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Background Detection of CMV by PCR is the preferred method for both diagnosing infection and monitoring therapy. The design of CMV PCR depends on analysis of all available nucleic acid sequences to maximize performance. We describe two patients in whom our in-house CMV PCR was falsely negative (FN) due to two recently emerged mutations in the DNA polymerase gene. Methods In-house CMV PCR targeting a specific 61 bp fragment of the polymerase gene (UL54) has been in use in our lab since 2003. Confirmatory CMV PCR was sent to a reference lab which uses PCR targeting US9 gene. Results Case 1: 4 months F with familial hemophagocytic lymphohistiocytosis (homozygous PRF1) underwent 10/10 MUD BMT (CMVD+/R−). Plasma CMV was not detected on admission and monitoring was performed weekly. She developed respiratory failure, intubated on D+13 with hemorrhagic respiratory secretions. Repeat PCR of tracheal secretions and plasma detected CMV on D+33, prompting ganciclovir and cytogam. She developed refractory hypoxemia and asystolic cardiac arrest on D+51 (Figure 1a). Case 2: Thirty-two-week F born via C-section for fetal distress noted to have SGA, microcephaly, thrombocytopenia and hyperbilirubinemia at birth, concerning for congenital CMV; urine CMV + (Ct 43.18). Repeat urine and blood PCRs on Day 5 of life were indeterminate. Given initial CMV detection and clinical stigmata, ganciclovir was started. Close analysis in Case 1 of the amplification curve (Figure 1b1) on the 21st sample submitted lead us to sequence the amplicon region and to discover two mutations (C-T) in the probe binding site affecting the sensitivity of UL54 PCR(Figure 1b2). These previous FNs delayed CMV diagnosis and the start of antivirals. For Case 2, the distinct curve was noted on the first sample and was sent for confirmation, resulting in no adverse clinical implications. We subsequently developed a CMV PCR targeting US9 that can detect these mutations. Conclusion Periodic assessment of all available CMV sequences and close review of amplification curves are essential to prevent FN PCR. With conflicting laboratory and clinical data, clinicians with a high suspicion for CMV should question negatives and if appropriate, ask for PCR using an alternate target. Disclosures A. Leber, Nationwide Children’s Hospital: Research Contractor, Research support.

2086. Perils of CMV PCR Primer/Probe Design: Emergence of Mutations in Clinical Samples from Two Pediatric Patients