Tracking and Pinpointing a Viral Invasion

MAJ Jeffrey Kugelman
Center for Genome Sciences

From Combat & Casualty Care, Summer 2021

Major Jeffrey R. Kugelman was born in California in 1976. He graduated in 1999 from Arizona State University with a Bachelor of Science degree in Design and from American Intercontinental University in 2001 with a Master’s degree in Information Technology. On 3 January 2003, he enlisted as an active duty medic in the U.S. Army. In 2007 he transferred to the Texas National Guard and began the simultaneous membership pro-gram as an ROTC cadet. In 2010, Major Kugelman graduated from The University of Texas at El Paso with a Ph.D. in Pathobiology and reentered active duty service as a Biochemist in the Medical Service Corps. After completing the AMEDD Officer Basic Course, Major Kugelman was as-signed to the United States Army Medical Research Institute of Infectious Diseases (USAMRIID).

During Major Kugelman’s assignment to USAMRIID, he served as a Biodefense Scientist and Assistant Director of the Center for Genome Sciences and now serves as the Division Chief of Molecular Biology. In 2014, he deployed to Liberia in support of Operation United Assistance, where he provided real-time sequencing identification of Ebola-virus infected human samples. In 2015, Major Kugelman served as a Medical Research and Materiel Command Liaison Officer to the Army G8 Office of Chemical and Biological Defense. In 2016, he was selected to serve as a Science and Technology Attaché in the Engineering and Science Exchange Program with France, located at the National Reference Center for Tropical Disease in Marseille, where he served until June 2018. In 2019, he rejoined USAMRIID as the Director of the Center for Genome Sciences.

Inside the U.S. Army Medical Research and Development Command’s (USAMRDC) United States Army Medical Research Institute of Infectious Diseases (USAMRIID), the effort to combat the novel coronavirus pandemic continues unabated. Since the very beginning of the pandemic, USAMRIID has been an integral player in developing the research required to fight COVID-19 and keep both U.S. Soldiers and the greater U.S. public safe. Specifically, scientists at USAMRIID have been tracking the so-called “evolutionary drift” of the virus across massive global databases containing information on hundreds of thousands of cases; additionally, they are tracking variants (viral changes) appearing in different parts of the world that may be affecting outbreak parameters. The Institute also recently launched a unique surveillance platform based on next-generation sequencing that has the potential to expand COVID-19 testing efforts, and make them more cost-effective while screening for other diseases of concern.

Heading this effort is Major Jeffrey R. Kugelman who has directed the Center for Genome Sciences at USAMRIID and served as Division Chief of Molecular Biology since 2019. Kugelman is a Medical Service Corps officer who holds a Ph.D. in Pathobiology from The University of Texas at El Paso. During his first assignment to USAMRIID, he served as a Biodefense Scientist and Assistant Director of the Center for Genome Sciences. In 2014, he deployed to Liberia in support of Operation United Assistance, where he provided real-time sequencing identification of Ebola-virus infected human samples. In 2015, Kugelman served as a Medical Research and Materiel Command Liaison Officer to the Army G8 Office of Chemical and Biological Defense before being selected for a scientific exchange program with France.

Combat & Casualty Care Magazine sat down with Kugelman to talk about USAMRIID’s continuing efforts in the fight against COVID-19, including new developments involving global surveillance of the virus.

C&CC: How are you progressing in charting this respiratory disease across the globe?

MAJ Kugelman: You hear a lot of talk about the India, U.K., and California variants – for SARS-CoV-2 it’s more of a geographic distribution. You can sample a population and see how much of a certain lineage is transmitting across an area. There are specific variants of concern that have a link to more severe disease and the ability to escape immune response – so it’s critical to understand what’s driving a local outbreak, or cluster.

C&CC: Is it incorrect then to refer to the country or location where an outburst spike occurs? Should we instead refer to a new variant by another metric?

MAJ Kugelman: No, you just have to understand the level of variance. For instance, you might have a cluster of 200 patients from a specific area and time. And there may be three viral lineages circulating in that group at that time, and so sequencing all 200 of them is not going to be fully beneficial. There’s just not a lot of diversifications in a closely-tied population for this virus. Whereas with Ebola virus disease (EVD), I can sequence all two hundred and it will establish a very clear person-to-person transmission chain. The World Health Organization has subsequently suggested a new Greek alphabet nomenclature to avoid regional virus association stigma.

C&CC: How does that knowledge translate to – or impact – the average citizen?

MAJ Kugelman: Some of the changes in the virus have been linked to changes in severity. Some have been linked to presentation in younger individuals post vaccination. So, it is important to know what’s driving that cluster in your area. It’s important to distinguish that it’s not [actually] tracking individual-to-individual [or] who-gave-what-to-whom. It’s more of ’70 percent of the cases in my area are presenting with any given variant.’

C&CC: Do you see a positive outlook for the U.S. given the currently climbing vaccination rate and the development of multiple vaccines?

MAJ Kugelman: We’re seeing the same. The number of samples is slowing down, and [so] we’re focusing now on cases that are post-vaccination – what we call “vaccine breakthroughs” – meaning they were fully immunized but still got sick. So yes, we are seeing the same general trend in our work. In the past year we’ve also developed a massively-pooled surveillance platform based on next-generation sequencing. All of these are good things.

C&CC: What more can you tell us about that surveillance platform?

MAJ Kugelman: A large part of diagnostics is based on real-time PCR [polymerase chain reaction] where you have a PCR target that’s amplified, and then as it’s amplified you measure the accumulation of a light signal. So, we’ve replaced that assessment of the light signal with a genetic sequencer. And in the preparation of the sample, in each sample we put on a unique sequence barcode at the front of the PCR target, and then that’s amplified. Then, because we’ve uniquely identified them in the first step, we’re able to then pool – as many as we can fit – into the sequencer.

Basically, we came up with a solution to the numbers problem because [in] the original protocols, we were only able to get up to 200 to 400 samples sequenced before the libraries became unstable and we were unable to sequence. We’ve now gotten to where we can run 12,000 samples concurrently on the same sequencing run. It takes 24 hours to get through the whole process, but at 12,000 concurrent tests presently, it’s now one of the most capable multi-sample approaches we have in the world to test for the presence or absence of COVID. We have submitted for a patent for this method.

C&CC: How does that translate into real-world usage?

MAJ Kugelman: Real world, we were also funded to do operationalization of this method. So we did several thousand nasal swabs and some saliva [samples] from the Naval Academy. And essentially, we were able to screen their asymptomatic population – because, obviously, if they had symptoms, they would be sent to a provider and handled a different way. And then any positives that we found were then referred for clinical testing so that it could be entered into the medical record and they could receive care. [This route] is also much cheaper. It’s between three and five dollars per test. We are able to start looking at populations that before weren’t being screened. One more small detail is that we’ve figured out how to do this process with neat saliva, so rather than doing a nasal swab, people can spit in a tube and we have a pretty good detection rate on that.

What this means is that you could take an entire company, an entire battalion, have them open ranks, spit in a tube, turn it in – and then, within 20 to 30 minutes, you’ve surveilled the entire company at the same point in time. So when those results come back, you can then pull out the infected individuals and maintain your organizational capacity.

While we were working with the Naval Academy, there was a week there – if you remember the news reports – where they went up to triple-digit cases. We were surveilling their asymptomatic population at that time, and we were able to identify eight asymptomatic and one pre-symptomatic case in their population. These all had very high viral loads, so they were not symptomatic but clearly infectious. The pooling assay detected those cases. Then, within a week, we turned it around and were able to do the whole genome sequencing and tell them that it was the new U.K. variant that had caused that spike in the population.

C&CC: What is the eventual plan for this new method? Is the goal to bring it to market?

MAJ Kugelman: The USAMRDC did file for the patent. What they decide to do with the licensure upon completion of the patent application is up to [the Command]. It may be sold; it may become commercially available. But my goal was to get it ready for the military. As I said, we were funded to do the operationalization; so we were actually using the test to support this outbreak.

C&CC: What else is taking place at USAMRIID that is worthy to note?

MAJ Kugelman: We also have done [work] with reverse genetics. As these samples are coming in, we are attempting to do isolates for any new lineages we see of variants of concern. We just finished characterizing the India variant, the 617.2, which took [about] two to three weeks. In the event that we cannot isolate virus from the sample, or we don’t have access to the sample, but we still have the sequence – then we have a reverse genetics system where we can make the changes observed and then reconstitute it. Using the tools of synthetic biology, we’re able to make a virus that looks like the one we want to acquire, and then grow that and create a stock from it. So, we’ve developed this method and we’ve been using it for new variants. The silent mutation system – or, the barcoding for the transmissibility –was built on that platform. We’ve also reconstituted all the known clade (viral family) members so that we can do a comparison study for transmissibility. We’ve been extremely busy at USAMRIID over the past year with our COVID-19 support.