Pathogen-Agnostic Testing Reveals Hidden Respiratory Threats in Negative Samples

PCR belongs to a group of diagnostic methods known as nucleic acid amplification tests (NAATs), which identify pathogens by detecting and amplifying their genetic material. While highly accurate, these tests can only detect pathogens that laboratories specifically look for. This limitation means infections caused by unexpected or emerging pathogens may go unnoticed. A new study now highlights how a broader, pathogen-agnostic testing approach can uncover hidden infections.

A study by scientists from Lawrence Livermore National Laboratory (LLNL, Livermore, CA, USA, in collaboration with the California Department of Public Health (CDPH, Sacramento, CA, USA), explored the use of untargeted metagenomic sequencing to identify pathogens in respiratory samples that previously tested negative using standard NAAT-based diagnostic panels. Unlike PCR testing, which targets specific genetic sequences, metagenomic sequencing analyzes all genetic material present in a sample and compares it with reference databases to identify any microorganisms present.

Researchers analyzed respiratory samples collected through a sentinel surveillance program monitoring patients with respiratory symptoms across counties in California. Many of these samples had previously tested negative for a panel of 22 pathogens, including SARS-CoV-2. Using untargeted metagenomic sequencing, the team discovered that about five percent of these negative samples actually contained different respiratory viruses that had not been included in the original testing panel. In addition, the analysis, published in Scientific Reports, identified bacterial and fungal species in several samples, revealing infections that would otherwise have gone undetected.

Although metagenomic sequencing provides a broader view of potential infections, it currently requires more time, specialized equipment, and higher costs than conventional NAAT methods. Researchers emphasize that pathogen-agnostic sequencing is not meant to replace PCR testing but rather to complement it. Routine diagnostics can continue relying on fast, targeted PCR tests, while broader sequencing approaches may play a key role in identifying emerging pathogens and strengthening disease surveillance systems. This combined strategy could help public health agencies detect new infectious threats earlier and improve preparedness for future outbreaks.

“Pathogen-specific assays do not provide comprehensive coverage of a broad spectrum of pathogens, while pathogen-agnostic tests provide much more information,” said Crystal Jaing, scientist at Lawrence Livermore National Laboratory. “Using the agnostic approach, you can detect any or all pathogens in a sample with a single test.”

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