In the endless war between humans and microbes, the discovery of a new pathogen is our first line of defense.
Imagine a world where a mysterious illness emerges, spreading rapidly with doctors unable to identify the culprit—no diagnostic tests, no treatments, no vaccines. This isn't science fiction; it's a scenario that has played out repeatedly throughout history and continues to threaten global health today. The field of new microbes and new infections represents science's frontier in the battle against infectious diseases, combining cutting-edge technology with brilliant detective work to identify and characterize previously unknown microbial threats.
The human body is a vast ecosystem teeming with trillions of microorganisms—bacteria, viruses, fungi, and other microbes—collectively known as the microbiome. This complex community resides in various parts of our body, including the gut, skin, oral cavity, and respiratory tract, living with us in a delicate, usually beneficial, balance 1 7 . In healthy states, our microbiota performs essential functions like aiding digestion, producing vitamins, and protecting against pathogens through a phenomenon called colonization resistance, where our native microbes outcompete invaders for resources and space 1 .
However, this balance is fragile. When disrupted—a state known as dysbiosis—our protective microbial shield can be compromised, potentially increasing our susceptibility to infections 7 . Simultaneously, factors including globalization, climate change, and human encroachment into wildlife habitats have accelerated the spread of previously unknown pathogens from animal reservoirs to human populations 2 . These zoonotic jumps—where pathogens move from animals to humans—have been responsible for more than 70 significant infectious disease outbreaks since 1980 2 .
| Pathogen | Year Emerged | Confirmed Deaths | Key Characteristics | Scientific Challenges at Time of Emergence |
|---|---|---|---|---|
| Influenza A (H1N1) | 1918 | 50-100 million | High mortality in young adults | Limited viral knowledge, no antibiotics, no vaccines |
| HIV | ~1900 (detected 1981) | Millions ongoing | Attacks immune system, long latency | Initially unrecognized, slow scientific response |
| SARS-CoV-2 | 2019 | Over 6.37 million globally | High transmission, variable severity | Rapid development of diagnostics and vaccines needed |
For over a century, the primary method for identifying pathogens relied on cultivation-based techniques—attempting to grow microbes in laboratory settings 2 8 . While this approach successfully identified many known pathogens, it came with significant limitations. Scientists now recognize that standard laboratory methods can only cultivate about 2-3% of microbial species, leaving the vast majority of microorganisms undetectable through these traditional means 2 8 .
Reliance on growing microbes in petri dishes and culture media. Limited to organisms that can be cultivated in laboratory conditions.
Introduction of PCR and other molecular techniques allowed detection without cultivation. Enabled identification of previously "uncultivable" pathogens.
Next-generation sequencing technologies provide comprehensive analysis of all genetic material in samples, revolutionizing pathogen discovery.
Gene-editing technology combined with sequencing to improve sensitivity and specificity of pathogen detection.
The turning point came with the development of molecular detection methods that identify pathogens based on their genetic signatures rather than their ability to grow in a lab 8 . The Polymerase Chain Reaction (PCR) became a revolutionary tool, allowing scientists to amplify tiny amounts of microbial DNA or RNA to detectable levels 2 . PCR-based methods, particularly reverse transcription PCR (RT-PCR), became the gold standard for diagnosing countless infections, including COVID-19 2 .
More recently, Next-Generation Sequencing (NGS) technologies have transformed pathogen discovery by enabling scientists to sequence all genetic material in a sample without prior knowledge of what might be present 2 4 . This "shotgun sequencing" approach is particularly powerful for identifying completely novel pathogens, as it requires no pre-existing assumptions about what to look for 4 . However, NGS has traditionally faced sensitivity challenges when pathogen levels are low amidst abundant human and commensal microbial genetic material 4 .
To overcome the sensitivity limitations of standard NGS approaches, researchers have developed an innovative solution that combines CRISPR gene-editing technology with next-generation sequencing 4 . This experiment, conducted by Chan et al. and published in 2023, aimed to create a more effective method for detecting low levels of pathogens in clinical samples 4 .
Researchers collected nasopharyngeal swabs from patients, similar to those used for COVID-19 testing 4 .
Total RNA was extracted from these samples, containing genetic material from human cells, commensal bacteria, and potential pathogens 4 .
The RNA was converted to DNA and prepared for sequencing by adding adapter sequences 4 .
The innovative step involved using the CRISPR-Cas9 system programmed with guide RNAs to specifically target and remove abundant, uninformative sequences—particularly human and bacterial ribosomal RNA (rRNA) sequences that dominate clinical samples and mask the signal from low-abundance pathogens 4 .
The cleaned-up libraries were sequenced using high-throughput NGS platforms, and the resulting data was analyzed for pathogen detection 4 .
The CRISPR-enhanced NGS method demonstrated remarkable efficiency, removing 70% of unwanted ribosomal RNA sequences compared to only 52% removal by previous methods 4 . This cleanup resulted in a significant increase in useful sequencing data, allowing detection of 2.04 times more bacterial species in tested samples 4 .
Most importantly, the method achieved sensitivity for SARS-CoV-2 detection comparable to the gold-standard RT-PCR test, even in samples with very low viral loads 4 . Beyond mere detection, the approach provided additional valuable information, including variant strain typing, identification of co-infecting pathogens, and insights into the human host response to infection—all from a single test 4 .
| Method | Sensitivity | Time to Result | Additional Information Provided | Limitations |
|---|---|---|---|---|
| Microbial Cultivation | Low (only detects cultivable species) | Days to weeks | Allows antibiotic susceptibility testing | Misses uncultivable pathogens; slow |
| PCR-based Methods | High | Hours to 1 day | Specific detection of targeted pathogens | Requires prior knowledge of what to target |
| Standard NGS | Moderate | 1-2 days | Can detect unexpected or novel pathogens | Less sensitive when pathogen abundance is low |
| CRISPR-enhanced NGS | High (comparable to PCR) | 1-2 days | Strain typing, co-infections, host response | More complex workstream; higher cost |
| Metric | Standard NGS (RiboZero Plus) | CRISPR-enhanced NGS | Improvement |
|---|---|---|---|
| rRNA Removal Efficiency | 52% at 5ng RNA input | 70% at 5ng RNA input | 18% more efficient |
| Bacterial Species Detected | 269 species at 5ng input | 462 species at 5ng input | 1.72x more species detected |
| SARS-CoV-2 Detection Sensitivity | Limited at high Ct values (low virus) | Comparable to RT-PCR up to Ct 35 | Approaches gold-standard sensitivity |
| Introduced Bias (correlation coefficient) | Not reported | r² = 0.898-0.994 | Minimal bias introduced |
The revolution in pathogen discovery relies on a sophisticated array of research reagents and technologies. Here are some of the essential tools enabling scientists to detect and characterize new infectious threats:
| Tool/Reagent | Function | Application in Pathogen Discovery |
|---|---|---|
| CRISPR-Cas Systems | Gene editing technology that can be programmed to target specific DNA/RNA sequences | Selective removal of abundant host sequences to enhance pathogen detection sensitivity 4 |
| Next-Generation Sequencers | High-throughput platforms for rapid DNA/RNA sequencing | Comprehensive analysis of all genetic material in clinical samples without prior target selection 2 4 |
| Broad-Range PCR Primers | Primers targeting conserved regions across microbial groups | Amplification of unknown pathogens by targeting universal genetic signatures 8 |
| Metagenomic Analysis Software | Computational tools for analyzing complex genetic mixtures | Identification and classification of microbial sequences from host and environmental background 4 |
| λRed Recombineering System | Phage-derived proteins enabling efficient genetic recombination in bacteria | Engineering of model organisms like E. coli for studying pathogen mechanisms 5 |
| Tn6677 Transposon System | Bacterial genetic element that facilitates DNA insertion | Site-specific integration of large DNA cargos into microbial genomes for functional studies 5 |
Programmable gene editing for selective removal of host sequences to enhance pathogen detection.
High-throughput platforms for comprehensive analysis of genetic material in samples.
Target conserved regions across microbial groups to amplify unknown pathogens.
The COVID-19 pandemic served as a stark reminder of our vulnerability to emerging infectious threats, but it also accelerated innovations in pathogen detection 2 9 . The development of testing strategies deployable at "day zero"—the time of the first reported case of an outbreak—represents a critical goal for pandemic preparedness 4 . The ideal pathogen discovery platform would be pathogen-agnostic (able to detect any infectious agent without prior knowledge), highly sensitive, and capable of providing comprehensive information for public health response 4 .
As we look to the future, the integration of artificial intelligence with multi-omics data, the development of portable sequencing devices for field deployment, and enhanced global collaborative networks for data sharing will further transform our ability to detect and respond to new microbial threats 2 .
The battle against infectious diseases is ongoing, but with these powerful new tools and technologies, scientists are better equipped than ever to identify new microbes, understand their potential impact on human health, and develop strategies to prevent the next pandemic before it begins. In the endless dance between humans and microbes, knowledge remains our most powerful defense.