AMPLICON-based sequencing may help improve the diagnosis of severe pneumonia in intensive care units (ICUs), according to new research suggesting the technique can uncover pathogens missed by standard tests. Severe pneumonia is a life-threatening lung infection that commonly affects critically ill patients and is especially difficult to diagnose accurately in those who are immunosuppressed.
The study assessed whether amplicon-based sequencing could add clinically useful information to routine microbiological testing in ICU patients with suspected severe pneumonia.
Why Severe Pneumonia Diagnosis Is So Challenging
In ICU settings, identifying the cause of severe pneumonia is often complicated by prior antibiotic use, low pathogen loads and overlapping clinical features. Standard of care testing typically relies on microbial cultures, targeted PCR tests and antigen detection, but these approaches can fail to detect mixed infections or unexpected organisms.
Amplicon-based sequencing offers a broader, culture-independent approach. It detects bacterial DNA through 16S rRNA sequencing and fungal DNA via internal transcribed spacer (ITS) sequencing, allowing simultaneous screening for multiple pathogens.
Comparing Amplicon-Based Sequencing with Standard Tests
Researchers analysed bronchoalveolar lavage samples from 24 ICU patients with severe pneumonia and 12 lung transplant recipients without infection. Results from amplicon-based sequencing were compared with standard diagnostic methods.
Overall, there was complete agreement between the two approaches in just over half of samples. Partial agreement was common for bacterial results, while full disagreement occurred in a minority of cases, mainly involving gram-negative bacteria. These discrepancies related to either how much bacteria were present or difficulties identifying organisms at species level.
Strong Performance for Fungal Pneumonia
The most consistent findings were seen with fungal ITS sequencing. Only one fungal result differed from standard testing, suggesting high reliability. Importantly, semi-quantitative ITS results helped clinicians distinguish true Pneumocystis jirovecii pneumonia from simple colonisation – a key challenge in ICU practice.
The study also found good correlation between fungal sequencing results and galactomannan testing, supporting the clinical relevance of the sequencing data.
Added Value in Immunosuppressed Patients
Amplicon-based sequencing appeared particularly useful in immunosuppressed patients, where infections are often complex and atypical. In several cases, the method identified additional pathogens not detected by routine tests, including co-infections and bacterial superinfections following viral pneumonia.
The authors caution that limitations remain, especially for bacterial identification using 16S rRNA sequencing. However, they conclude that amplicon-based sequencing could support severe pneumonia diagnosis when standard tests are inconclusive, particularly for suspected fungal disease, helping guide more targeted treatment decisions without overstating causality.
Reference
Michel C et al. Amplicon-based sequencing as a diagnostic tool for severe pneumonia in the ICU. Sci Rep. 2026;16(1):2845.
