Voice series: Interview with Dr. Doris Di, University of Hawaii at Manoa; frontier in COVID-19 detection from wastewater treatment

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antibiotics including those used as last resort treatments [3]. This bacterium became a critical emerging issue after it was first identified in an environmental sample, and it received local attention and was the subject of interviews, features, and broadcasts on a Korean television program. Dr. Di has extensive microbial and molecular experience in the analysis of various types of environmental samples, including soil, fresh water, seawater, wastewater, bio-waste, and air, as well as samples influenced by natural events such as hurricanes, typhoons, floods, and seasonal changes. Over the years, she has received numerous awards and honors including the Outstanding Student and Strengthening Research Capacity Award, Top Student (2 nd Prize) of the Global University Project, outstanding student presentation awards (oral and posters), Brain Korea 21 Research Fellowships, a POSCO Foundation Scholarship, and a Korean government scholarship. She has published 11 internationally recognized research articles and has four manuscripts currently in revision.
Dr. Di's most recent research focuses on the wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the coronavirus strain causing the disease COVID-19. Since the start of the COVID-19 pandemic, she has worked on developing rapid and sensitive methods for detecting SARS-CoV-2 in municipal wastewater, to provide early detection of COVID-19 disease signals at the community level. She and her team worked with the City and County of Honolulu during a statewide lockdown at the beginning of the pandemic to investigate the trends in SARS-CoV-2 in samples from nine wastewater treatment plants, which process wastewater for approximately 1 million people on the island of Oahu. This surveillance report, published in Water Research, described tracking the daily dynamics of SARS-CoV-2 RNA in the wastewater from two large wastewater treatment plants in Honolulu during a rapidly expanding COVID-19 outbreak and a 4-week lockdown that resulted in a rapid decrease in daily new clinical COVID-19 cases [4]. The wastewater SARS-CoV-2 RNA concentrations exhibited significant inter-day fluctuations and an overall downward trend over the lockdown period, and the solid wastewater fraction contained the majority of the SARS-CoV-2 RNA mass. Dr. Di's team also worked with UH Manoa for early detection of the spread of COVID-19 in a student dormitory during a lockdown period. This project P. E. Saw: DOI: 10.15212/bioi-2022-0012 139 was aimed at detecting an early SARS-CoV-2 RNA signal in the wastewater from the dormitory, to enable rapid isolation of students before any apparent symptoms or positive tests for COVID-19, and prevent further viral spread. Currently, she is working on comparing rapid and efficient methods of SARS-CoV-2 recovery in wastewater and on developing a nested amplicon sequencing method to rapidly detect SARS-CoV-2 variants (Delta, Omicron, and others) in wastewater.
EE: Congratulations on your exciting episode of The Conversation, "Hawaii is ramping up wastewater surveillance for COVID-19," which was broadcast on Hawaii Public Radio. Could you please tell us a little bit of background on how this whole idea started? EE: What was your major concern during the progress of this research? Did you face major stumbling blocks along the way? How did you overcome these problems? DYW Di: There were a lot of struggles and difficulties along the way in this research, especially at the beginning. As you know, SARS-CoV-2 is a new emerging virus that is spreading rapidly in the community from airborne water droplets, person-to-person contact, or indirect contact with contaminated objects. Safety issues were the priority in initiating this research. Even though we knew that the rates of contagion of COVID-19 from wastewater samples were very low or near zero, we still had to be extremely cautious when handling and processing the samples. I had to go through a series of safety trainings (including respirator training, laboratory safety training, and so on); get medical clearance from physicians; be fully immunized against all possible exposure diseases, including influenza, hepatitis A and B, and Tdap (before vaccines for COVID-19 were introduced); and get permission and approval from the UH Manoa Institutional Biosafety Committee before I could start working. These took some time. When my team and I were developing the viral concentration and detection methods in wastewater, we also faced a lot of challenges. Although this virus is a strain of SARS coronavirus (severe acute respiratory syndrome coronavirus 1), the study of SARS coronavirus in wastewater was very limited, because this virus emerged and disappeared in a very short time (November 2002to May 2004, and had a lower infection rate than SARS-CoV-2. The physical properties, survivability, partitioning behavior, and many other characteristics of SARS-CoV-2 in wastewater were not very well studied at that time. We had to find references and methods that were used to detect and characterize enveloped viruses (like human coronavirus and avian influenzas), because SARS-CoV-2 is an enveloped virus, and adapt viral concentration methods that were used to recover non-enveloped viruses (more common viruses present in wastewater). We utilized the molecular detection methods developed by the Centers for Disease Control and Prevention (CDC) and World Health Organization (WHO), but we had to optimize these assays and compare which assays were more reliable and applicable to SARS-CoV-2 in wastewater, because these assays were developed for clinical detection in human specimens. We also faced some problems in getting supplies, such as experimental reagents, chemical materials, laboratory supplies, and also personal protective equipment (including face masks, face shields, and gloves). Because that was a global lockdown period, the whole world was prioritizing these materials for hospitals. Deliveries were delayed as logistics supply chains were interrupted, and mostly every material needed had to be imported into Hawaii. I just did my best with everything I had. Every day had new challenges, and I just had to deal with it with patience. When we started processing wastewater samples massively and consistently, we also faced some manpower issues. First, not every person was authorized to handle wastewater samples. Second, we were processing wastewater samples, and at the same time enhancing and increasing the detection sensitivity of the methods we were developing. Plus, the research on SARS-CoV-2 is still developing and evolving rapidly every day through the efforts of scientists all over the world, and we have to get the latest information and adopt it in our research. It was like burning candles at both ends back then. While most of the people were working from home, we had to come to the laboratory every day-just like the essential workers in the hospitals and clinical facilities-which also increased our risk of getting  There are a lot of methods, devices, and machines developed and introduced in the industry today for the same purpose, which is to detect SARS-CoV-2 in wastewater samples. However, you need to have funding and a budget to purchase the devices or machines, the supplies needed would cost money, and ordering and delivering these items would take time. The method that we developed uses reagents, chemicals, and supplies that are easily found in a microbiology laboratory. The turnaround time is short, and the data that we obtained are reliable. The cost is very low, too. The method is simple and easy to conduct. Of course, we are still trying to improve the detection sensitivity and limit of detection, because wastewater samples have a complex component and contain many unknown inhibitors that limit the detection sensitivity. If I had a chance to revamp, I would still choose an easy method that would provide accurate and reliable results with a short turnaround time. Plus, I would probably hire and train more people to improve the work efficiency and reduce the workload. We were not a big organization, so we basically did everything, from setting up autosamplers, collecting wastewater samples, processing wastewater samples, analytical measurement, data interpretation, report writing, and so on. All of this takes time, and what we were fighting with was time. We used to have two projects going on at the same time, so that was probably the thing that I would want to do differently if I had a chance.
EE: From your CV, we understand that you came from a microbiology background. When did you start venturing into wastewater treatment? Are there difficulties in the integration of these two fields?

DYW Di: Well, I had experience with wastewater research during my graduate study. One of the projects was wastewater surveillance of antibiotic-resistant bacteria (ARB) and
antibiotic-resistance genes (ARG). We collected and processed the wastewater from the influent, which is the stage where the wastewater flows into a treatment plant from the communities, and identified the ARB and ARG. Surprisingly, we were able to capture a whole lot of ARB and ARG in the wastewater from the local community. I was also involved in a global sewage surveillance project with WHO that aimed to collect ARG distribution patterns in wastewater all over the world. Another project was to investigate the fate of ARG during wastewater treatment, specifically the inactivation efficiency of plasmid-encoded ARG in both extracellular and intracellular (within bacteria) forms during water treatment with chlorine, UV, and UV/H 2 O 2 [5]. I have been doing research on microbiology in various kinds of environmental samples; wastewater is another environmental sample for me. I did not find it difficult. The things that I needed to know were the research purposes, hypothesis, targeting microbes, and methods before initiating the research.

In fact, I think that wastewater is a very interesting and valuable environmental sample, which provides a lot of information, especially related to public health. With my experience in working with the pandemic virus, I really think that integration in various fields is important for resolving global issues like COVID-19, especially in wastewater research. I know that a singlediscipline investigation is inadequate to respond to the global outbreak of COVID-19. Basically, it needs pathogen identification (epidemiology and virology), virus screening (omics and imaging/genetics), vaccine development (biomedicine and molecular biology), and diagnosis and therapeutics
(clinical medicine and pharmacy) [6]. We need information, experts, and professionals from different fields (physicians, policymakers, researchers/scientists, health officers, and communities) to work together and try to find out more about this virus, to contain the spread of this virus, to develop detection methods for this virus, to develop vaccines for this virus, and so on. In the research field itself, I also encountered that having interdisciplinary integration really helps in speeding up research on SARS-CoV-2. For example, we needed information from the medical fields about the survival, spreading, and shedding rate of the SARS-CoV-2 in order to incorporate the information into our research designs and concerns. We also required actual