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Detection of 16S rRNA gene for rapid identification of bacterial pathogens causing peritonitis in patients on
C Sheela Devi1, P Vivian Joseph Ratnam1, S R Ramya1
1Department of Microbiology, Pondicherry Institute of Medical Sciences, Kalapet, Puducherry - 605014, India.
Purpose:
Peritonitis is the most important complication with high rate of morbidity and mortality in patients on continuous ambulatory peritoneal dialysis (CAPD) despite the success and advances. Rapid and accurate identification of pathogens causing peritonitis in a CAPD patient is essential for early targeted treatment. The aim of the study was to evaluate the role of 16S rRNA gene and ITS region PCR and sequencing in detecting bacterial and fungal pathogens from the dialysate of patients undergoing CAPD.
Methods:
Fifty eight peritoneal dialysate from suspected cases of peritonitis on CAPD were subjected to conventional culture as per the ISPD guidelines and automated culture system. A conventional PCR was performed to detect the 16S rRNA gene and ITS region. Sequencing and analysis were performed to identify the etiological agent from the remaining dialysate.
Results:
Among the 58 dialysate fluid, the etiological agents were identified in 8(14%) samples by conventional culture, 28(48%) by automated culture and 47(81%) by 16S rRNA sequencing and analysis. In 8 samples there was discordance in the results of the culture and 16S rRNA PCR. BLAST search of nine sequences obtained from 16S rRNA PCR revealed that these sequences matched best with uncultured bacterial clones. In eleven samples the sequence failed.
Conclusion:
The molecular tool 16S rRNA gene and ITS region PCR and sequencing cannot be used as a standalone test as it lacks sensitivity to identify some bacterial species due to high genetic similarity in some cases and inadequate database in GenBank. However, it could be used as a supplementary test to the culture method especially in the diagnosis of culture negative peritonitis.
Insights
16S rRNA gene and ITS region PCR sequencing shows promise for diagnosing peritonitis in continuous ambulatory peritoneal dialysis (CAPD) patients. While not a standalone test, it aids in identifying pathogens, especially in culture-negative cases.
Area of Science:
- Medical Microbiology
- Molecular Diagnostics
Background:
- Peritonitis is a critical complication in continuous ambulatory peritoneal dialysis (CAPD) patients, leading to significant morbidity and mortality.
- Timely and precise pathogen identification is crucial for effective, targeted peritonitis treatment in CAPD.
Purpose of the Study:
- To assess the utility of 16S rRNA gene and ITS region PCR and sequencing for detecting bacterial and fungal pathogens in CAPD peritonitis dialysate.
- To compare the diagnostic yield of molecular methods with conventional and automated culture techniques.
Main Methods:
- Fifty-eight peritoneal dialysate samples from suspected CAPD peritonitis cases were analyzed.
- Conventional and automated culture methods were employed alongside 16S rRNA gene and ITS region PCR and sequencing.
- Sequencing and BLAST analysis were performed for pathogen identification.
Main Results:
- Molecular methods identified etiological agents in 81% of samples, significantly higher than automated (48%) and conventional (14%) cultures.
- Discordant results between culture and PCR were observed in 8 samples.
- BLAST searches revealed some 16S rRNA sequences matched uncultured bacterial clones, and 11 sequencing attempts failed.
Conclusions:
- 16S rRNA gene and ITS region PCR and sequencing is a valuable supplementary diagnostic tool for CAPD peritonitis.
- It is particularly useful for diagnosing culture-negative peritonitis but cannot replace culture methods as a standalone test.
- Limitations include sensitivity issues due to genetic similarity and database inadequacies.
