENTiNAT® 200 instrument, offer a complete, automated, sample to result solution for the simultaneous detection and quantification of BKV and JCV in different human matrices, providing sensitive, precise and reproducible results.
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5. CLSI Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures (EP17-A2); Evaluation of Linearity of Quantitative Measurement Procedures (EP06); Evaluation of Precision of Quantitative Measurement Procedures (EP05-A3); Molecular Diagnostics Methods for Infectious Diseases (MM03 Quantitative Molecular Methods for Infectious Diseases (MM06-A 2).