Supportive Data

The following validation data support the use of this assay for clinical testing.

Accuracy:

A total of 107 previously tested (n=78) and prospective (n=29) lower respiratory clinical specimens submitted to our reference laboratory for routine cytomegalovirus (CMV) real-time polymerase chain reaction (PCR) (CMVPV / Cytomegalovirus (CMV) Molecular Detection, PCR, Varies) were tested by this test.

The overall qualitative positive percent agreement was 96% (48/50). The overall qualitative negative percent agreement was 82% (47/57).

Additional CMV testing on 8 of the 12 discrepant specimens suggests these are true CMV positive specimens, which increased the overall percent agreement to 96% (103/107). Result discrepancies were notable for specimens retrospectively collected that were subjected to extended frozen storage prior to concurrent testing by routine CMV real-time PCR and the new CMV lower respiratory assay (Diasorin Simplexa CMV). Results suggest increased assay sensitivity over the routine CMV real-time PCR assay for lower respiratory specimens, especially for those undergoing extended frozen storage (≤-70° C).

Analytical Sensitivity/Limit of Detection:

To evaluate the analytical sensitivity, whole virus control (ZeptoMetrix) was spiked into residual analyte-negative lower respiratory fluid specimens between 0.1 and 1000  copies/mL and tested in six replicates per dilution. The lowest concentration detected in all six replicates was confirmed by spiking 20 unique residual analyte-negative lower respiratory fluid specimens. A positive result was obtained in 19/20 (95%) replicates, and the analytical sensitivity (limit of detection) was determined to be 90 copies/mL.

Analytical Specificity:

The Simplexa Congenital CMV Direct assay is not US Food and Drug Administration-authorized for lower respiratory specimens. However, the kit instructions for use (Simplexa Congenital CMV Direct, IFUK.US.MOL2255, Rev. 01, 11/2022) indicate no cross-reactivity was observed with a panel of 41 organisms spiked into an upper-respiratory matrix. Inclusivity was confirmed with CMV strains AD-169, Merlin, Towne and Toledo. Additionally, in silico BLAST analysis demonstrated that the assay should detect at least 327 CMV sequences available in the NCBI database, including the 4 CMV genotypes gB1, gB2, gB3 and gB4.

Additionally, a panel of 7 organisms commonly found in the lower respiratory tract were tested by this method as part of this validation, and no was signal detected.