Analysis of human cytomegalovirus strain populations in urine samples of newborns by ultra deep sequencing

Abstract

Background: Different human cytomegalovirus (HCMV) strains may persistently coexist in the human host. In immunosuppressed patients infection with mixed HCMV populations was associated with a more severe course of infection. Congenital HCMV infection may lead to severe fetal disease and possibly mixed HCMV strain infections might have also impact on the clinical consequences for the newborn. Mixed HCMV strain populations were so far detected in saliva but only rarely in urine of congenitally infected newborns.

Objectives: We have therefore analyzed the extent of mixed HCMV genotype populations in urine of congenitally infected newborns using a highly sensitive deep sequencing method.

Study design: Twenty urine samples (17 initial and 3 follow-up samples) from 17 congenitally infected newborns with a median HCMV DNA load of 7.5log10 copies/ml were included. Deep sequencing was applied for gO (UL74) genotyping and quantitative real-time PCR assays were used for gB (UL55) and gH (UL75) genotyping.

Results: In none of the urine samples a gO genotype mixture was detected, although a mean of 10.000 sequence reads per amplicon was analyzed, which allows to explore gO genotypes down to less than 1% of the total gO sequences. Also only one gB genotype was detected in the patients' initial samples, while a gH genotype mixture was detected in one case using real time PCR with a sensitivity of 5% for minor populations.

Conclusion: Mixed HCMV genotype populations are only rarely found in urine of congenitally infected newborns even when highly sensitive HCMV genotyping methods are applied.

Keywords: Congenital infection; Cytomegalovirus; Deep sequencing; Genotypes; Mixed infection.