Development and validation of a nanoplate-based digital PCR assay for absolute MPXV quantification.

Quantification of mpox virus (MPXV) across different human body anatomical sites can provide insights about the most likely transmission routes, so methods able to release absolute and exact quantitative values of MPXV DNA are crucial. Here, we optimized a new QIAcuity digital PCR (dPCR) protocol for the detection and quantification of MPXV DNA in clinical samples and assessed the performance of the assay by comparing the results obtained in 144 biological samples with those resulting from the use of an in-house real-time PCR (qPCR). Overall, the concordance between the two assays was 95%, with samples identified concordantly as MPXV DNA positive and having a mean number of copies per µl of 1708 (95% CI: 107-2830 copies/µl). The remaining samples gave discordant results, with 5 out of 7 detected with the QIAcuity dPCR assay but not with the in-house qPCR. MPXV DNA levels measured by QIAcuity dPCR were strongly correlated with the Ct values detected by in-house qPCR and with those detected by another dPCR assay previously developed in our laboratories. The QIAcuity dPCR assay may be a robust and easy-to-perform method for MPXV DNA quantification in several biological samples.

Performance evaluation of the Aptima® HCV Quant Dx assay for hepatitis C virus (HCV) RNA detection and quantification in comparison to the Abbott RealTime HCV assay.

BACKGROUND: The Aptima HCV Quant Dx assay (Aptima) is a real-time transcription-mediated amplification assay CE-approved for the diagnosis and monitoring of hepatitis C virus (HCV) infection. OBJECTIVE: Aptima's analytical performance was compared to the Abbott RealTime HCV assay (RealTime) in a clinical routine setting. STUDY DESIGN: Overall 295 clinical plasma samples (117 prospective/fresh; 178 retrospective/frozen) from HCV-infected patients were tested in Aptima and RealTime to determine concordance on qualitative and quantitative results. Linearity and precision at low viral loads (VLs; 0.8-3.3LogIU/mL) was tested using dilutions of the 5th WHO standard, in 10 and 20 replicates in the two assays, respectively. The ability to measure different HCV genotypes and accuracy were analyzed using the Seracare EQA panel. RESULTS: Inter-assay agreement for qualitative results (prospective samples) was 88% (kappa=0.78). For the 127 samples with quantitative results in both assays, Aptima yielded on average slightly higher values (by 0.24LogIU/mL; Bland-Altman method) than RealTime. Concordance between assay results was excellent (R=0.98). At low VLs (0.8-3.3LogIU/mL), Aptima demonstrated good linearity and precision, similar to RealTime. Aptima detected and accurately quantified all main HCV genotypes. CONCLUSIONS: Aptima demonstrated excellent precision, linearity, and accuracy in all genotypes tested. Good concordance was observed between Aptima and RealTime assays in clinical samples. The performance of the Aptima assay, on the fully automated Panther platform, makes it an excellent candidate for the detection and monitoring of HCV RNA in plasma and serum samples.