False positive congenital cytomegalovirus saliva screening results with an FDA-approved assay at two institutions

•Elevated false-positive rates were noted in July 2022 by two institutions using the FDA-approved alethia CMV Assay Test System as part of local congenital CMV screening programs, leading to implementation of prospective quality management strategies.•A minority of initially-positive saliva swab specimens confirmed as positive after repeat Alethia CMV testing on the same sample, orthogonal PCR testing on the same sample, and/or clinical adjudication, with false-positive rate estimated at 4.5–6.2% (substantially higher than the 0.2% reported in FDA claims).•Laboratories using the Alethia CMV assay may consider closely monitoring positivity/confirmation rates, implementing stringent decontamination, and requiring repeat testing of positives as needed. Congenital cytomegalovirus (CMV) infection is a significant cause of childhood hearing loss and developmental delay. Congenital CMV screening was implemented at two large hospital-affiliated laboratories using the FDA-approved Alethia CMV Assay Test System. In July 2022, an increase in suspected false-positive results was noted, leading to implementation of prospective quality management strategies. The Alethia assay was performed per manufacturer-provided instructions on saliva swab specimens. After discovery of possible elevated false-positive rates, all positive results were confirmed by repeat Alethia testing on the same specimen, orthogonal polymerase chain reaction (PCR) on the same specimen, and/or clinical adjudication. Additionally, root cause analyses were conducted to pinpoint the source of false-positive results. At Cleveland Clinic (CCF), 696 saliva specimens were tested after initiation of the prospective quality management strategy, of which 36 (5.2%) were positive for CMV. Five of 36 (13.9%) were confirmed CMV positive by repeat Alethia testing and orthogonal PCR. Vanderbilt Medical Center (VUMC) tested 145 specimens, of which 11 (7.6%) were positive. Two of 11 (18.2%) confirmed as positive by orthogonal PCR or clinical adjudication. The remaining specimens (31 from CCF and 9 from VUMC) were negative for CMV by repeat Alethia and/or orthogonal PCR testing. These findings suggest a false positive rate of 4.5–6.2%, higher than the 0.2% reported for this assay in FDA claims. Laboratories using Alethia CMV may consider prospective quality management to evaluate all positive results. False-positive results can lead to unnecessary follow-up care and testing, and decreased confidence in laboratory test