A-303 Validation of a Quantitative and Qualitative Molecular Test for the Detection of Epstein-Barr Virus by Real-Time qPCR

Abstract

Abstract Background The Epstein-Barr virus (EBV, HHV-4) is a human virus of the Herpesviridae family, mainly associated with infectious mononucleosis in adolescence, but it can also be related to other tumors. About 90% of the world's population has been infected by this virus, although the vast majority of these people are asymptomatic. In immunocompromised individuals, such as transplant recipients and HIV-infected patients, high EBV replication is a major predisposing factor to the development of a wide range of B-cell lymphoproliferative disorders such as Burkitt's lymphoma, nasopharyngeal carcinoma, Hodgkin's and non-Hodgkin lymphoma. Early detection of EBV is crucial for the effective management of patients on immunosuppressive therapy after transplantation and immunoproliferative disease. EBV-related diseases, such as in suspected cases of chronic fatigue syndrome and after transplants can be identified by EBV-DNA quantification. Molecular assays, such as Real Time qPCR, have high sensitivity and specificity and are a useful tool for the early diagnosis of EBV infection. This assay differentiates healthy carriers, with low levels of viral load, from ill carriers with a high rate of viral replication. In this way, this work aimed to validate and verify the performance of the alinity m EBV assay. Methods To evaluate the molecular detection of the EBV virus, we used the automated EBV alinity m assay (Abbott). Were evaluated 141 samples from the Molecular Genetics routine of the Pardini Group. Of those, 91 samples (63 plasma and 28 cerebrospinal fluid) were previously tested by in-house PCR for qualitative EBV detection and 50 samples (plasma) were previously tested for quantitative EBV detection by Real-Time PCR (Seegene platform). At the end of the tests, the previous qualitative and quantitative results were compared with the Alinity m EBV test results. Results The range of Alinity m EBV assay detection was <20 IU/mL or <1.30 log(IU/mL) to 200 000 000 IU/ml or 8.30 log(IU/ml). For qualitative analysis, the positive agreement was 100% in both plasma (7/7) and cerebrospinal fluid (6/6) samples, with quantification variation from 2.07 to 4.60 Log(IU/mL) and <1 .30 to 6.30 Log(IU/mL), respectively. The negative agreement was 89% (50/56) for plasma samples, with 6 discordant samples between 1.13 to 3.39 Log(IU/mL) and 91% for cerebrospinal fluid samples (21/23), with two disagreements (1.69 and 2.23 Log(IU/mL), considering the results below the detection range of the assay (<20 IU/mL or <1.30 Log (IU/mL)) as negative because they did not clinical relevance. In quantitative analysis, the negative agreement between comparative methodology (Seegene platform) was 81.0% (33/42), considering as discordant only the samples with a quantifiable detected result. Discordant samples detected by Alinity—and not detected by Seegene platform—presented quantification between 21–791 IU/mL, indicating greater sensitivity of the analyzed methodology. Positive agreement was 100% (5/5). Conclusion The Alinity m EBV Assay demonstrated good performance for molecular detection of EBV. This test can be used as an important tool for clinicians to identify patients with infection, especially in immunosuppressed cases that require a fast, accurate, and reliable diagnosis.