A standard negative test result may offer false reassurance when a patient is actually infected with a pathogen the test was never designed to find. That is the central finding of a new study from researchers at Lawrence Livermore National Laboratory (LLNL) and the California Department of Public Health, published in Scientific Reports, which demonstrates how an untargeted sequencing method can identify dangerous respiratory pathogens lurking in samples that conventional diagnostic panels have cleared.
The study arrives at a time when public health systems are under sustained pressure to improve early warning capabilities for novel and emerging infectious diseases. Its findings highlight a structural limitation in the diagnostic tools that form the backbone of clinical and surveillance testing across the United States.
The Core Problem With Targeted Testing
Polymerase chain reaction, or PCR, became a household term during the COVID-19 pandemic. As a type of nucleic acid amplification test (NAAT), PCR works by detecting and amplifying specific genetic sequences from a known pathogen. It is fast, relatively inexpensive, and highly accurate for what it is designed to find. But that precision is also its weakness: if a pathogen is not on the test’s list, the test cannot detect it.
“If a patient is infected with a life-threatening pathogen that is not covered by these tests, then this would cause a serious health risk,” said LLNL scientist Crystal Jaing, an author on the study. “Pathogen-specific assays do not provide comprehensive coverage of a broad spectrum of pathogens, while pathogen-agnostic tests provide much more information.”
The study examined respiratory samples drawn from a sentinel surveillance program that collected specimens from patients with respiratory symptoms across multiple California counties. The samples were first tested against a standard NAAT-based panel covering 22 pathogens, including SARS-CoV-2. The majority returned negative results.
Hidden Pathogens in “Negative” Samples
The researchers then applied untargeted metagenomic sequencing to those negative samples. Unlike PCR, this approach reads all genetic material present in a sample, then compares the results against a broad reference database to identify what organisms are present, known or otherwise.
The results were striking. Five percent of the samples that tested negative on the standard panel contained a different, previously undetected respiratory virus. Additional cases revealed the presence of bacterial or fungal species that the NAAT panel had also missed.
“Pathogen-agnostic tests provide the advantage that you don’t have to know what pathogens are in a sample,” Jaing said. “Using the agnostic approach, you can detect any or all pathogens in a sample with a single test.”
A Complement to Standard Testing, Not a Replacement
The research team is careful not to overstate the case for replacing existing diagnostic infrastructure. Metagenomic sequencing requires more sophisticated equipment, takes longer to process, and costs significantly more than conventional NAATs. For routine clinical diagnosis, PCR and similar tools remain the practical standard.
“NAAT can be used for routine tests because they are faster and cheaper,” Jaing said. “However, NAAT is not sufficient for surveillance of new and emerging pathogens and for future pandemic forecasting.”
The authors argue instead for a tiered approach: standard NAATs for frontline clinical use, with pathogen-agnostic methods integrated into surveillance systems designed to detect novel threats before they reach pandemic scale.
Integrating metagenomic sequencing into sentinel surveillance programs would give public health agencies a broader and more sensitive net for catching emerging pathogens, including those not yet characterized or named. The researchers express hope that their findings will prompt public health entities at the state and federal level to evaluate how pathogen-agnostic approaches can be incorporated into existing detection and surveillance frameworks.
Sources and further reading:
Pathogen-agnostic testing reveals hidden respiratory threats in negative samples – LLNL
Metagenomic sequencing identifies potential respiratory pathogens in PCR-negative subset of surveillance samples. Scientific Reports, February 16, 2026