Abstract
Human cytomegalovirus (CMV) infections comprise a leading cause of newborn impairments worldwide and are pervasive concerns among the immunocompromised. Quantification of CMV viral loads is increasingly used to guide definitions of CMV disease but standardization of CMV quantitation remains problematic, mostly due to differences in qPCR amplicon sizes between clinical laboratories. Here, we used plasma cfDNA sequencing data from 2,208 samples sent for non-invasive prenatal aneuploidy screening to detect CMV and precisely measure the length of CMV fragments in human plasma. CMV reads were identified in 120 (5.4%) samples. Median cfDNA fragment size derived from CMV was significantly shorter than cfDNA derived from human chromosomes (103 vs 172 bp, p < 0.0001), corresponding to the 3rd percentile of human cfDNA. Sequencing of cfDNA from seven plasma samples from transplant patients positive for CMV confirmed the extraordinarily short nature of CMV cfDNA fragment size with a median length of 149 bp. We further show that these high-resolution measurements of CMV DNA fragment size accurately predict measured discrepancies in serum viral load measurements by different qPCR assays. These results highlight the exceptionally fragmented nature of CMV cfDNA and illustrate the promise of plasma cfDNA sequencing for quantitating viral loads through detection of fragments that would be unrecoverable by qPCR.
Highlights
Human cytomegalovirus (CMV) is the most common cause of congenital defects in the United States and affects roughly 0.5–1.3% of live births worldwide[1,2], causing more congenital disease than all disorders tested for in newborn screening combined[3]
In order to compare cell-free DNA (cfDNA) detection, quantitation, and fragment length in the maternal plasma to another betaherpesvirus to control for CMV-specific biology, we looked for HHV-6 in our Non-invasive prenatal testing (NIPT) cfDNA sequencing data. 18 maternal cfDNA samples from 17 different individuals had reads aligning to HHV-6A or HHV-6B and were classified as HHV-6 positive
We show that approximately 5% of samples sent for NIPT had detectable CMV-specific reads, which routinely outstripped the analytical sensitivity of our CMV qPCR assay when performed on cfDNA
Summary
Human cytomegalovirus (CMV) is the most common cause of congenital defects in the United States and affects roughly 0.5–1.3% of live births worldwide[1,2], causing more congenital disease than all disorders tested for in newborn screening combined[3]. Non-invasive prenatal testing (NIPT) via sequencing of maternal cell-free DNA (cfDNA) has revolutionized the ability to screen for fetal aneuploidies, subchromosomal copy number alterations, and other genetic diseases. A major determinant of variation is the size of the PCR amplicon used in the assay design, as assays with amplicon sizes ≤86 bp have higher results than those with amplicons sizes ≥105 bp[22,24] While this intuitively makes sense – PCR reactions cannot amplify templates shorter than their targeted amplicon size – to date, there exists no precise measurement of the fragment length of CMV DNA in plasma. We have performed clinical screening for fetal aneuploidies by cfDNA since May 2017 These methods include paired-end sequencing that allows us to measure cfDNA fragment length to accurately determine fetal fraction. We examined cfDNA sequencing data from 2,208 samples for CMV and a related betaherpesvirus, human herpesvirus 6, as well as 7 additional known CMV-positive plasma specimens taken from solid organ transplant patients
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