551 - Nanotechnology-directed Rapid Identification of Cytomegalovirus in Urine

Poster Number: 551
Publication Number: 551.328

Cedric Pritchett, Nemours Children's Health System, Orlando, FL, United States; Peter Phelan, Nemours Children's Hospital, Orlando, FL, United States; Rajarajeshwari Venkataraman, Nemours Children's Hospital, Orlando, FL, United States; Kenneth A. Alexander, University of Central Florida College of Medicine, Orlando, FL, United States; Qun Huo, University of Central Florida, Orlando, FL, United States

Background: Cytomegalovirus (CMV) is the most common congenital infection and the most common infectious cause of congenital hearing loss. Universal newborn screening for cCMV is costly, and conventional methods fail to provide timely diagnosis. We describe a novel method for rapid CMV identification in children.

Objective: Development of a rapid nanotechnology-based immunoassay (D2Dx) to detect CMV in the urine of children.

Design/Methods: A non-randomized, prospective experimental design with control group. Urine from 40 children < 8 years of age (20 with a diagnosis of cCMV) was collected and stored at -80°C prior to analysis. Each specimen (40 µL) was mixed with a gold nanoparticle (AuNP) probe in the presence or absence of an anti-CMV monoclonal antibody (NanoDiscovery Inc.; LifeTechnologies). The presence of CMV in the urine was determined by measuring the change in max UV-Vis spectral absorbance at 568 nm as a function of aggregation of CMV virions, anti-CMV antibody, and the AuNP probe. Colorimetric change was evaluated over a 5 min period using a hand-held, fixed-wavelength CT-100 UV-Vis spectrometer (NanoDiscovery, Inc.). Detection of CMV was determined by comparison of “Test A” - absence of antibody vs. “Test B”- presence of antibody. Confirmatory quantitative real-time PCR (qPCR) testing was performed using purified DNA from each sample and published primer and probe sequences targeting the CMV genes pp65, AD-1, and IE2ex5.

Results: The results of 19 samples with clinical CMV diagnosis tested positive with matching D2Dx and qPCR test results. There were no false-negative D2Dx test results. In the remaining 21 samples (all negative by qPCR analysis), 13 CMV-negative samples were consistent by clinical diagnosis, qPCR detection, and D2Dx analysis; 3 positive D2Dx test results matched positive clinical diagnoses; 1 positive D2Dx test result was associated with a negative clinical diagnosis. Four clinically diagnosed samples did not match with positive CMV detection by either qPCR or D2Dx. The sensitivity of D2Dx test was comparable with qPCR technique and noted to reliably detect the virus at a concentration of 500-1000 virion or genome copies/mL.Conclusion(s): We found D2Dx test results highly correlative with the clinical diagnosis and qPCR testing. In comparison to qPCR, the estimated sensitivity and specificity of the D2Dx assay are 100% and 81%, respectively. This urine-based nanotechnology immunoassay can offer an alternate and comparable option for rapid ( < 10 min), inexpensive point-of-care population screening for CMV.