2086. Perils of CMV PCR Primer/Probe Design: Emergence of Mutations in Clinical Samples from Two Pediatric Patients
Session: Poster Abstract Session: Diagnostics: Virology
Saturday, October 6, 2018
Room: S Poster Hall

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 2 patients in whom our in-house CMV PCR was falsely negative (FN) due to 2 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 mo 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. (Fig 1a)

Case 2: 32 wk 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 (Fig 1b1) on the 21st sample submitted lead us to sequence the amplicon region and to discover 2 mutations (C-T) in the probe binding site affecting the sensitivity of UL54 PCR(Fig 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 mutation.

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.


Amy Leber, PhD1,2, Douglas Salamon, MB(ASCP)SV2, Monica I. Ardura, DO, MSCS3 and Huanyu Wang, PhD2, (1)The Ohio State University, Columbus, OH, (2)Department of Laboratory Medicine, Nationwide Children's Hospital, Columbus, OH, (3)Pediatrics, Infectious Diseases and Immunology, Host Defense Program, The Ohio State University and Nationwide Children’s Hospital, Columbus, OH


A. Leber, Nationwide Children's Hospital: Research Contractor , Research support .

D. Salamon, None

M. I. Ardura, None

H. Wang, None

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