False-negative CMV PCR results due to viral sequence variation - a diagnostic pitfall with the potential for serious consequences

Sep 29, 2026

Cytomegalovirus (CMV) remains a major cause of morbidity and mortality in immunocompromised patients, where nucleic acid amplification tests (NAATs) are used for diagnosis and monitoring antiviral response. A 4-month-old patient with familial hemophagocytic lymphohistiocytosis underwent allogeneic hematopoietic cell transplant (HCT). Weekly in-house quantitative CMV PCR targeting UL54 produced false-negative results due to nucleotide mutations in the probe-binding site, delaying detection of CMV DNAemia and postponing preemptive antiviral therapy after transplant. The case emphasizes routine, rigorous evaluation of CMV NAAT performance, critical clinical interpretation of negatives, and consideration of alternate PCR assays targeting different genes when discordance occurs.

LanguageEnglish

CategoryHealthcare

FormatPDF

Pages7

Reading Time18 min

AccessFree to download

Abstract

Case Presentation

Pre-HCT assessment and post-transplant monitoring

Respiratory deterioration and expanded infectious workup

Unusual amplification curves and confirmatory testing

Treatment initiation and subsequent monitoring

| Editor’s Pick | Clinical Microbiology | Case Report False-negative CMV PCR results due to viral sequence variation: a diagnostic pitfall with the potential for serious consequences Huanyu Wang,1,2 Monica I. Ardura,3 Sophonie J. Oyeniran,1,2 Amy L. Leber1,2,4 AUTHOR AFFILIATIONS See affiliation list on p. 5. ABSTRACT Background Cytomegalovirus (CMV) continues to be a significant cause of morbidity and mortality in immunocompromised patients. Nucleic acid amplification tests (NAATs) are the preferred method for both diagnosing CMV infection and monitoring response to antiviral therapy in these patients. The use of a sensitive and specific CMV NAAT is essential to ensure early and reliable detection. Case Summary A 4-month-old patient with familial hemophagocytic lymphohistiocyto sis received an allogeneic hematopoietic cell transplant (HCT) . Weekly CMV monitoring before and after HCT was performed using an in-house quantitative CMV PCR assay that targets the CMV UL54 gene. Review of the amplification curves of PCR runs raised concerns about potential false-negative results. Sequencing of the patient sample identified nucleotide mutations within the probe-binding site, confirming the cause of the assay failure. These false-negative results led to delayed detection of CMV DNAemia before HCT and delayed initiation of CMV preemptive antiviral therapy after transplant. Conclusion This case underscores the critical importance of rigorous and routine evaluation of CMV NAATs by clinical laboratories. Laboratories should recognize the limitations of NAATs and implement strategies to address them. In situations where laboratory findings conflict with clinical data, clinicians should critically assess a negative NAAT result. If CMV infection remains a concern, testing with an alternate PCR assay targeting a different gene should be considered. KEYWORDS transplant, CMV, NAAT, immunocompromised, false negative C ytomegalovirus (CMV), a beta-herpes virus, causes significant morbidity and mortality in immunocompromised patients and is the leading cause of congenital infection and hearing loss in children in industrialized countries (1) . Nucleic acid amplification tests (NAATs) are preferred for diagnosis and monitoring response to CMV-directed therapy. Primers and/or probes typically target highly conserved regions of the genome, such as glycoprotein B (UL55), DNA polymerase (UL54), matrix phosphoprotein (UL83), or major immediate-early (MIE) gene (2–4) . However, nucleotide variability within UL55 and MIE genes causing false-negative results has been reported (2, 5–7) . The Clinical Microbiology Laboratories at Nationwide Children’s Hospital has offered a laboratory-developed quantitative real-time PCR assay targeting a 61 bp fragment of the UL54 gene from plasma as the standard-of-care molecular assay since 2003 (SOC-PCR) (3) . Here, we describe a pediatric hematopoietic cell transplant (HCT) patient in whom this assay produced false negatives due to mutations in UL54, delaying initiation of CMV therapy. Editor Nicholas M. Moore, Rush University Medical Center, Chicago, Illinois, USA Address correspondence to Amy L. Leber, leber@nationwidechildrens CASE PRESENTATION A 4-month-old patient with familial hemophagocytic lymphohistiocytosis (homozygous PRF1 mutation) received myeloablative conditioning and a 10/10 matched unrelated allogeneic HCT from a CMV-seropositive donor (recipient was CMV seropositive) . Pre-HCTCMV NAAT testing was negative. The infant was considered at high risk of CMVreactivation, and per institutional protocol, preemptive prophylaxis management with twice-weekly CMV monitoring by SOC-PCR on plasma was performed post-transplant and was not detected. Thirteen days post-HCT (D + 13), the patient developed respiratory failure requiring intubation and progressive bilateral multifocal ground-glass opacities on imaging, consistent with alveolar hemorrhage and potential CMV pneumonitis. Infect

FAQs

Why can CMV PCR tests return false-negative results in immunocompromised patients?

CMV nucleic acid amplification assays can fail when nucleotide variability introduces mutations within the probe-binding site, preventing detection despite true viral presence.

What was the clinical consequence of the false-negative CMV PCR in this case?

False-negative results delayed detection of CMV DNAemia before HCT and delayed initiation of CMV preemptive antiviral therapy after transplant.

How should clinicians respond when lab NAAT results conflict with clinical findings?

Clinicians should critically assess the negative NAAT result and, if CMV infection remains a concern, consider testing with an alternate PCR assay that targets a different gene.

Sep 29, 2026

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