M.A. De Lorenzis, C. Pultrone, R. Bosotti, S. Zonca, A. Ursino, I. Merli, F. Cilenti, M.L. Incandela, L. Spinelli
The infections caused by herpes viruses represent an issue for the healthcare systems worldwide due to their widespread Furthermore, it is often difficult to differentiate the type of infection based on the symptoms alone, because of their similarity, thus Real Time PCR assays are used to identify the specific virus infection.
The aim of this study is to evaluate the analytical performances of STAT-NAT® SN 200 HSV-1&2 and STAT-NAT® SN 200 VZV kits (Sentinel Diagnostics) to respectively detect and quantify Human alphaherpesvirus 1 and 22 1 3 (HSV-1&2) and Human alphaherpesvirus 3 4 5 VZV Varicella Zoster Virus) on different human matrices, using the fully automated system SENTiNAT® 200 (Sentinel Diagnostics).
Human plasma, blood and swabs were analyzed to evaluate the performances of STAT-NAT® SN 200 HSV-1&2 and STAT-NAT ® SN 200 VZV kits to determine the analytical sensitivity (LoD, LoQ) linearity, precision and specificity on 22 pathogens, following the CLSI 6 (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, samples to result platform.
For the STAT-NAT® SN 200 HSV-1&2 and STAT-NAT® SN 200 VZV kits, the analytical sensitivity was evaluated by calculating the Limit of Detection LoD and the Limit of Quantitation LoQ using different concentrations of the EDX viral particles (Exact Diagnostics, Bio Rad Laboratories) HSV-1, HSV-2 and VZV.
Results showing a 95 probability of having a positive LoD and LoQ result were calculated on multiple sample replicates (Table A).
Table A.
LoD and LoQ in different matrices (whole blood, plasma and swab). The tested dilution range is from 2 x 102 cps/mL to 1 x 103 cps/mL for HSV-1 and HSV2 and from 1 x 102 cps/mL to 2 x 103 cps/mL for VZV.
Linearity of the STAT-NAT® SN 200 HSV-1&2 kit was investigated using a 10 level panel of EDX HSV-1 and EDX HSV-2 virus particles.
The assay shows a linear trend from 2 5 x 102 to 1 x 107 cps/mL for HSV-1 (Figure 1) and from 2 x 102 to 1 x 108 cps/mL for HSV-2 (Figure 2). Linearity of the STAT-NAT® SN 200 VZV kit was investigated using a 9 level EDX VZV panel The assay shows a linear trend from 2 x 102 to 1 x 107 cps/mL (Figure 3).
Figures 1 and 2. Linearity plots of HSV-1 and HSV-2 in plasma matrix. Linearity of the STAT-NAT® SN 200 HSV-1&2 kit has been investigated for HSV-1 in the range from 2 5 x 102 to 1 x 107 cps/mL; while for STAT-NAT® SN 200 HSV-2 in the range from 2 x 102 to 1 x 108 cps/ mL.
Figures 2.
Figure 3. Linearity plot of VZV in plasma matrix. Linearity of the STAT-NAT® SN 200 VZV kit was investigated in the range from 2 x 102 to 1 x 107 cps/ mL.
The precision of the kits was evaluated by estimating the degree of agreement between the quantities obtained on the same analyte in multiple replicates. The %CV of reproducibility and repeatability is <10% for HSV-1&2 and VZV Analytical specificity is 100 and has been demonstrated using a large panel of different pathogens No cross reactivity was observed with any of the organisms tested (Table B, Table C).
The STAT-NAT ® SN 200 HSV-1&2 and STAT-NAT ® SN 200 VZV assays were also evaluated for the presence of exogenous and endogenous interfering substances in selected human samples (Table D).
| Pathogens | Cross-reactivity (Yes/No) |
| Enterovirus | NO |
| Adenovirus | NO |
| Streptococcus pneumoniae | NO |
| Varicella-Zoster virus | NO |
| Human herpesvirus 6 | NO |
| Human herpesvirus 8 | NO |
| Human immunodeficiency virus 1 | NO |
| Human immunodeficiency virus 2 | NO |
| Cytomegalovirus | NO |
| Staphylococcus aureus | NO |
| Streptococcus pyogenes | NO |
| Staphylococcus epidermidis | NO |
| BK polyomavirus | NO |
| Epstein Barr Virus | NO |
| Hepatitis B virus | NO |
| Enterococcus faecalis | NO |
| Klebsiella pneumoniae | NO |
| Human betaherpesvirus 7 | NO |
| JC polyomavirus | NO |
| Parvovirus B19 | NO |
| Toxoplasma gondii | NO |
Table B – Evaluation of in vitro cross reactivity. A panel of 21 pathogens was used for the STAT-NAT® SN 200 HSV-1&2 kit.
| Pathogens | Cross-reactivity (Yes/No) |
| Enterovirus | NO |
| Adenovirus | NO |
| Streptococcus pneumoniae | NO |
| Herpes Simplex Virus 1 | NO |
| Herpes Simplex Virus 2 | NO |
| Toxoplasma gondii | NO |
| Human herpesvirus 6 | NO |
| Human herpesvirus 8 | NO |
| Human immunodeficiency virus 1 | NO |
| Human immunodeficiency virus 2 | NO |
| Cytomegalovirus | NO |
| Staphylococcus aureus | NO |
| Streptococcus pyogenes | NO |
| Staphylococcus epidermidis | NO |
| BK polyomavirus | NO |
| Hepatitis B virus | NO |
| Enterococcus faecalis | NO |
| Klebsiella pneumoniae | NO |
| Human betaherpesvirus 7 | NO |
| JC polyomavirus | NO |
| Epstein Barr Virus | NO |
| Parvovirus B19 | NO |
Table C – Evaluation of in vitro cross reactivity. A panel of 22 pathogens was used for the STAT-NAT® SN 200 VZV kit.
| HSV-1&2 e VZV | |
| Interfering substances | Tested concentrations |
| Valganciclovir | 10 µg/mL |
| Prednisone | 22,2 µg/mL |
| Cidofovir | 20 µg/mL |
| Cefotaxime | 214 µg/mL |
| Mycophenolate mofetil | 40 µg/mL |
| Vancomycin | 50 µg/mL |
| Tacrolimus | 100 ng/mL |
| Famotidine | 200 µg/mL |
| Valacyclovir | 100 µg/mL |
| Leflunomide | 100 µg/mL |
| Triglycerides | 500 mg/dL |
| Conjugated bilirubin | 0,25 g/L |
| Unconjugated bilirubin | 0,25 g/L |
| Albumin | 58,7 g/L |
| Hemoglobin | 0,25 g/L |
| Human genome | 2 mg/L |
Table D – Exogenous and endogenous interferents tested for STAT-NAT® SN 200 HSV-1&2 and STAT-NAT® SN 200 VZV 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 HSV-1&2 and VZV in different human matrices, providing sensitive, precise and reproducible results.
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