M.A. De Lorenzis, C. Pultrone, S. Battaglia, R. Abis, M. Gilardi, V. Pipoli, I. Merli, S. Zonca, M.L. Incandela, L. Spinelli
There are 8 Herpes viruses able to infect humans they remain latent in the host even after the primary infection has resolved and can reactivate in physical conditions like immunodepression and transplantations.
Related infections can be diagnosed by multiplex real time PCR 1 assays. Aim of this study is to evaluate the analytical performances of STAT-NAT® SN 200 HHV-6 STAT-NAT® SN 200 HHV-7 and STAT-NAT® SN 200 HHV-8 (Sentinel Diagnostics) for the detection and quantification respectively of Human betaherpesvirus 6, Human betaherpesvirus 7 e Human herpesvirus 8 2 3 4 5 on different human matrices, using the fully automated system SENTiNAT® 200 (Sentinel Diagnostics).
Human plasma and blood samples were analyzed to evaluate the performances of STAT-NAT® SN 200 HHV-6 STAT-NAT® SN 200 HHV-7 and STAT-NAT® SN 200 HHV-8 kits relatively to 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, sample to result platform.
For STAT-NAT® SN 200 HHV-6 STAT-NAT® SN 200 HHV-7 and STAT-NAT® SN 200 HHV-8 kits, the analytical sensitivity was evaluated by calculating the Limit of Detection (LoD) and the Limit of Quantitation (LoQ) using different concentrations of EDX virus particles (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). The linearity of the STAT-NAT® SN 200 HHV-6 kit was investigated using an 8 level panel of the HHV-6 EDX virus particles.
The HHV-6 assay presents a linear trend from 5 x 101 to 1 x 10 8 IU/mL (Fig. 1). Linearity of the STAT-NAT® SN 200 HHV-7 kit was investigated using an 8 level panel of EDX HHV-7.
The STAT-NAT® SN 200 HHV-7 assay shows a linear trend from 1 x 102 to 1 x 107 cps/mL (Fig. 2). For the STAT-NAT® SN 200 HHV-8 kit, linearity was investigated using a 9 level panel of EDX HHV-8.
Table A. LoD and LoQ in different matrices (whole blood, plasma). The tested dilution range of HHV-6 viral particles is from 1 x 102 IU/mL to 1 x 103 IU/mL, while for HHV-7 and HHV-8 it’s from 1 x 102 cps/mL to 1 x 103 cps/mL.
The STAT-NAT® SN 200 HHV-8 assay presents a linear trend from 2 x 102 to 1 x 107 cps/mL (Fig. 3). The precision of the kits was performed by evaluating the degree of agreement between the quantities obtained on the same analyte in multiple replicates. The CV of reproducibility and repeatability is 10 for HHV-6 HHV-7 and HHV-8. Analytical specificity is 100 and has been demonstrated using a panel of 22 different pathogens for each assay.
No cross reactivity was observed with any of the organisms tested (Table B).
The various STAT-NAT® SN 200 HHV-6 STAT-NAT® SN 200 HHV-7 and STAT-NAT® SN 200 HHV-8 assays were also evaluated for the presence of exogenous and endogenous interfering substances in selected human samples (Table C).
Figures 1. Linearity plot of HHV 6 in plasma matrix. Linearity of the STAT-NAT® SN 200 HHV-6 kit has been investigated in the range from 5 x 101 to 1 x 108 IU/ mL.
Figure 2. Linearity plot of HHV-7 in plasma matrix. Linearity of the STAT-NAT ® SN 200 HHV-7 kit was tested in the range of 1 x 102 to 1 x 107 cps/ mL.
Figure 3. Linearity plot of HHV-8 in plasma matrix. Linearity of the STAT-NAT® SN 200 HHV-8 kit was evaluated in the range from 2 x 102 to 1 x 107 cps/ mL.
| Pathogens | HHV-6 Cross-reactivity (Yes/No) |
HHV-7 Cross-reactivity (Yes/No) |
HHV-8 Cross-reactivity (Yes/No) |
| Enterovirus | NO | NO | NO |
| Adenovirus | NO | NO | NO |
| Streptococcus pneumoniae | NO | NO | NO |
| Herpes Simplex Virus 1 | NO | NO | NO |
| Herpes Simplex Virus 2 | NO | NO | NO |
| Varicella-Zoster virus | NO | NO | NO |
| Epstein-Barr virus | NO | NO | NO |
| Human herpes virus 8 | NO | NO | – |
| Human immunodeficiency virus 1 | NO | NO | NO |
| Human immunodeficiency virus 2 | NO | NO | NO |
| Cytomegalovirus | NO | NO | NO |
| Staphylococcus aureus | NO | NO | NO |
| Streptococcus pyogenes | NO | NO | NO |
| Staphylococcus epidermidis | NO | NO | NO |
| BK polyomavirus | NO | NO | NO |
| Hepatitis B virus | NO | NO | NO |
| Enterococcus faecalis | NO | NO | NO |
| Klebsiella pneumoniae | NO | NO | NO |
| Human betaherpesvirus 7 | NO | – | NO |
| JC polyomavirus | NO | NO | NO |
| Parvovirus B19 | NO | NO | NO |
| Toxoplasma gondii | NO | 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®
SN200 HHV-6, STAT-NAT® SN200 HHV-7 and STAT-NAT® SN200 HHV-8 kits.
| 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 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, combined with the SENTiNAT® 200 instrument, offer a complete, automated, sample to result solution for the simultaneous detection and quantification of HHV-6 HHV-7 and HHV-8 in different human matrices, providing sensitive, precise and reproducible results.
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