Modern Biosurveillance Methods: A Lay Introduction to Effective Use of Nucleic Acid Methods

The devastating impact of the coronavirus disease 2019 (COVID-19) pandemic has led governments, health care institutions, and researchers to intensify their focus on developing improved means to detect, contain, and manage potential future outbreaks. As part of these efforts, it is useful for policymakers and other nontechnical personnel to understand the promise of available biosurveillance technologies, in particular nucleic acid testing for genetic markers and the technologies that support them. Biosurveillance systems are used to detect, monitor, and characterize health threats, or pathogens, in human and animal populations, food, wastewater, and the environment.

Nucleic Acid Testing Approaches

Various nucleic acid testing approaches can be used to identify if a pathogen's deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) is present in a sample. These approaches vary in terms of cost, speed, accuracy, and other features.

Pathogen-Specific Tests

Some testing approaches target a specific pathogen (or pathogens), allowing a scientist to ask, “Is pathogen X present in my sample?”:

• Polymerase chain reaction (PCR) is a rapid, accurate method to detect and copy very small amounts of genetic material, which can then be used to determine if a pathogen's DNA is present. First developed in the 1980s, PCR approaches have been enhanced in recent years.
  • Quantitative PCR (qPCR) is a variation of PCR that enables much faster results and relative quantification of the target DNA than traditional PCR.
  • Digital PCR (dPCR) enables better quantification of the nucleic acids present, especially when there are small amounts of the DNA of interest available.
• Loop-mediated isothermal amplification (LAMP). LAMP is not a PCR-based technique, but, like PCR, it uses primers and special enzymes to amplify the nucleic acid in a sample to indicate the presence of the pathogen of interest. LAMP is a versatile, inexpensive technique.

There are several advantages to PCR and LAMP. PCR techniques are widely used in research and diagnostic facilities, so the equipment and skills required to perform these tests are common. LAMP offers some advantages with respect to establishing nucleic acid detection capabilities in facilities with minimal scientific staff and equipment.

Pathogen-Agnostic Tests

Other forms of nucleic acid testing are “pathogen-agnostic” (or “unbiased”), which means that the scientist can use these techniques to discover the genetic material present, without first having a list of suspected pathogens in mind. Currently, pathogen-agnostic nucleic acid techniques involve next generation sequencing (NGS). Nucleic acid sequencing is a process that determines the exact composition of the nucleic acids in a sample. Historically, sequencing methods used primers and were therefore only useful if the researcher had some information about the pathogens that might be present. However, the latest nucleic acid sequencing methods, NGS methods, are pathogen-agnostic and enable researchers to sequence all the nucleic acids found in the sample simultaneously. The final step of NGS methods involves advanced data analysis techniques that can search the sequences found in the sample against known genomic sequences to lead to identification of the genomes present in the original sample. NGS techniques allow scientists to ask, “What pathogens are present in my sample?”:

• Illumina sequencing is a type of “short read” sequencing technology developed and sold by Illumina, Inc.; this means that it is only capable of producing very short fragments of a sequence and relies heavily on data analysis to generate complete sequences. Many parts of this method are starting to lose patent protections and we may begin to see alternatives using this basic chemistry hit the market under new names.
• Nanopore sequencing, such as the systems being developed and sold by Oxford Nanopore Technologies, enables “very long read” sequencing, which greatly simplifies the assembly process. Nanopore sequencing uses specialized flow cells that contain numerous nanopores—that is, microscopic holes that can be monitored for changes in the electric current.

One known issue with NGS on complex mixtures of nucleic acids is that species present in smaller amounts may be missed. In these cases, target enrichment can be used. Target-enrichment sequencing involves enriching the sample for a specific agent or type of agent and can be used with either Illumina or nanopore sequencing. Target enrichment can occur using PCR-like amplification of targets or through selective purification techniques to try to eliminate other contaminating species (e.g., filtering out larger bacterial species to study the viruses present). Target enrichment prioritizes the detection of previously identified pathogens or classes of pathogen over new or unexpected microbes. Target enrichment is useful if a large number of other, less relevant, microbes are expected in the sample; however, it does narrow the aperture on the pathogens that can be identified from the enriched sample.

The major advantage of using any NGS approach is that, because NGS enables researchers to detect and identify whichever species are present in the sample, it is capable of identifying truly novel agents, novel variants of known agents, and unexpected agents. Additionally, NGS can provide more information about the pathogen beyond its presence in the sample. For example, NGS can indicate whether a pathogenic bacterium contains known antibiotic resistance genes that may affect the clinical or public health response.

However, in samples with many microbes and therefore a large mixture of DNA sequences, PCR and LAMP technologies are more sensitive than NGS in detecting agents that are not present in high concentrations. In addition, NGS can be complicated to use. Not every laboratory has the hardware to run the sequencing, limiting its accessibility and prolonging the time needed to obtain results. Also, detecting an microbe's genome in a clinical sample does not necessarily imply causation of disease, which is important to keep in mind if a novel agent is detected. NGS can also be limited by characteristics of the sample; samples teeming with microbes, for example, require additional processing.

Metagenomic Analysis Using Next Generation Sequencing

NGS can be used to support metagenomic analysis, which uses advanced computing to simultaneously analyze genomic sequences from multiple species in a single sample. Metagenomics involves sequencing the nucleic acids from the mixed sample to identify the genetic sequences that are present and assembling the sequences into genomes to determine which species are present.

In recent years, the cost of sequencing a full genome has been driven down significantly because of the high data output from NGS technologies. For these reasons, metagenomic studies are now almost exclusively performed using NGS. However, metagenomic sequencing approaches have some important limitations, including longer time requirements for analysis, difficulty in characterizing novel agents as potential threats, and dilution of smaller genomes by larger genomes. Thus, strong experimental design—which takes into account the research questions of interest and any resource constraints (e.g., time, funding)—is critical to obtaining the best-quality data when choosing a biosurveillance approach.

Selecting a Technology for Wastewater Biosurveillance

We examined a scenario involving testing for pathogens in wastewater using biosurveillance technology. Wastewater offers a method for collecting samples without individuals presenting in clinics for care and thus can offer data prior to symptoms occurring and from those who have lower access to clinical care. During the COVID-19 pandemic, this field showed promise in identifying hotspots for COVID-19 outbreaks before other traditional epidemiological indicators.

Policymakers interested in establishing a wastewater biosurveillance (WBS) program should evaluate their specific research questions and their resource limitations to determine which technology is best suited for their needs. Selecting a single technology that can accomplish all biosurveillance goals is not necessary. Table 1 provides some information and guidance toward selecting the best technique; however, relevant stakeholders, including laboratory personnel and wastewater utilities, should be included in these discussions.

What is your goal?	Technologies Available	Speed (time from collection to analyzed data)	Restrictions
Identifying whether known agents are present in wastewater	Illumina NGS	Slow	Target enrichment may be required prior to sequencing for reliable detection of pathogens.
	Nanopore	Slow
	PCR	Fast	Agent mutation may reduce or eliminate efficacy of the test.
	LAMP	Very fast
Monitoring a known agent for mutations	Illumina NGS	Slow	Best-use case involves PCR-based amplification for target enrichment prior to sequencing.
	Nanopore	Slow
Monitoring a known agent for levels of agent in the population	Illumina NGS	Slow	Results are less quantifiable than PCR/LAMP, especially in cases with target enrichment prior to sequencing.
	Nanopore	Slow
	PCR	Fast	Agent mutation may reduce or eliminate test efficacy.
	LAMP	Very fast
Identifying novel agents that are present in wastewater	Illumina NGS	Slow	Target enrichment may be required prior to sequencing for reliable detection of pathogens; however, these methods do limit the scope of the resulting analysis.
	Nanopore	Slow
Identifying unexpected agents that are present in wastewater	Illumina NGS	Slow	Target enrichment may be required prior to sequencing for reliable detection of pathogens.
	Nanopore	Slow
	PCR	Fast	Large numbers of individual reactions will be needed to accomplish this goal. Agents not specifically tested for will be missed.
	LAMP	Very fast	Large numbers of individual reactions will be needed to accomplish this goal. Agents not included in the test will be missed. Speed may not be the same for all agents; only those with tests that work on raw sewage will be very fast.