Diagnosis of Human Congenital Cytomegalovirus Infection by Amplification of Viral DNA from Dried Blood Spots on Perinatal Cards

Abstract

Congenital human cytomegalovirus (HCMV) infection affects 1% of children and is the most common infectious cause of sensorineural hearing loss. Due to the difficulty of diagnosing deafness and other neurological disorders in infants , affected individuals may not be recognized until much later when active infection has resolved and culture is no longer informative. To overcome this problem , congenital HCMV infection was diagnosed retrospectively by testing residual blood samples collected from newborns and dried on perinatal cards as part of the North Carolina Newborn Screening Program. We modified the Qiagen method for purifying DNA from dried blood spots to increase the sample size and recovery of the lysate. A multiplex , real-time TaqMan polymerase chain reaction assay on an ABI 7900 instrument measured a highly conserved segment of the HCMV polymerase gene and the APOB human control gene. HCMV DNA was detected in blood dried on perinatal cards from all seven infants with culture-proven congenital infection , and all 24 negative control cases lacked detectable HCMV DNA. Our findings suggest that it is possible to diagnose congenital HCMV infection using dried blood collected up to 20 months earlier.

Evaluation of Cytomegalovirus (CMV) DNA Quantification in Dried Blood Spots: Retrospective Study of CMV Congenital Infection

“…The threshold values of the CMV DNA-specific PCR in DBS reported here, following any of our two DNA extraction protocols, was in the same range as those (e.g., 2,000 or 4,000 genome copies/ml) reported by two other groups (3,14). These threshold values are much higher than those obtained using fresh whole-blood samples; indeed, the latter values usually range from 50 to 500 genome copies/ml.…”

“…Policies for screening during pregnancy and at birth have not been implemented in European countries or in the United States, essentially because there is no well-established treatment for pregnant women or for newborns with CMV infection (7). Retrospective diagnosis of congenital infection has been achieved by PCR detection of the CMV DNA in dried blood spots (DBS) stored on perinatal Guthrie cards (2,5,6,8,9,14,16,17). Only one protocol (heat DNA extraction, followed by nested PCR) has been extensively evaluated in a clinical setting, with excellent sensitivity and specificity compared to that of viral isolation in the urine (1,2).…”

“…However, lower sensitivities (63 and 71%) were reported using the same protocol (5,16). Alternative methods based on either phenol-chloroform or silica extraction protocols have been proposed, but their sensitivities (81% and 100%, respectively) were studied only with small numbers of patients (9,14).…”

Hearing Loss and Congenital CMV Infection: A Systematic Review

“…Cross-contamination of adjacent stored cards has been reported. 54,[59][60][61] To understand the importance of cCMV as a cause of childhood hearing loss, we reviewed studies that conducted retrospective testing in a group of hearingimpaired children. Requirements were testing by real-time PCR for quantitative analysis of CMV DNA on DBS or on dried umbilical cords.…”

Active retinitis in an infant with postnatally acquired cytomegalovirus infection

“…Nevertheless, CMV DNA extraction from the Guthrie cards can help in ascertaining whether a baby was congenitally infected or not. 3 Regarding the timing of infection, in the case we describe we excluded congenital CMV infection because of the result of maternal serology, and because of the negative result of CMV DNA amplification performed on the Guthrie card. The baby was not breast-fed, therefore, the most probable source of infection was horizontal transmission by an infected individual.…”