Poster Y7 - Evaluation of Rapid Point-of-Care Assay to Detect Congenital Cytomegalovirus in Kenyan Infants

Friday, October 25, 2024

3:30 PM - 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Anastasia Giyoun Kim, RSE (they/them/theirs)

Research Scientist/Engineer
University of Washington, Seattle, WA, USA
Seattle, Washington, United States
LC

Lesley Chan (she/her/hers)

Undergraduate Student Researcher
University of Washington, Seattle, WA, USA
Seattle, Washington, United States

Co-Author(s)

KG

Kerusha Govender

Researcher
CHU Sainte-Justine Research Centre, Montreal, Canada, United States
JH

Jing Hu

Researcher
CHU Sainte-Justine Research Centre, Montreal, Canada, United States
BC

Bhavna Chohan, PhD

Clinical Assistant Professor
University of Washington, Seattle, WA, USA, United States
GJ

Grace John-Stewart

Professor
University of Washington, Seattle, WA, USA, United States
EB

Emily Begnel, MPH, PhD (she/her/hers)

Researcher
University of Washington, Seattle, WA, USA, United States
DL

Dara Lehman

Researcher
Fred Hutchinson Cancer Center, Seattle, Washington, United States
JK

John Kinuthia

Head of Research and Programs
Kenyatta National Hospital, Nairobi, Kenya, United States
DW

Dalton Walmalwa

Professor
University of Nairobi, Nairobi, Kenya, United States
SG

Soren Gantt, MD

Director of Clinical Research
CHU Sainte-Justine Research Centre, Montreal, Canada, United States
JS

Jennifer Slyker

Associate Professor
University of Washington, Seattle, WA, USA, United States

Primary Investigator(s)

Nuttada Panpradist, PhD

Research Engineer
University of Washington, Seattle, WA, USA, United States

Introduction: Congenital human cytomegalovirus (cCMV) infections among infants is a significant global threat, with prevalence of cCMV being as high as 6-14% in low and middle-income countries (LMICs). If a mother is seropositive, there is a 20% chance that the infant will be affected by cCMV. Of those affected, approximately half will suffer from long-term health issues, including hearing loss, vision loss, and neurodevelopmental delay. Diagnosis of cCMV within 3 weeks after birth can be crucial to prompt early antiviral treatment to infants, which can stabilize or prevent permanent deafness. The current gold standard for CMV diagnosis is polymerase chain reaction (PCR), which can detect CMV DNA from saliva, blood, or urine samples. The use of traditional PCR requires laboratory infrastructure and trained personnel, often necessitating samples to be shipped to centralized laboratories for analysis. In LMICs, such as Kenya, this process can take 3 weeks or more, creating complications regarding follow-up and treatment that could delay treatment and cause long-term health issues. To address this need, we have developed a proof-of-concept for a novel, equipment-free, isothermal molecular test for screening cCMV infections using human body temperature. Here we present our progress in further evaluating this assay with urine specimens collected from Kenyan infants.

Materials and

Methods: Our assay was designed to be a single-step, one-pot CMV DNA amplification by Recombinase Polymerase Amplification (RPA) and detection by fluorescence using Clustered Regularly Interspaced Short Palindromic Repeats associated (CRISPR-Cas12a) as previously described. To enable a single-tube reaction, CRISPR-Cas12a guide RNA was designed with protospacer adjacent motif (PAM)-free guide RNA going the opposite direction of the RPA forward primer. Urine has known PCR inhibitors such as urea and dietary metabolites, thus we first added different volumes of pooled human urine up to 20% to our RPA/CRISPR-Cas12a reactions and observed the amplification of spiked-in synthetic CMV DNA (GenBank: MT044485.1, 83069 to 83227 bases). The maximum volume of urine that can be added without RPA/CRISPR-Cas12a inhibition was determined and used to analyze a panel of urine specimens covering a range of specimen concentrations (median: 17444.8). These specimens were collected as part of the Linda Kizazi Project, tested for the presence of CMV using a commercial PCR kit (AltoStar® CMV PCR Kit 1.5), and de-identified prior to use in this study. To evaluate our assay, 4µL of heat-treated urine specimens (95 °C for 10 minutes and cooled down to room temperature) was added to our assay components to achieve a final volume of 20µL. The reaction was then subjected to 37°C, and fluorescence was recorded every 1 minute for 150 minutes. Specificity and sensitivity were reported along with 95% CI (Binomial Exact). All specimens were tested in triplicates.

Results, Conclusions, and Discussions: To develop a rapid sample-to-result workflow, we opted to bypass traditional DNA extraction. Instead, we heated the urine specimens to lyse the CMV virus and directly analyzed the CMV DNA from the lysate using our isothermal RPA/CRISPR-Cas12a assay. We found up to 20% of the lysate can be added into our RPA/CRISPR-Cas12a reaction with minimal delay in amplification. Using this protocol, we could detect CMV DNA down to 5 copies/µL of reaction spiked in negative human urine lysate. Applying this protocol to analyze 41 clinical urine specimens (10 negative, 31 positives), our isothermal RPA/CRISPR-Cas12a assay did not falsely detect CMV in negative samples and had 100% specificity [95%CI: 69.15-100]. Among 31 positive samples tested, our assay detected at least 1 out of three replicates in 29 samples (sensitivty 93.5% [95%CI: 82.59-98.63]. The two positive specimens missed by our assay were 750 and 9000 IU/mL. This difference in detection suggests that our isothermal assay is not quite as sensitive as the PCR assay comparator (limit of detection: 711 IU/mL with the 95 % CI: 420 - 1,625 IU/ml). However, our method is likely sufficient as a tool to detect cCMV in urine specimens from infants, which has the viral load of 1.79 x 106 copies/mL (95% CI: 7.97×105 to 4.02×106) or 1.63 x 105 IU/mL [3]. The time to detection of our RPA/CRISPR-Cas12a assay for samples above 105 IU/mL was 24 minutes on average, which is faster and simpler than traditional PCR, with 100% sensitivity. These findings demonstrate that our RPA/CRISPR-Cas12a assay provides a rapid and relatively sensitive method sufficient for detecting clinically-relevant concentrations of CMV DNA in urines from infants with cCMV infection. The simplicity of the protocol, requiring minimal sample preparation, makes it a practical tool for point-of-care testing and early detection of cCMV in infants, potentially improving clinical outcomes through timely treatment initiation. Our next steps include further assay optimization to increase speed and sensitivity in order to expand use of this assay in other CMV diagnostic applications.

Acknowledgements (Optional): We thank our collaborators, Dr. Barry Lutz, Dr. Eric Klavins, and Dr. Karl Bohringer, for providing access to the laboratory and equipment used to support our studies.This work was funded by the 2022 UW/Fred Hutch CFAR New Investigator Award (PI: Dr. Panpradist), 2022 Thrasher Early Career Award (PI: Dr. Panpradist) and 2023 UW ITHS Catalyst Award (PI: Dr. Panpradist). Ana Giyoun Kim was funded by the 2022 Mary Gates Research Scholarship. The funders have no role in study design, experimental results, and interpretations.