Cytomegalovirus (CMV) GN genotypes in newborns and their potential for symptoms at birth and sequelae.

Introduction CMV is the leading cause of congenital infection with morbidity and mortality at birth and psychomotor and sensorineural sequelae. Strain virulence may depend on genetic variability in "key-genes", such as UL73, encoding the envelope glycoprotein N(gN) (1). Aims The aim of this research is to study the link between gN variants and pathological findings at birth and during long-term follow-up. M/M The eligible subjects, enrolled from January 1998 to May 2008 were: newborns from women with ascertained acute infection, confirmed as congenitally infected by virus isolation within the first 2 weeks and all newborns with clinical signs of infection and presence of virus in their urine within the first 2 weeks. 74 infants were investigated and 64 of them monitored at 1, 3, 6, 12 and 24 months and then annually. The parameters evaluated were: a) intrauterine growth retardation,hepatosplenomegaly,petechiae/purpura,jaundice,pneumonia,neurologic involvement; b) neuroimaging as a result by UltraSound and/or Magnetic Resonance Imaging, and/or Computed Tomograph ; c) sensorineural defects (hypoacusia, visual impairment); d)abnormal transaminase ,thrombocytopenia , hyperbilirubinemia. Cranial US was performed within the first 2 weeks and at 1 and 3 m. Neonates with abnormal US findings were re-examined weekly with Cranial CT, MRI during the 1st month and then monthly until 6th m. Ophthalmology was undertaken in the neonatal period and at 6 and 12 m. Hearing was assessed by brainstem auditory evoked responses at 3 m, 6 and 12 m and yearly by behavioral audiometry until school age. Psychomotor development was assessed at 6, 12 and 24 m by Brunet-Lezine test rating posture, coordination, speech and socialization. The newborns were finally classified as asymptomatic or symptomatic at birth with a favorable outcome (healthy) or adverse long-term outcome (sensorineural defects, psychomotor disabilities). After DNA extraction on urine samples, UL73 was amplified by a nested PCR (2). Automated DNA sequencing assigned each CMV isolate to one of the seven gN genotypes. Viral DNA load in blood was quantified by Cobas Amplicor CMV Monitor test. The investigators who genotyped and those who assembled clinical data, were blinded to reciprocal findings. Two-step cluster analysis was used to divide cases into uniform groups on the basis of either categorical or continuous data (3). The clustering algorithm is based on a distance measure that gives the best results if all variables are independent, continuous variables have a normal distribution, and categorical variables have a multinomial distribution. This is seldom the case, but the algorithm is thought to behave reasonably well when the assumptions are not met. The first step of the two-step procedure is the formation of preclusters that are clusters of the original cases based on the distances between all possible pairs of cases; when preclustering is complete, all cases in the same precluster are treated as a single entity. In the second step, a standard hierarchical clustering algorithm on the preclusters was used. The choice of a similarity measure and the determination of the number of clusters were based on the Log-likelihood distance and Schwarz’s Bayesian Criterion,respectively. Chi-square test with Bonferroni correction was used to test the statistical significance. A validation data set of 64 subjects was analyzed in order to verify independently whether the genotype was significantly associated with the outcomes (symptoms, sequelae, neuroimaging abnormalities) by using logistic regression. All p-values are 2-sided and if <0.05 were considered statistically significant. Results CMV congenitally-infected newborns fall into two sub-populations based on definite parameters of CMV disease: the first population with no symptoms at birth, negative imaging findings and a favorable outcome was significantly associa..