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Updated: May 2, 2026
Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS
Published on: July 11, 2016
Nested polymerase chain reaction (PCR) targeting 16S rDNA for bacterial identification in empyema
Background:
Empyema in children causes significant morbidity and mortality. However, identification of organisms is a major concern.
Objective:
To detect bacterial pathogens in pus specimens of children with empyema by 16S rDNA nested polymerase chain reaction (PCR) and correlate it with culture and sensitivity.
Method:
Sixty-six children admitted to the paediatric ward with a diagnosis of empyema were enrolled prospectively. Aspirated pus was subjected to cytochemical examination, culture and sensitivity, and nested PCR targeting 16S rDNA using a universal eubacterial primer.
Results:
Mean (SD) age was 5·8 (1·8) years (range 1-13). Analysis of aspirated pus demonstrated total leucocyte count >1000×10(6)/L, elevated protein (≧20 g/L) and decreased glucose (≤2·2 mmol/L) in 80·3%, 98·5% and 100%, respectively. Gram-positive cocci were detected in 29 (43·9%) and Gram-negative bacilli in two patients. Nested PCR for the presence of bacterial pathogens was positive in 50·0%, compared with 36·3% for culture.
Conclusion:
16S rDNA PCR improves rates of detection of bacteria in pleural fluid, and can detect bacterial species in a single assay as well as identifying unusual and unexpected causal agents.
Insights
16S rDNA nested PCR significantly improves bacterial detection in pediatric empyema pus specimens compared to traditional culture methods. This molecular technique enhances pathogen identification, aiding in better diagnosis and treatment of childhood empyema.
Area of Science:
- Pediatric Infectious Diseases
- Molecular Microbiology
- Clinical Diagnostics
Background:
- Childhood empyema presents significant health challenges, with difficulties in identifying causative organisms.
- Accurate and timely pathogen detection is crucial for effective management of pediatric empyema.
Purpose of the Study:
- To evaluate the efficacy of 16S ribosomal DNA (rDNA) nested polymerase chain reaction (PCR) for detecting bacterial pathogens in pediatric empyema pus.
- To compare the diagnostic yield of 16S rDNA nested PCR with conventional culture and sensitivity methods.
Main Methods:
- Prospective enrollment of 66 children diagnosed with empyema.
- Analysis of aspirated pus included cytochemical examination, bacterial culture and sensitivity testing, and 16S rDNA nested PCR using universal eubacterial primers.
Main Results:
- Nested PCR detected bacterial pathogens in 50.0% of samples, surpassing the 36.3% yield from culture.
- Conventional methods identified Gram-positive cocci in 43.9% and Gram-negative bacilli in 2 patients.
- Pleural fluid analysis showed elevated white blood cell counts, protein, and low glucose levels.
Conclusions:
- 16S rDNA nested PCR offers a superior method for detecting bacteria in pleural fluid from children with empyema.
- This molecular approach can identify a broader range of bacterial species, including unexpected or unusual pathogens, in a single assay.
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