(I-358) Point-of-care assay for screening congenital cytomegalovirus

Friday, October 13, 2023

3:30 PM – 4:30 PM PDT

Location: Exhibit Hall - Row I - Poster # 358

Presenting Author(s)

Anastasia Giyoun Kim

Undergraduate Student Researcher
University of Washington, United States
LC

Lesley Chan (she/her/hers)

Undergraduate Student Researcher
Department of Bioengineering, University of Washington
Seattle, Washington, United States

Co-Author(s)

Xuanchang Hu

Undergraduate Student Researcher
University of Washington, United States

Primary Investigator(s)

Nuttada Panpradist, PhD (she/her/hers)

Postdoctoral Fellow
Department of Global Health, University of Washington
Seattle, Washington, United States

Introduction:: Congenital human cytomegalovirus (cCMV) infections pose a silent yet significant global threat. In low and middle-income countries (LMICs), the prevalence of cCMV can be alarmingly high, ranging from 6 to 14%. As much as 20% of infants born to seropositive mothers are affected by cCMV. Even more concerning, about half of these infants born with cCMV will suffer from long-term health issues, including hearing loss, vision loss, and neurodevelopment delay. The key to combating the devastating effects of cCMV lies in early diagnosis, ideally occuring within 3 weeks after birth. This early detection is crucial to distinguish between cCMV infections and CMV acquisition that may happen during or after delivery. With timely diagnostics, physicians can promptly initiate antiviral treatment in infants, potentially stabilizing or preventing permanent deafness. Currently, cCMV is typically diagnosed using polymerase chain reaction (PCR), which detects DNA of thde CMV from either saliva, blood, or urine samples. Performing traditional PCR necessitates laboratory infrastructure and trained personnel. Thus, specimens are often shipped to the centralized laboratory for PCR analysis, which can take 3 weeks or more to obtain the results in LMICs such as Kenya. The delay in obtaining the results can lead to loss-to-follow-up and lack of treatment. To address this unmet need, we envision developing a novel equipment-free molecular test for screening congenital CMV infections that provides accurate test results within the mother's delivery stay at the health facilities. Here we present our progress in assay development and evaluation using human body temperature.

Materials and Methods:: We designed primers and guide RNA to enable a single-step CMV DNA amplification by Recombinase Polymerase Amplification (RPA) and DNA detection by fluorescence using Clustered Regularly Interspaced Short Palindromic Repeats associated (CRISPR-Cas12a). To develop the RPA assay, we adapted previously validated PCR primers making necessary modifications to the 5’ sequences to meet RPA requirements. For CRISPR-Cas12a, we designed guide RNA with protospacer adjacent motif (PAM)-free guide RNA to have opposite directions of RPA primers. The guide RNA was systematically screened in a high-throughput manner using a commercial qPCR machine across 33 to 42 °C. We assessed their performance using 0 and 1000 copies of CMV DNA, a clinically relevant concentration for cCMV (with four technical replicates). To explore the feasibility of the assay’s operation under resource-limited settings, we conducted reactions using human body temperature, capturing heat from the palm. The reactions contained 0 or 1000 copies of CMV DNA and were allowed to proceed for 30 minutes. After completion, the reaction tubes were imaged using a cell phone and a low-cost transilluminator. To analyze the images efficiently, we developed an in-house Python-based algorithm that automatically classifies the tubes as either positive or negative results.

Results, Conclusions, and Discussions:: We have successfully developed an isothermal RPA/CRISPR-Cas12a assay to detect CMV DNA. This assay can detect 1000 copies of CMV DNA within a rapid timeframe of 12-13 minutes (Fig. 1A) when incubated at 37°C. Among the temperatures tested, 37 °C exhibited the fastest reaction rate, yielding significantly higher fluorescence signals than other conditions (i.e., 33 °C and 42 °C). Next, we investigated the feasibility of utilizing body temperature to facilitate the reactions to replace the need for equipment. Encouragingly, the reactions containing 0 and 1000 copies of DNA were differentiable to the naked eye and our in-house software (Fig. 1B). However, we noted that using body temperature required approximately 25-30 minutes before observing visible differences, slightly delaying the detection process. This delay suggests that body temperature variations from the optimal 37 °C may not be ideal for RPA/CRISPR-Cas12a, potentially requiring slightly longer reaction times. For real-world context, we intend to incorporate a feedback loop, including a body temperature measuring unit. This addition will automatically guide the required reaction time based on each user’s body temperature. By personalizing the reaction time according to individual body temperatures, we will ensure that the results will not be read prematurely. In summary, we have successfully demonstrated the development of a new isothermal amplification for cCMV DNA detection. The rapid detection and equipment-free operation hold great promise for cCMV diagnostics in low-resource settings.

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We thank our collaborators: Dr. Jennifer Slyker, Dr. Bhavna Chohan, and Dr. Grace John-Stewart for their clinical insights regarding cCMV. This work was funded by 2022 Thrasher Early Career Award (PI: Dr. Panpradist) and 2023 UW ITHS Catalyst Award (PI: Dr. Panpradist). The funders have no role in study design, experimental results, and interpretations.

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