1H NMR Urinary Metabolomics Profiling of Newborns with Congenital Human Cytomegalovirus Infection: Insights into Metabolic Alterations

Abstract

Human cytomegalovirus (HCMV) is the leading cause of congenital infections resulting in severe morbidity and mortality among newborns worldwide. Currently, the most significant prognostic factor of congenital cytomegalovirus (cCMV) infection is the time of maternal infection, with a more severe clinical phenotype if the mother's first outbreak occurs during the first trimester of pregnancy. Nonetheless, the pathogenesis of cCMV infection has still to be completely characterized. In particular, little is known about the metabolic response triggered by HCMV in congenitally infected newborns. As such, urinary metabolic profiling by ¹H nuclear magnetic resonance (NMR) might represent a promising tool to be exploited in the context of cCMV. This study aims to investigate the impact of HCMV infection on the urine metabolome in a population of congenitally infected newborns and uninfected controls by ¹H NMR spectroscopy combined with multivariate statistical analysis. The ¹H NMR spectra of patients (n = 35) and controls (n = 15) allowed the identification of an overall amount of 55 metabolites. Principal Component Analysis (PCA) and clustering correctly assigned 49 out of 50 newborns into the infected and control groups. Partial Least-Squares-Discriminant Analysis (PLS-DA) revealed that newborns with cCMV resulted in having increased betaine, citrate, 3-hydroxybutyrate, 4-hydroxybutyrate, acetoacetate, formate, glycolate, lactate, succinate, and threonine levels in the urine. On the other hand, healthy controls showed increased 4-aminohippurate, creatine, creatinine, fumarate, mannitol, taurine, and dimethylamine levels. These results showed a clear difference in metabolomic fingerprint between newborns with cCMV infection and healthy controls. Thus, metabolomics can be considered a new, promising diagnostic and prognostic tool in the clinical management of cCMV patients.

Keywords: HCMV; NMR spectroscopy; congenital; cytomegalovirus; infection; metabolomic.

Figure 1

Representative urine 1D ¹H NMR spectra (600 MHz) from (A) controls and (B) patients. Selected metabolites annotations: 1 lactate; 2 acetate; 3 adipate; 4 alanine; 5 betaine; 6 urea; 7 citrate; 8 mannitol; 9 creatinine; 10 cis-aconitate; 11 trimethylamine; 12 dimethylamine; 13 methylguanidine; 14 taurine; 15 myo-inositol; 16 xanthosine; 17 glucose; 18 N-methylnicotinamide; 19 formate; 20 glycine; 21 hippurate; 22 acetone; 23 oxypurinol; 24 glycolate; 25 creatin-phosphate; 26 N,N-dimethylglycine; 27 succinate; 28 propylene glycol; 29 histidine.

Figure 2

PCA scores plot (PC1 vs PC2) of ¹H NMR spectra obtained from cCMV infected patients (red squares, “P”), or healthy controls (green rhombs, “C”).

Figure 3

(A) Prediction plot obtained from the first PLS-DA model. The samples are colored according to the two modeled classes (red squares, “P” or green rhombs, “C”). (B) Prediction plot obtained from the second PLS-DA model built using only those variables selected with the VIP scores and SR combined approach. The samples are colored according to the two modeled classes (red squares, “P” or green rhombs, “C”).