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Congenital cytomegalovirus (cCMV) infection is the leading infectious cause of mental retardation, developmental delay and sensorineural deafness. Nonprimary infection plays a major role in transmission of this infection in countries with high maternal seroprevalence. Noninvasive sampling and testing is a useful alternative to traditional methods of laboratory detection of congenital CMV infection. The present study was conducted to understand birth prevalence of cCMV infection using molecular techniques, in an urban setting of a developing country with evidence of high maternal seroprevalence.Universal newborn screening for cCMV was performed for 750 infants born at a tertiary care center in Western India. Real-time polymerase chain reaction was directly carried out on saliva samples. Follow-up laboratory testing of saliva, urine and blood was performed for neonates identified as positive. Sequential clinical follow-up was offered to the affected infants.A birth prevalence of 0.4% (95% CI: 0.13–1.2) was observed with 3 of 750 babies confirmed to be positive for cCMV infection. All 3 babies were born to seropositive mothers (anti-CMV immunoglobulin G positive). One of the babies detected was symptomatic with sepsis like features. All of them survived and did not develop any sequelae up to 1 year of age.The use of direct real-time polymerase chain reaction of saliva samples can be considered as a feasible option for newborn screening of congenital CMV infection in developing countries. Relatively low birth prevalence of cCMV infection was observed in our study, which needs to be corroborated through further studies.