Diagnosing and Tracking COVID-19 Infections Leveraging Next-Gen Sequencing

by Dr. Christiane Scherer and Dr. Andreas Scherer

The COVID-19 pandemic is reaching historic proportions. We are dealing with an infectious disease that is caused by a novel coronavirus we discovered just a few months ago. Since then, it has brought healthcare systems to the brink, it altered how we work, it changed how we socialize, and it impacted the world economy in a major way.

While a global response has been mobilized to defeat the virus, there are currently no good solutions available. The current goal is to reach a sufficiently high level of immunization in the global population and to develop treatment options. In the meantime, we have to be efficient in diagnosing infections, isolating COVID-19 cases, and studying this virus by understanding its subtypes, epidemiology, routes of transmission, and clinical manifestation. In this article, we summarize our current understanding of COVID-19 and the virus SARS-Cov-2 causing it. Most of the papers we reference were published since the beginning of this year. The body of knowledge is quickly expanding and evolving. Next-Generation Sequencing (NGS) can deliver significant insights into this process. This article outlines how NGS can be used for diagnosing and tracking COVID-19 infections in the clinic.

Chapter 1: Introduction

At the end of 2019, a virus appeared somewhere in the Chinese city of Wuhan. For most individuals infected with the virus, symptoms were limited to mild cold and flu-like symptoms, but in a minority of cases, the virus resulted in severe pneumonia and death. It proved  to be highly contagious. The disease it caused would soon be coined COVID-19, standing for coronavirus disease of 2019. It quickly emerged as a global phenomenon mobilizing resources in every country to defeat it.

At the time of this writing, we are in the midst of a global pandemic. COVID-19 has arrived in many countries: Asia, Europe, and Latin America. There are cases reported in Australia, the Middle East, Africa, and Canada. The United States was hit especially hard, trying to contain the exponential spread in a country that is based on individual freedom and liberty. As the country adopts social distancing measures, based on federal and state guidance, we are facing the reality that the reported case numbers are climbing undeterred (see Figure 1).

As we are writing this sentence, there have over 4.3M confirmed cases of the coronavirus globally. The death toll is quickly approaching 300,000. This will obviously be out of date by the time you are reading this paragraph. We are dealing with exponentially growing numbers. There are estimations that put the number of infections in the tens of millions and the number of deaths in the hundreds of thousands. The Johns Hopkins University has developed a website, the Coronavirus Resource Center, that gives up-to-date information on this pandemic with updated information multiple times a day.

There are other informative resources that help to quantify the spread of the virus. Dr. Edward Parker, from the Vaccine Centre at the London School of Hygiene & Tropical Medicine, is maintaining a website that allows viewers to visualize current trends with his COVID-19 tracker (see Figure 2).

In addition, the website gives information about other recent outbreaks, such as the epidemic of Severe Acute Respiratory Syndrome (SARS) in 2003, the 2009 Swine Flu Pandemic, and the 2014 Ebola outbreak. The virus behind COVID-19 is called SARS-CoV-2. It is a pathogen that has unique characteristics turning it into a threat to our lives and the global economy. According to Fang et al. 2020, the preliminary estimate of R0, which estimates the expected number of new cases produced by each infected individual in a population, is 2.2 – 3.7 (see also Qun Li et al., 2020). It could be shown that it is able to persist for days on uncleaned surfaces.

Chapter 2: COVID-19 Key Facts

SARS-CoV-2 has just been recently discovered. The knowledge about this virus is fairly new, and certain aspects of it are still under review or in-flux entirely as we learn more about this virus on a daily basis (see Di Wu et al., 2020 and Scherer, 2020a).

Epidemiology

The virus has rapidly spread from Wuhan to China’s other areas and reached global proportions as it is now present on all continents except Antarctica. According to the European Centre for Disease Prevention and Control (ECDC), the latest daily risk assessment is moderate to high level. The case fatality rate of the currently reported cases in China is less than 4%, which implies that so far, this novel coronavirus does not seem to cause the high fatality rates previously observed for SARS-CoV and MERS-CoV. However, it has a higher R0 (2.2 -3.7) value than either of these viruses. SARS-CoV has an R0 of 0.67 – 1.23. MERS-CoV has an R0 of 0.29-0.8 (see Trilla, 2020).

Reservoir Hosts

Bats and other species can function as so-called reservoir  hosts. They have  played a critical role in transmitting various viruses, including Ebola. Cui et al. 2019 describes the origins of SARS-CoV  and  MERS-CoV  likely to be in bats as there is a strong genetic overlap between the viruses extracted from bats and their human-transmissible versions.
In fact, recent research showed that SARS- CoV-2 is 96% identical at the whole-genome level to a bat coronavirus. Understanding the origins of a virus in addition to when and how exactly the jump to humans occurred helps  us in understanding and eventually controlling its spread.

Route of Transmission

There are a number of ways in which the virus can transmit from human to human. The main transmission route is through droplet infection. Someone comes  into direct contact with a carrier when coughing or sneezing. There are also indications that transmission occurs during general social interaction in limited spatial surroundings (e.g., restaurants, school, sports events, etc.). Asymptomatic infections can lead to a wider spread as the hosts are unaware of their ability to transmit.

It is possible that the virus is able to persist for days on uncleaned surfaces. Recently, the new coronavirus was also found in the feces of confirmed patients in Wuhan, Shenzhen, and even in the U.S. Also, neonatal infections (i.e., mother-to-child transmission) have been observed but need to be confirmed (see Fuk- Woo et al., 2020, Phelan et al., 2020, Jin et al., 2020, Shen et al., 2019, Zhu et al., 2020).

Clinical Manifestation

Let’s look in further detail at what we know about the clinical manifestation of this novel pathogen.

Incubation Period and Symptoms

A number of publications based on smaller enrollment numbers suggest an incubation period from 1 to 12 days with a mean of 5-7.5 days. In a larger study with 1,099 patients extracting data from laboratory-confirmed cases from 552 hospitals in 30 Chinese provinces, researchers reported that the estimated mean incubation period of  a  SARS-CoV-2  infection was 4.0 days (Guan et al. 2020).

The same study (Guan et al., 2020) reported the following data. The median age of the patients was 47 years. 41.9 % of the patients were female. 5% were admitted to the I.C.U. 2.3% underwent invasive mechanical ventilation, and 1.4% died. Only 1.9% of the patients had a history of direct contact with wildlife. The most commons symptoms were the following:

• Fever: 43% on admission, 88.7% during hospitalization
• Cough: 8%
• Diarrhea: 8%

Coincidentally, the SARS-CoV-2 infected cases have symptoms like fever, fatigue, dry cough, dyspnea, etc., with or without nasal congestion, runny nose, or other upper respiratory symptoms. There are also reports of loss of smell and taste in otherwise non-symptomatic cases.

Diagnosis

From a diagnostic standpoint, the available options are as follows:

Physical Examination

Some patients may not present any noteworthy clinical symptoms despite being infected with the virus except perhaps the loss of smell or taste. Patients in severe condition may have shortness of breath, moist rales in lungs, weakened breath sounds, dullness in percussion, and increased or decreased speech tremor.

C.T. Imaging Examination

In the early stage of pneumonia, chest  images show multiple small patchy shadows and interstitial changes, remarkable in the lung periphery. Severe cases can further develop to bilateral multiple ground-glass opacity, infiltrating shadows, and pulmonary consolidation, with infrequent pleural effusion. While chest C.T. Scan pulmonary lesions are shown more clearly by C.T. than x-ray examination, including ground-glass opacity and segmental consolidation in bilateral lungs, especially in the lung periphery.

In a study of 41 patients, 40 (98%) had bilateral involvement. The typical findings of chest C.T. images of I.C.U. patients on admission were bilateral multiple lobular and subsegmental areas of consolidation (Figure 3A). The representative chest C.T. findings of non-ICU patients showed bilateral ground-glass opacity and mental areas of consolidation (Figure 3B). Later, chest C.T. images showed bilateral ground- glass opacity, whereas the consolidation had been resolved (see Figure 3). This data was extracted from Huang et al., 2020.

Laboratory Diagnosis

Current diagnostic strategies involve the exclusions of other known viral causes of pneumonia, such as influenza virus, parainfluenza virus, adenovirus, respiratory syncytial virus, rhinovirus, or SARS-CoV. Also, bacterial infections such as mycoplasma pneumonia, chlamydia pneumonia, and bacterial pneumonia should be tested for prior to conducting a COVID-19 test. A variety of specimens such as nasal swabs, nasopharynx or trachea extracts, sputum or lung tissue, blood, and feces are commonly used for testing.

Should those causes be ruled out, samples can then be collected from the upper respiratory tract (oropharyngeal and nasopharyngeal) or lower respiratory tract (endotracheal aspirate, expectorated sputum, or  bronchoalveolar lavage). The standard diagnosis is the C.D.C. 2019-nCov Real-Time RT-PCR Diagnostic Panel, a molecular in vitro diagnostic test, based on the widely used nucleic acid amplification technology.

Next-Gen Sequencing (NGS) is an alternative testing paradigm that has significant advantages over the RT-PCR method. We discuss this in  detail in the next chapter.

Treatment and Prevention

At this time, there is no vaccine or antiviral treatment for human and animal coronavirus. The World Health Organization (WHO) has announced that a vaccine for SARS- CoV-2 should be available in 18 months. The currently available clinical treatment options essentially focus on dealing with the symptoms arising from the infection. This ranges from bed rest, antiviral therapy, antibiotics application, immunomodulating therapy, organ function support, respiratory support, bronchoalveolar lavage (B.A.L.), blood purification, and extracorporeal membrane oxygenation (ECMO). Prevention  is  mostly about self-isolation, social distancing, and minimizing the exposure to a potential infection while living a healthy lifestyle.

Development of Future Treatment Options

Kupferschmidt and Cohen (2020) have reviewed four of the most promising therapies that the WHO has identified: an experimental antiviral compound called remdesivir, the malaria medication chloroquine and hydroxychloroquine, a combination treatment consisting of lopinavir and ritonavir and lastly a combination of lopinavir, ritonavir, and interferon-beta. All drugs are being tested, although the WHO opted not to conduct randomized, double-blind studies in the interest of time.

Remdesivir: It was initially tested to treat Ebola with no confirmed efficacy.  There is a reported case in the U.S. with a positive health outcome after treatment with the drug. More data is required. It is a drug that is being administered intravenously.

Chloroquine    and    Hydroxychloroquine: Studies in cell cultures have suggested that there is some effect on SARS-Cov-2 at very high doses close to toxic dose ranges. Multiple smaller studies in various countries such as

China and France have been conducted that showed some encouraging results. However, there is overall insufficient evidence that warrants a broad usage as of today.

Lopinavir and Ritonavir: This is  a  drug  that was approved in 2000 to treat H.I.V.  It has shown efficacy in treating MERS virus infections. Rigorous data collection is required. The drug can cause severe liver damage.

Lopinavir,  Ritonavir,  and   Interferon-Beta: This combination is already in trials to treat MERS. It could be potentially helpful to treat a COVID-19 infection, although experts point out that a late application of interferon-beta could actually lead to worse tissue damage.

In the meantime, there is a substantial global effort underway to develop a vaccine that could provide population-level protection against this novel virus. However, there is a major concern. It is a known risk that coronavirus vaccines potentially make the disease worse. The mechanism that causes that risk is not fully understood and is one of the stumbling blocks that has prevented the successful development of a coronavirus vaccine. Normally, researchers would take months to test for the possibility of vaccine enhancement in animals. Given  the urgency to stem the spread of the new coronavirus, some drug makers are moving straight into small-scale human tests, without waiting for the completion of such animal tests (see Steenhuysen 2020).

In the U.S., the National Institute of Allergy and Infectious Diseases (NIAID) within the National Institutes of Health (N.I.H.) is overseeing the funding of federal research and response to COVID-19. There are also some companies in the U.S. that are conducting their own COVID-19 research. Internationally, the U.K. Medicines and Healthcare products Regulatory Agency (MHRA) and the European Medicines Agency (E.M.A.) are supporting efforts to develop therapies against COVID-19. In general, it is expected that it takes 12-18 months to develop a vaccine. This tracker lists the major vaccine candidates currently in development.

Chapter 3: Leveraging NGS-Technology in the Fight Against COVID-19

The constellation that made SARS-CoV-2 a pandemic agent lies in its high transmissibility during the incubation period in combination with a mild to an asymptomatic course in most cases of its disease COVID-19 (see Rothe et al. 2020, Chen et al.). As a novel virus, the pathogen meets naive human immune systems worldwide, so that there is no disruption of the

infection chains by a significant proportion of immune individuals in the population. However, the mostly mild course of the disease contrasts with a not inconsiderable proportion that have a severe or critical disease. According to a report on 73,314 cases of the Chinese Centre for Disease Control and Prevention, about 14% of cases are severe with dyspnea and viral pneumonia and 5% critical with a need for intensive care (Wu et al. 2020).

In order to avoid the collapse of health systems, unprecedented, drastic measures are being taken around the world to reduce human contacts to a minimum in order to curb the number of new infections. The downside  of these efforts is a freeze in social life with unforeseeable consequences for society. The SARS-CoV-2 pandemic thus presents us with the historic choice of either drastically overburdening our healthcare capacity or driving the global economy into a global recession with far-reaching socio-economic consequences.

With the prospect of such a scenario, the question, “how can we reduce the number of new infections and return to social life,” is crucial to get back to our socio-economic balance.

Answers to this must already be found today, where measures based on contact isolation cannot be consistently implemented. This applies in particular to medical and social community facilities, hospitals, elderly homes, and social housing for people in need. Contact barriers cannot be implemented consistently as personal interactions and assistance are an essential component of the care provided for these facilities.

The role of  hospitals  in  SARS-CoV-2 infection chains has not yet been systematically described, but putting together the facts, it is clear that there is a high potential of

transmission to vulnerable groups of patients who are at risk of severe infection. Systematic testing of patients and staff even without clinical symptoms, depending on a patient group adapted risk assessment, is, therefore, an essential cornerstone for the rapid detection of infections. Especially in the hospital, patients can develop a veiling mix of symptoms due to their underlying disease and in some cases cannot be asked about important symptoms of lighter respiratory infections such as sore throat. However, comprehensive testing alone only makes sense if, preferably, every case leads to an infection-chain search in both patients  and employees with the consistent  isolation and placing in quarantine critical contacts. A pandemic pathogen provides a unique challenge in tracing the source of an infection. When there are multiple, several competing sources of infection are possible: has patient A really infected employee B or patient C or is    there possibly an unrecognized co-patient and employee B has acquired the infection in the home environment? The central hospital hygiene question is always the same: Have I ever seen this pathogen in another case before? In this respect, NGS, together with smart, customized bioinformatics tools, enables us to enter a new dimension of infection surveillance.

With 12 cases of COVID-19 sequenced with NGS, the complete genome and unique variants of each patient’s virus can be compared. With a dendrogram analysis, as  shown  in Figure 4, clustering is performed on all samples comparing variants by euclidian distance. The visible grouping helps to separate the cases into two main clusters. Patient A and Patient B are unlikely to belong to the same infection chain. With Patient C, no other case is associated. Instead, Patient D-G came from the same long-term care facility, giving a strong hint at an outbreak there.

Together with classic instruments of tracking infection chains such as timelines of the patient’s hospital stay and the identification of all their contacts, hygienists are able to clarify the entire infection process in their facility.

Dendrogram analysis is particularly suitable for distinguishing infections that have been detected in a short period of time. Since it lies in the nature of a virus to acquire new random mutations over time, having complete sequences allows for the identification of known isolates and novel variants to discover unknown isolates. Genetic information also provides insight into the evolutionary lineage of isolates, which can identify a temporal dimension in the relationship between samples. The scientific community publishes SARS-CoV-2 sequences on a regular basis, along with the date and location the sample was collected. Golden Helix’s NGS software is able to determine to what particular previous occurrence a particular sample is most similar. In this way, chains of infection can be presented in a regional, national, or international context beyond their local context (Figure 5). This supports highly specific and accurate contact-tracing and identification of potential hotspots.

With today’s sequencing methods, complete virus genomes can be sequenced very easily from preparations for PCR testing so that the tracking of infection chains at the molecular level is able to be accomplished practically in real-time.

In this way, the comparative analysis of NGS data can help to improve treatment processes in relation to their transmission risk. This can lead to the identification of training deficiencies, e.g., in the use of personal protective equipment. It can also be used to detect infection rates in other facilities: for example, when infections occur in patients who are taken from a particular nursing home or other hospitals.

By sharing this data between institutions (connected hospitals, associated long term facilities, etc.), even novel networks can be created to share data and improve response times to current and future infection events and even the next pandemic.

Clinical Testing with NGS Sequencing

In concert with the rapid-diagnosis capabilities of RT-PCR tests currently in use, the capabilities of NGS machines may be employed to capture the complete genomes of the virus as it spreads and evolves as well as confirm the virus presence. Longer-term, these collected virus samples will provide critical data to understand the evolution of the virus, its biological properties, and to aid in the development of therapeutic drugs and even vaccines.

The key to scaling up any NGS pipeline for clinical diagnosis and rapid turnaround is the automation of as many parts of the bioinformatics process as possible. This not only reduces time-to-result but also removes the potential for human error in otherwise manually performed steps. Today’s state of the art, commercial pipeline tools can be used as the high-throughput automation tool enabling repeatable workflows of the NGS analysis capabilities previously discussed.

With molecular barcoding, the throughput  of NGS machines allows for many samples to be multiplexed on a single run. The automated analysis workflow after sequencing run may look like:

• De-multiplex raw reads into per-sample sequence files
• Align sequence reads to the SARS-CoV-2
• Remove duplicate reads
• Call variants from the reference sequence
• Run customized workflow with VSPipeline to:

◆    Call Positive/Negative for SARS-CoV-2 based on coverage analysis

◆    Analysis of strain based on variants

◆    Output PDF clinical report

While these workflows presented here are certainly not comprehensive, they provide examples of common analysis strategies for different use cases of COVID-19 NGS sequencing.

Chapter 4: Summary

COVID-19 challenges the healthcare system, governments, and the global economy in an unprecedented way. As the novel coronavirus SARS-CoV-2 has just recently been discovered, vast resources are being directed towards understanding this new disease and the virus that causes it.

This article outlines the current state of our understanding of COVID-19 and SARS-CoV-2. Chapter 1 gives a brief introduction to this topic. Chapter 2 summarizes key facts about COVID-19; it reviews the epidemiology, reservoir hosts, transmission routes, and clinical manifestation. Chapter 3 answers the question of how Next- Generation Sequencing can be utilized in the clinic for diagnostic and tracking purposes.

By the time you read this sentence, there will be new findings, studies, and research papers. Also, any of the population-level statistics that have been cited will be dwarfed by the numbers that are reported by the time you read these words. But one thing is certain: Next-Gen Sequencing technologies will allow us to gain a deeper understanding of this virus and to develop advanced diagnostic capabilities to help patients and provide us the ability to conduct research at the same time.

Dr. Christiane Scherer is a medical microbiologist and senior hospital hygienist at the Evangelischen Klinikum Bethel, a German hospital with around 1,750 beds in 26 specialist clinics, three institutes and 11 interdisciplinary centers. Since the beginning of the COVID-19 pandemic, she has been part of the hospital’s task force for managing the crisis and is a member of the pandemic working group of the city of Bielefeld. She heads the clinic’s microbiological laboratory, which offers laboratory diagnostics and advice in the fields of serology, bacteriology, parasitology, virology, and molecular biological diagnostics. Dr. Scherer completed her specialist training at the Institute of Medical Microbiology at the University of Essen, where she received her doctorate in the Department  of Microbiology. She also holds a Master’s degree in Health Administration from Bielefeld University. Since 2004, she has held courses for medical students, doctors, and nurses, in particular on topics of infection diagnosis, antimicrobial resistance, and infection prevention. She participates in various working groups in the curriculum development of the medical faculty of Bielefeld University, which is in the process of being founded.

Dr. Andreas Scherer is the C.E.O. of Golden Helix. The company has been delivering industry-leading bioinformatics solutions for the advancement of life science research and translational medicine for over two decades. Its innovative technologies and analytic services empower clinicians and scientists at all levels to derive meaning from the rapidly increasing volumes of genomic data produced from next-generation sequencing and microarrays. With its solutions, hundreds of the world’s hospitals, testing labs, academic research organizations, and governments are able to harness the  full potential of genomics to identify the cause of disease, develop genomic diagnostics, and advance the quest for personalized medicine. Golden Helix products and services have been cited in thousands of peer-reviewed publications. He is also Managing Partner of Salto Partners, a management consulting firm headquartered in the D.C. metro area. He has extensive experience successfully managing growth as well as orchestrating complex turnaround situations. Dr. Scherer holds a Ph.D. in computer science from the University of Hagen, Germany, and a Master of Computer Science from the University of Dortmund, Germany. He is author and co-author of over 20 international publications and has written books on project management, the Internet, and artificial intelligence. His latest book, “Be Fast Or Be Gone,” is a prizewinner in the 2012 Eric Hoffer Book Awards competition, and has been named a finalist in the 2012 Next Generation Indie Book Awards!.

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