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Targeted Next-Generation Sequencing Takes On Growing Role in Pathogen Detection

Earth is now home to some 7.2 billion people, but in and around each individual are an estimated 100 trillion bacteria, viruses and other microorganisms. These variable species are responsible for many human ailments, ranging from mild and asymptomatic, to fatal infections. Re-emergence of the deadly Ebola virus in West Africa early last year is the most recent high-profile example. However, less publicized infections, such as bacterial sepsis, continue to claim hundreds of thousands of lives each year and drain billions of healthcare dollars.

The impact can be partially attributed to the methods historically used to root out these pathogens. In the past, scientists identified bacterial species by isolating and growing them in culture, a sometimes unreliable process. Since most require different environments for optimized growth, only a few microbial species can be detected at one time. Perhaps the greatest limitation to this approach is the time it takes for cultures to generate results. Doctors who are treating patients with a critical infection must wait at least two days or more for information – time that can mean the difference between life and death. This is precisely why many clinicians begin treating their patients with antibiotics before results are available. It’s a hit-or-miss approach, as the prescribed treatment may not have an effect on the bacterial strain present. Furthermore, if results eventually point to a virus, the battery of antibiotics will have been prescribed in vain, as they don’t have any effect on viral infections.

Pathogen detection is an emerging application where targeted, next-generation sequencing (NGS) holds great potential. Not only can it quickly identify all species in a given sample and guide targeted treatments, it can also be useful to track the evolution of microorganisms, allowing public health officials to develop proactive strategies to help them stay ahead of deadly outbreaks.

NGS in the field

The 2014-2015 Ebola outbreak in West Africa served as a sobering reminder of the ongoing threat that viruses pose. The deployment of NGS platforms, complemented by traditional qPCR instruments, by several groups is demonstrative of the valuable data that can be rapidly generated to better understand the molecular nature of a deadly pathogen.

While infection rates have peaked, the virus has persisted in Sierra Leone. To bring much needed assistance and public health vigilance to that region, Thermo Fisher Scientific embarked on a collaborative effort with the University of Cambridge, Wellcome Trust Sanger Institute, Public Health England, International Medical Corps, Ministry of Health and Sanitation Sierra Leone. The goal is to leverage NGS to monitor mutations in the virus and upload that data to the web in real-time. Until now, researchers had to wait for the data to be published in peer-reviewed journals. This approach is designed to expedite research among the global scientific community, potentially delivering solutions faster. The team in Sierra Leone has since released the first data set, enabling researchers to begin developing a deeper understanding of where and how the virus is spreading. With improved surveillance, progress may be made toward preventing future outbreaks.

Speed to results is key in pathogen detection applications and it’s an area where targeted NGS can deliver. However, access to a large body of genomic data that supports bacterial and viral identification is also critical. To date, millions of human genes have been sequenced, which provide a point of reference for scientists to routinely isolate mutations and pinpoint their contribution to disease. Microorganisms, on the other hand, remain elusive. Some have never been sequenced. For those that are, it can be extremely difficult to cross-reference microbe databases to find the species of interest. Until recently, much of this process was performed manually.

Central database key to effective mitigation

Rapid microbial identification is an invaluable tool for public health officials as they track infectious disease outbreaks throughout the world. The challenge has been in building the curated databases that allow identification and real-time microbe monitoring. To address this need, the Centers for Disease Control and Prevention (CDC) have developed MicrobeNet, an online library of known microbial sequences. In 2015, the CDC adopted MicrobeBridge, a software platform designed to seamlessly connect sequencing results to the CDC’s database.

The novel tool enables researchers to more easily access the public health information stored in MicrobeNet. It simultaneously informs laboratory-based surveillance and improves the general understanding of disease outbreaks with the ability to cross-reference data. Through MicrobeNet and similar initiatives, including the Human Microbiome Project, science can now make greater strides applying genomics to pathogen detection and mitigation.

Blood-borne pathogens

Diseases such as lung cancer remain in the crosshairs of research scientists and oncologists alike, and therapeutic advancements are being made. Yet in the United States, more people die each year from a largely unknown condition called sepsis. Triggered by a bacterial or fungal infection of the blood, sepsis mortality can be as high as 30 percent in certain patient demographics. Each hour that passes without treatment increases mortality by 8 percent; yet traditional culture-based methods to identify the causal organism and determine drug susceptibility can take at least two days or longer.

Using targeted NGS technology, researchers can isolate and characterize a pathogen directly from blood, bypassing culture steps and delivering results in less than one working day. Blood-based sequencing definitively identifies microbe species and can provide an indirect phenotype characterization to inform treatment decisions. The presence or absence of virulence factors, for example, can provide information about how easily the virus spreads, and certain genes can also help predict drug susceptibility.

Scientists are beginning to maximize the potential of targeted NGS to scale up and characterize multiple species in tandem. Researchers at the University of Arizona Cancer Center have drawn on this strength to classify unknown bacteria strains swabbed from diabetic foot ulcers. In any particular patient, multiple bacteria communities may be contributing to disease progression, which could explain why some wounds fail to heal. With NGS technology, researchers can clearly distinguish between the populations by uncovering the small snippets of ribonucleic acid (RNA) unique to each microbial species. Once again, the sequencing data can help guide a targeted and more effective treatment.

While targeted NGS has proven to be an effective and still-evolving tool in the field of cancer genomics, science is starting to realize its potential in the field of pathogen detection. Access to real-time sequencing data is currently driving mitigation strategies in even more recent global cases, such as the Middle East respiratory syndrome (MERS) and avian influenza virus (AIV) outbreaks. As in cancer, which has benefited from vast amount of genomic data collated through years of research, the field of pathogen detection will strengthen as central databases continue to build and inform public health labs with critical data to help identify and characterize the microorganisms around us. With an estimated 100 trillion of them in our midst, we have our work cut out for us.

Source: Bioscience Technology

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