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Cytomegalovirus (CMV) is the leading cause of viral congenital infection. Some viral factors have been proposed to be CMV pathogenicity markers. The objective of this study was to investigate the frequency of congenital CMV infection in symptomatic patients and the possible association with the CMV glycoprotein B (gB) genotype and viral load.A total of 361 newborns (NB) and 158 pregnant women (PW) with clinically suspected CMV infection were enrolled. Studied samples included urine, saliva, serum, vaginal swabs and amniotic fluid. CMV infection was diagnosed by multiplex nested PCR. CMV gB genotyping was performed on infected samples, followed by viral load determination.Overall, 18.7% of the tested patients were positive for CMV infection, 19.7% of NB were congenitally infected and 16.5% of PW showed active CMV infection. gB-2 was the most prevalent genotype detected (39/97 patients). gB CMV mixed infections were detected in 12 patients. gB-2 was associated with mono-infections (P < 0.01). Mixed infections showed higher levels of viral load compared with gB mono-infection (P = 0.03). Hepatomegaly, splenomegaly, jaundice, sepsis-like syndrome and malformations were the most prevalent clinical findings. gB-4 was more frequently associated with sepsis-like syndrome than other gB genotypes (P = 0.04, odds ratio = 4.3, confidence interval: 0.9 to 21.6). The difference in medians of CMV load was statistically significant among patients presenting different clinical signs (P = 0.04).This study showed that CMV is a frequent cause of congenital infection in symptomatic Cuban patients. Despite gB2 being the most frequently detected, gB-4 was the only genotype associated with clinical features (sepsis-like syndrome in NB). No other associations among specific genotypes and clinical characteristics were found. Further studies are needed to clarify the role that viral load and genotype play in the outcome of congenital infection.