*Purpose: Solid organ transplant recipients are predisposed to opportunistic infections, and current viral diagnostic methods focus on sensitive detection of single pathogens by qPCR. However, a method that detects common viral pathogens with sensitivity and specificity comparable to current methods allows a comprehensive approach for detection.

*Methods: Whole-genome sequencing for donor-derived cell-free DNA (dd cfDNA), in which all cfDNA in a sample is sequenced, allows both quantification of % dd cfDNA and detection of viral DNA sequences from the same sample. A total of 605 plasma samples from 255 CTOT-08 subjects were analyzed for dd cfDNA by NGS as described (CJASN 16: 1539-1551, 2021). The NGS output files were further analyzed to provide read-assembly (contig) length and read depth for common DNA viruses. In this study, significant detections were defined as those from the Adenoviridae, Annelloviridae, Herpesviridae and Polyomaviridae with a contig lengths >150 bp.

*Results: Of the 605 samples tested, 188 (31.1%) yielded 257 viral detections from 137 subjects. Torque Teno virus was the most common detection (48.6%), followed by BK virus (BKV, 19.8%), cytomegalovirus (CMV, 10.9%) and Epstein Barr virus (EBV, 3.1%). Adenovirus, JC virus, human herpesvirus 6, herpes simplex virus 1/2, human polyomavirus 6 and human polyomavirus 8 all represented less than 2% of viruses detected. A subset of 40 samples was tested by BKV and CMV qPCR assays with 92.1% concordance to NGS results (78.1% positive agreement, 97.7% negative agreement). The mean dd cfDNA values differed significantly (P=0.0004, Mann Whitney test) between samples with viral detection (mean=0.64%, 95% CI=0.43-0.80%) compared to those without a viral detection (mean=0.97%, 95% CI=0.80-1.14%). Concordance of NGS and standard-of-care (SOC) diagnostic results was 84.6% (22 of 26 samples tested by both methods); however NGS detected 3 BKV, 3 CMV, and 2 EBV infections in samples drawn prior to SOC testing. Additionally, 4 BKV, 3 CMV, and 2 EBV infections with high viral loads were detected by NGS but not by SOC testing.

*Conclusions: In this retrospective study metagenomic viral detection combined with dd cfDNA quantification demonstrated sensitivity and concordance with qPCR while detecting some infections both earlier and when not identified by SOC testing. Prospective studies are needed to assess clinical utility.

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