§ Our method of collection is easier and more practical than traditional urine CMV tests.

§ Our method of detection detects >1 log[CMV] (IU/mL) below salivary CMV concentration levels found in infants infected by congenital Cytomegalovirus.10

§ Our method has excellent sensitivity, specificity, and positive predictive values as determined in actual practice and therefore facilitates an effective universal newborn screening program.

§ Affecting 20,000-30,000 infants per year, congenital Cytomegalovirus infection (cCMV) is the most common congenital viral infection in the United States.1

§ cCMV is the leading cause of non-genetic sensorineural hearing loss (SNHL) and is the most common identifiable cause of mental retardation in the U. S.2,3

§ 85-90% of infants born with cCMV show no indication of disease and therefore receive no treatment.1,4

§ Appropriate treatment for cCMV (PO valganciclovir) has been shown to minimize or prevent SNHL and other cCMV associated morbidities.5,6

Background: Congenital CMV infection (cCMV) is the most common identifiable cause of mental retardation in the United States but requires early diagnosis to define the infection and to institute effective antiviral therapy. Traditional identification strategies including hearing screens and physical exams likely miss many patients with cCMV. We therefore developed and evaluated the performance of a PCR assay optimized for low cost, specimen collection at time of dried blood spot collection, and detection thresholds below the salivary CMV concentrations known to occur in cCMV patients.

Methods: We utilized a real-time CMV PCR assay (SimplexaTM CMV)(DiaSorin, Cypress CA) amplifying the UL83 gene and the 3M Integrated Cycler. Saliva was collected from volunteers (Copan swab), and spiked with known concentrations of CMV culture supernatant quantified by COBAS AmpliprepTM. (Roche Diagnostics). Additionally, saliva was collected by COPAN swab from all births within a single multi-hospital system from 3/21/16 –5/4/17. Newborns who were initial screen PCR positive were subsequently evaluated by urine CMV PCR by an outside laboratory for confirmation of cCMV.

Results: Analytical threshold of detection was well below 4 log copies/ML, with 100% of samples testing positive at 3.5 log copies/ML (Fig 1). 6127 newborn saliva samples were evaluated and 61 were PCR positive (≤40 CT). 47 of these tests were confirmed by urine PCR (Fig 2) (PPV 0.9792, NPV 0.9988, Sens 0.8704, Spec 0.9998). Screen positive tests which were not confirmed by urine PCR had CT values ≤36. Adjusting the definition of a positive to CT ≤36 further improved the performance (PPV >0.9999, NPV 0.9997, Sens 0.9592, Spec >0.9999). Conclusion: We demonstrate good performance of a congenital CMV methodology thus facilitating an effective universal newborn screening program

Abstract

Background

Conclusions

Future Directions

Analytical and Clinical Performance of a real-time screening PCR assay identifying congenital CMV infection Benjamin R. Rohde1,3, Maria Carrillo-Marquez, MD1,2, Ryan H. Tomlinson1,2, Jacqueline Blanch1,2 , Young-in Kim-Hoehamer, PhD1,2, Hema Patel3, Chirag Patel3, Gita Zanalian3, Gertrude Osborne3, Joseph Davis, MBA3, Anami Patel,

PhD1,3, John P. Devincenzo, MD1,2,3,4 1Children’s Foundation Research Institute, Le Bonheur Children’s Hospital, Memphis TN 2Department of Pediatrics, University of Tennessee School of Medicine, Memphis TN 3Molecular Diagnostics Laboratory, Le Bonheur Children’s Hospital, Memphis TN 4Department of Microbiology, Immunology, and Molecular Biology, University of Tennessee School of Medicine, Memphis TN

§ Real Time PCR:Ø All reactions were performed using a modified extraction

protocol and proprietary reagents (DiaSorin). Ø Known log[CMV] samples were prepared by spiking

cultured CMV into CMV negative pooled saliva (quantified using IU/mL standard).

Ø Quantification of spiked saliva performed on the COBAS AmpliprepTM (Roche) with International Unit standards.

Ø Patient samples ≤ 36 CT were called positive and reported.

Ø Patient samples ≤ 40 CT were repeated in duplicate, if 2 of 3 were positive it was reported.

§ Follow up Visit:Ø All positive patients referred for follow-up.Ø Patients re-swabbed for repeat PCR and urine collected

for confirmation by external lab.

References1. Britt WJ., et al., Cytomegalovirus. In: Infectious diseases of the fetus and newborn infant. Philadelphia: Elsevier Saunders; 2011. pp. 706–55. 2. Dollard SC, et al. New estimates of the prevalence of neurological and sensory sequelae and mortality associated with congenital cytomegalovirus infection. Rev Med Virol. 2007;17:355–63. 3. Fowler KB, et al. Congenital cytomegalovirus (CMV) infection and hearing deficit. J Clin Virol. 2005;35:226–31. 4. Boppana SB, et al. Symptomatic congenital cytomegalovirus infection: neonatal morbidity and mortality. Pediatr Infect Dis J. 1992;11:93–9. 5. Kadambari S, et al. Evidence based management guidelines for the detection and treatment of congenital CMV. Early Hum Dev. 2011;87(11):723–728. doi: 10.1016/j.earlhumdev.2011.08.021. 6. Kimberlin DW, et al. Valganciclovir for symptomatic congenital cytomegalovirus disease. N Engl J Med. 2015;372(10):933–943. doi: 10.1056/NEJMoa1404599. 7. Williams EJ, et al. Feasibility and acceptability of targeted screening for congenital CMV-related hearing loss. Archives of Disease in Childhood - Fetal and Neonatal Edition 2014;99:F230-F236. 8. Cannon MJ, et al. Repeated measures study of weekly and daily cytomegalovirus shedding patterns in saliva and urine of healthy cytomegalovirus-seropositive children. BMC Infectious Diseases. 2014;14:569. doi:10.1186/s12879-014-0569-1. 9. SALIVA VS. URINE PCR: THE IDEAL SAMPLE FOR CONGENITAL CMV SCREENING AND DIAGNOSIS. ID week 2017 https://idsa.confex.com/idsa/2014/webprogram/Paper46726.html 10. Luck et al (Sr. Author is Paul Griffith). ESPID 2007 (European society for pediatric infectious disease) 11. Vauloup-Fellous C, et al. Evaluation of Cytomegalovirus (CMV) DNA Quantification in Dried Blood Spots: Retrospective Study of CMV Congenital Infection . Journal of Clinical Microbiology. 2007;45(11):3804-3806. doi:10.1128/JCM.01654-07. 12. Marianne Leruez-Ville, et al,. Prospective Identification of Congenital Cytomegalovirus Infection in Newborns Using Real-Time Polymerase Chain Reaction Assays in Dried Blood Spots, Clinical Infectious Diseases, Volume 52, Issue 5, 1 March 2011, Pages 575–581, https://doi.org/10.1093/cid/ciq241

Results

Objective§ Develop a screening method that detects CMV below the

initial threshold of presentation to allow for universal newborn screening at a multi-hospital system (5,834 births/yr).

Methods

§ The screen demonstrated an exceptional quantitative analytical detection threshold, detecting 100% of samples at a concentration of 3.5 log[CMV] (Fig. 1).

§ Parental education and consent:Ø CMV educational materials describing outcomes and

significance of results were created and included in system-wide postpartum informational packets.

Ø Screen was offered at no charge to all infants born in the Methodist Le Bonheur Healthcare system from 3/21/16 –5/4/17.

§ Sample Collection:Ø Liquid saliva samples were collected orally using swabs.

Ø Nurse-collectors instructed not to collect saliva samples within 30 minutes after infant feeding.

Ø All samples were collected before 7 days of age.Ø Samples were shipped at room temperature and stored

at 4oC for 1-3 days prior to extraction and real-time PCR amplification.

§ The average CT of positive patient specimens (Fig. 2) was well below 36CT and is consistent with [CMV] between 5 and 6 log[CMV] IU/mL (Table 2).

3000 genomes/ml is equivalent to 2520 IU/ml. Personal communication, Dr. Paul Griffiths and as is referenced in the paper: American Journal of Transplantation 2012; 12: 2457–2464

0 1 2 3 4 5 6 70

20

40

60

80

100

120

Log [CMV] (copies/ml) Whole virus spiked in saliva,

quantified by COBAS

% o

f det

ectio

ns b

y sc

reen

ing

PCR

ass

ay

N=3 N

=3N

=15

N=3

3N

=15

N=1

3

N=1

3

N=3

Figure 1

Screen-pos, confirmed by

urine PCR

Screen-pos (All CT ≤ 40)

10

15

20

25

30

35

40

45

Scr

een

PC

R C

T va

lue

N = 61*N = 47*

Figure 2

Log [CMV] N mean CT range CT6 3 21.8 21.6-22.1

5 13 30.3 30-30.8

4 13 33.8 33.1-34.7

3.5 15 35.7 34.3-37.7

3* 33 37.2 35.6-39.9

2.5* 15 37.7 36.6-38.9

2* 3 - -

1* 3 - -*