Mark McCann

Detection of Cytomegalovirus DNA in Dried Blood Spots of Minnesota Infants Who Do Not Pass Newborn Hearing Screening

Background: Up to 15% of infants with asymptomatic congenital cytomegalovirus (CMV) infection will experience some degree of sensorineural hearing loss. Many infants who fail newborn hearing screening (NHS) are likely to have congenital CMV infection, but may escape definitive virologic identification because diagnostic evaluation may not commence until several weeks or months of age, making differentiation between congenital and postnatal CMV infection difficult. Early diagnosis linking virologic identification of congenital CMV infection to infants failing NHS may improve diagnostic precision and enhance opportunities for therapeutic intervention. Methods: The goal of this study was to compare newborn dried blood spots from Minnesota infants who had failed NHS, and were designated for referral, with control infants who passed NHS, for the presence of CMV DNA by real-time PCR, using hybridization probes for the CMV gene UL54. Results: Of 479 infants with a failed NHS (bilateral failure), 13 had CMV DNA present in the blood spot (2.7%). This compared with only 2/479 positive results from a control group of infants who passed the NHS (0.4%; P = 0.007, Fisher exact test). Comparisons of the glycoprotein B (gB) genotype as well as direct DNA sequencing of selected positives revealed that PCR positive samples represented unique clinical isolates. The mean viral load among the 15 positive samples was 1.6 × 103 genomes/microgram of total DNA. Conclusions: Newborn bloodspot CMV screening by real-time PCR may be a useful and rapid adjunct to functional NHS and may enable more rapid etiologic diagnosis of sensorineural hearing loss in newborns.

Comparison of One-Tier and Two-Tier Newborn Screening Metrics for Congenital Adrenal Hyperplasia

BACKGROUND: Newborn screening (NBS) for the classic forms of congenital adrenal hyperplasia (CAH) is mandated in all states in the United States. Compared with other NBS disorders, the false-positive rate (FPR) of CAH screening remains high and has not been significantly improved by adjusting 17α-hydroxyprogesterone cutoff values for birth weight and/or gestational age. Minnesota was the first state to initiate, and only 1 of 4 states currently performing, second-tier steroid profiling for CAH. False-negative rates (FNRs) for CAH are not well known. METHODS: This is a population-based study of all Minnesota infants (769 834) born 1999–2009, grouped by screening protocol (one-tier with repeat screen, January 1999 to May 2004; two-tier with second-tier steroid profiling, June 2004 to December 2009). FPR, FNR, and positive predictive value (PPV) were calculated per infant, rather than per sample, and compared between protocols. RESULTS: Overall, 15 false-negatives (4 salt-wasting, 11 simple-virilizing) and 45 true-positives were identified from 1999 to 2009. With two-tier screening, FNR was 32%, FPR increased to 0.065%, and PPV decreased to 8%, but these changes were not statistically significant. Second-tier steroid profiling obviated repeat screens of borderline results (355 per year average). CONCLUSIONS: In comparing the 2 screening protocols, the FPR of CAH NBS remains high, the PPV remains low, and false-negatives occur more frequently than has been reported. Physicians should be cautioned that a negative NBS does not necessarily rule out classic CAH; therefore, any patient for whom there is clinical concern for CAH should receive immediate diagnostic testing.

Evaluation of newborn screening bloodspot-based genetic testing as second tier screen for bedside newborn hearing screening

Purpose: Bedside newborn hearing screening is highly successful in identifying deaf or hard-of-hearing infants. However, newborn hearing screening protocols have high loss to follow-up rates. We propose that bloodspot-based genetic testing for GJB2 alleles can provide a means for rapid confirmation in a subset of infants who fail bedside newborn hearing screening.Methods: We performed a case-control study comparing the prevalence of common GJB2 mutations from deidentified bloodspots designated as “refer” by newborn hearing screening and contemporaneously selected randomly chosen controls designated as “pass.” Between March 2006 and December 2007, 2354 spots were analyzed for common alleles, c.35delG, c.167delT, c.235delC, and p.V37I in GJB2 with a subset reanalyzed by conventional Sanger sequencing to search for additional alleles.Results: The prevalence of biallelic GJB2 mutations in bloodspots from infants who referred by newborn hearing screening is approximately 1 in 50 (23/1177). In contrast, one bloodspot from an infant who passed newborn hearing screening was identified to harbor biallelic GJB2 mutations.Conclusions: These findings show that when a newborn refers by newborn hearing screening, there is a significant chance that GJB2-related hearing loss is present. Bloodspot-based genetic testing for common GJB2 alleles should be considered as second tier testing for bedside newborn hearing screening.

Analysis of archived newborn dried blood spots (DBS) identifies congenital cytomegalovirus as a major cause of unexplained pediatric sensorineural hearing loss

PURPOSE Congenital cytomegalovirus (cCMV) infection is the most common non-genetic cause of sensorineural hearing loss (SNHL). However, accurate diagnosis of cCMV as the etiology of SNHL is problematic beyond the neonatal period. This study therefore examined whether cCMV infection could be identified retrospectively in children presenting with unexplained SNHL to a multidisciplinary diagnostic outpatient otolaryngology clinic at an academic medical center in Minnesota. METHODS Over a 4-year period, 57 patients with an age range of 3months to 10years with unexplained SNHL were recruited to participate in this study. Informed consent was obtained to test the archived dried blood spots (DBS) of these patients for cCMV infection by real-time PCR, targeting a highly conserved region of the CMV UL83 gene. Results were normalized to recovery of an NRAS gene control. Chart review was conducted to identify subjects who underwent genetic testing and/or neurodiagnostic imaging to investigate possible genetic, syndromic, or anatomical causes of SNHL. RESULTS In total, 15 of the 57 children with unexplained SNHL tested positive for CMV DNA in their DBS (26%). A mean viral load of 8.3×104 (±4.1×104) [range, 1×103-6×105] copies/μg DNA was observed in subjects retrospectively diagnosed with cCMV. No statistically significant correlation was found between viral load and SNHL severity. CONCLUSIONS A retrospective DBS analysis demonstrated that 26% of patients presenting with unexplained SNHL in childhood had cCMV. DBS testing is useful in the retrospective diagnosis of cCMV, and may provide definitive diagnostic information about the etiology of SNHL.