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Back in 2015, when Bill Gates famously declared we were “not ready for the next outbreak,” few people could have predicted how true his words would be and what kind of global impact that next outbreak would have. But the COVID-19 pandemic has not only altered most of our lives for more than a year and a half already, it has transformed approaches to tracking and testing for infectious disease overall. Importantly, it has raised the profile of Next-generation sequencing (NGS).

Garret Hampton
Garret Hampton, Thermo Fisher Scientific

“COVID-19 has rapidly driven the field of infectious disease sequencing forward,” said Justin O’Grady, Senior Director in the Applications group at Oxford Nanopore.  Garret Hampton, president of clinical next-generation sequencing and oncology at Thermo Fisher Scientific concurs. “With the calls for increased sequencing efforts to detect new and emerging SARS-CoV-2 strains, an increasing number of labs have adopted NGS—bringing molecular profiling capabilities to local communities to inform local public health decisions,” he said.

NGS provides a culture-free method for faster and more accurate identification of viruses, bacteria, fungi, and parasites. It can replace the need for multiple tests, and it could be applied to many conditions, not just the widely known diseases such as pneumonia, flu, and norovirus, but also less-common infections, such as “Lyme disease and food born illnesses, including e-coli, listeria, and salmonella,” said Madhuri Hegde, senior vice president and CSO at PerkinElmer Genomics.

PerkinElmer lab
In response to the COVIID-19 pandemic, PerkinElmer launched two research use only (RUO) tests—one PCR-based and the other NGS-based for the detection of the SARS-CoV-2 virus and its variants.

One example comes from the U.K., which has led the way in sequencing for COVID-19 (See Sidebar): “Going beyond SARS-CoV-2, MTB (mycobacterium tuberculosis) is the best example in the U.K. of sequencing being used to guide treatments,” said Andrew Page, head of informatics, Quadram Institute Bioscience.  “All MTB samples are sequenced by Public Health England, usually in under 3 weeks to get a full AMR profile. This is much faster than trying to culture. This then guides the treatment, which is particularly important for MTB given the widespread resistance.”

Smaller enterprises are also jumping into the game. Fry Labs reports it offers NGS for a much wider range of indications. “The big demand is in urology, gynecology, wound infections, CNS, and myocarditis,” said Stephen E. Fry, founder and director of the company. The company offers comprehensive kits that cover key microbial genome areas.  “We are now looking at drug resistance,” he added.

But hurdles remain. “It needs to be cheaper and we need better sample prep approaches,” said Hegde. She explained that while sample prep is relatively cheap, currently, several thousand samples need to be run on most NGS systems to be affordable. In addition, she’d like to see more automation. “The more plug in it becomes, the more widely it will be adopted.” Another problem that she and other experts highlight is quality and easy-to-use analytics, which have been a longstanding hold-ups in the field.

COVID-19 appears to have jump-started the use and interest in NGS, resulting in progress on all these fronts.

The COVID wake up call

A big problem with SARS-CoV-2, the coronavirus virus that causes COVID-19, is how quickly and broadly it has spread.  From the first case in Wuhan China in December 2019, it took about a month for the disease to infect several thousand people, mostly in China but also reached more than a dozen countries. By the end of January, WHO declared a Public Health Emergency of International Concern (PHEIC).

COVID-19 has impacted the public health and clinical medicine landscape to the greatest degree since the emergence of HIV in the early 1990s. It has led to a rapid uptick in sequencing for tracking purposes, the development of several vaccines that have come into widespread use, and dozens of diagnostic tests—including over-the counter products—that test for antibodies, nucleic acids (e.g. RT-PCR) tests, and antigens.

NGS has played a key role in all of this. Determining the virus’s sequence enabled vaccine development and the application of NGS in testing has given public health officials a powerful tool for monitoring the viruses’ spread and the emergence of new variants.

“The first SARS-CoV-2 genome was sequenced using a metagenomic approach, which was necessary as patients in Wuhan were clearly infected with a pathogen, but none of the existing diagnostic tests could identify it,” said O’Grady.  This sequence was then used to design targeted tiling PCR approaches (e.g. ARTIC) for faster, cheaper viral genome sequencing. qPCR-based diagnostic tests were perfect for the rapid diagnosis of SARS-CoV-2 infection.

So perhaps it is with diagnostics that COVID-19 has had its biggest impact on the clinical use of NGS.  In June of this year, the first NGS-based COVID-19 diagnostic—Illumina’s COVIDseq test, was approved for use by the FDA. It employs 98 DNA fragments, or amplicons, to cover the roughly 30 kilobases in the SARS-CoV-2 genome. By multiplexing the reaction, upwards of 3,000 tests of nasopharyngeal or oropharyngeal samples can be run in one go with a 24-hour turnaround. Company literature states that the test requires at least 1,000 copies of the viral genome per milliliter and displays 98% sensitivity and 97% specificity.

Others are also keeping up with the virus. For example, PerkinElmer launched two research use only (RUO) tests to identify SARS-CoV-2 variants—the PKamp VariantDetect SARS-CoV-2 RT PCR Assay and the NGS-based NEXTFLEX Variant -Seq SARS-CoV-2 Kit. Once a person has a positive COVID-19 diagnosis, these tests can be used to identify genomic mutations, a growing concern now that about one-third of cases are estimated to involve a variant.

Clearly, the response to COVID-19 has been spectacular. But can we use NGS to the same remarkable effect to contain other diseases?  Is this a one-off, a blueprint, or at the very least the beginning of a trend that will see NGS becoming more widely used in the clinic and in public health.

Guillermo Pimentel
U.S. Navy Capt. Guillermo Pimentel, Defense Health Agency, DoD

“The silver lining of the pandemic has been to recognize the importance of NGS in the near-real time tracking/monitoring of the evolution of a pathogenic virus to detect variants. The pandemic has allowed the GEIS NGSBC to provide sequencing support to the DoD since March 2020.  As part of the lessons learned, whole genome sequencing will be included in future,” said U.S. Navy Capt. Guillermo Pimentel, chief of the Global Emerging Infections Surveillance Branch, Armed Forces Health Surveillance Division, an agency under DoD’s Defense Health Agency.

Expansion to other diseases

O’Grady noted that the key to implementing sequencing for potential new diagnostics is speed and flexible throughput. Oxford Nanopore’s rapid library preparation, he says, approaches (15min-3 hours), real-time sequencing technology and real-time analysis tools (Epi2Me). That means that existing workflows could deliver clinically actionable results within 4 hours of sample collection.

“The possibilities that we are looking at here are broad,” he said.  A single sample could be processed on a Flongle flow cell (costing $90) for applications where speed is particularly important, such as nosocomial pneumonia or sepsis, and where batching samples would cause major delays. Tens to hundreds of samples could be processed together on MinION and GridION in examples where turnaround time isn’t critical, such as characterizing urinary tract infections.

He added that Oxford Nanopore expects “metagenomic approaches will be useful for diseases that can be caused by multiple pathogens, allowing the unbiased characterization of any pathogen and associated antimicrobial resistance in a sample. Targeted approaches could be taken for diseases caused by single or a small number of pathogens such as tuberculosis or sexually transmitted diseases.”

O’Grady believes sequencing is fast becoming a permanent tool for pathogens detection. “I think sequencing-based diagnostic technologies in the field of infectious diseases are here to stay, so training in the principles of sequencing and how to correctly interpret the data should be provided to biomedical scientists and clinicians,” he said.

Duncan MacCannell, CSO for CDC’s Office of Advanced Molecular Detection, sees metagenomic sequencing, which allows analysis of DNA or RNA recovered directly from the environment, without the intervening steps of culture and isolation as an important method of detection going forward. “This approach has the potential to generate rapid tests for diagnosis and surveillance. It is also the basis for studies of microbial ecology in diverse environments, from farms to the human body,” he explained.

He added that detection of DNA and RNA directly from patient samples also underlies the development of culture-independent diagnostic tests (CIDTs), which can be used in hospitals and doctors’ offices. Some are designed to test simultaneously for multiple organisms (for example, for several bacterial causes of gastrointestinal illness). CIDTs can return a precise diagnosis within hours, instead of days; however, he pointed out, they do not produce an isolate that can be subtyped.

PerkinElmer’s Hegde is also optimistic. “It took ten years for NGS to become a mainstay in cancer, and now it is commonplace for rare diseases, I think in another three-to-five years we will see it play an even more widespread role in infectious diseases,” she said.

Orther challenges are on the horizon. Capt. Pimentel noted: “We should continue worldwide surveillance of respiratory viruses (e.g., influenza viruses, coronaviruses, etc.) as well as vector-borne viruses such as arboviruses sensu lato—they are just as important as the others, since global warming and climate change have impacted areas where these vectors are expanding their geographic range.”

Tracking and Testing Initiatives

The realization that to stay ahead of the virus we would need to track it took off in the U.K., with the COVID-19 Genomics UK (COG-UK) Consortium established in March 2020.  That group includes testing centers, hospitals, and academic labs. By the time it was announced, the consortium had already sequenced more than 250 SARS-CoV-2-genomes.  Since then, it has done more than 200,000.

It’s already having a profound effect, both in tracking spread and the emergence of mutations and on the clinical front, said Andrew Page, PI for the COG-UK sequencing centre at the Quadram Institute.  “For one hospital in our region we were able to identify they had 2 different lineages circulating within the hospital and investigations revealed one was being spread by patients moving around wards, and the other by staff moving. Normal IPC practice said outbreaks on different wards were treated as different outbreaks, but rapid genomic epidemiology based on sequencing every case in the hospital identified that 11 wards had identical or near identical genomes.”

The COG-UK’s most high-profile finding was the discovery that a surge in cases was associated with one particular strain of SARS-CoV-2, dubbed B.1.1.7. Scientists soon determined that this caused the virus to become significantly more contagious.

PerkinElmer stepped in to help boost the U.K. Government’s NHS Test and Trace program in fall of 2020 as part of its collaboration with the Department of Health and Social Care (DHSC).  The company’s Lighthouse Lab in Charnwood, England, can test up to 50,000 samples daily and 24 hours per day.

Page noted that the COG-UK consortium began looking at how to expand surveillance by bringing together a collection of groups studying other bacteria and viruses. “We know the value in expanding surveillance to all pathogens since we’ve been doing this for many years as retrospective academic studies. It is however a herculean effort at enormous cost, but eventually we will get there.  Nosocomial infections are the next obvious low hanging fruit, particularly pathogens such as Klebsiella pneumonia which is increasingly multi-drug resistant,” he said.

In the US, meanwhile, the Advanced Molecular Detection (AMD) program has been promoting the integration of modern laboratory and computing technologies into infectious disease surveillance, investigation, and response throughout the public health system, including CDC and state and local health departments. The program has focused on building capacity for next-generation sequencing and bioinformatics into existing public health programs, such as those dedicated to preventing foodborne disease outbreaks, tuberculosis transmission, antibiotic-resistant infections, vaccine-preventable diseases, and many others.

Duncan MacCannell
Duncan MacCannell, CSO, Office of Advanced Molecular Detection, CDC

“One of the most exciting and yet challenging aspects of AMD is the ever-expanding technology horizon,” said Duncan MacCannell, CSO for CDC’s Office of Advanced Molecular Detection. “Nanopore sequencing and cloud computing have only became feasible options for public health since the AMD program began. Newer developments will offer opportunities as well as challenges for public health.”

Next-generation sequencing and computing to support bioinformatics analysis of sequence data are the core technologies of AMD, he says. The number of state public health laboratories with sequencing capacity increased from just 11 in 2013 to all 50 in 2020.

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