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Rapid isolation and diagnosis of live bacteria from human joint fluids by using an integrated microfluidic system
Wen-Hsin Chang1, Chih-Hung Wang, Sung-Yi Yang
1Department of Power Mechanical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan. gwobin@pme.nthu.edu.tw.
Abstract:
Arthroplasty is a general approach for improving the life quality for patients with degenerative or injured joints. However, post-surgery complications including periprosthetic joint infection (PJI) poses a serious drawback to the procedure. Several methods are available for diagnosing PJI, but they are time-consuming or have poor sensitivity and specificity. Alternatively, reverse-transcription PCR can detect live bacteria and reduce false-positive results but cannot avoid the cumbersome RNA handling and human contamination issues. In response, an integrated microfluidic system capable of detecting live bacteria from clinical PJI samples within 55 minutes is developed in this study. This system employs an ethidium monoazide (EMA)-based assay and a PCR with universal bacterial primers and probes to isolate and detect only the live bacteria that commonly cause PJI. The experimental results indicated that the developed system can detect bacteria in human joint fluids with a detection limit of 10(4) colony formation unit mL(-1). Furthermore, nine clinical samples were analyzed using the microfluidic system. The results obtained from the microfluidic system were negative for all culture-negative cases, indicating that the proposed system can indeed reduce false-positive results. In addition, experimental results showed that the EMA sample pre-treatment process was crucial for successful detection of live bacteria. The culture-positive cases were diagnosed as positive by the proposed system only when the clinical samples were treated with EMA immediately after being sampled from patients. Based on these promising results, the developed microfluidic system can be a useful tool to detect PJI and potentially be applied in other clinical situations.
Insights
A new microfluidic system rapidly detects live bacteria causing periprosthetic joint infection (PJI) using an ethidium monoazide (EMA) assay. This innovative approach significantly reduces false positives and improves diagnostic speed for PJI.
Area of Science:
- Biomedical Engineering
- Infectious Disease Diagnostics
- Microfluidics
Background:
- Periprosthetic joint infection (PJI) is a severe complication following arthroplasty, impacting patient quality of life.
- Current PJI diagnostic methods often suffer from long turnaround times, low sensitivity, or specificity issues.
- Existing molecular techniques like RT-PCR for live bacteria detection involve complex sample handling and contamination risks.
Purpose of the Study:
- To develop an integrated microfluidic system for rapid and accurate detection of live bacteria in clinical PJI samples.
- To improve upon existing diagnostic methods by reducing false positives and simplifying the detection process.
- To validate the efficacy of an ethidium monoazide (EMA)-based assay within a microfluidic platform for PJI diagnosis.
Main Methods:
- An integrated microfluidic system was designed, incorporating an ethidium monoazide (EMA) assay and PCR with universal bacterial primers.
- The system isolates and detects only live bacteria by targeting DNA from viable organisms.
- Clinical joint fluid samples were analyzed, with and without EMA pre-treatment, to assess system performance.
Main Results:
- The microfluidic system demonstrated a detection limit of 10(4) CFU/mL for bacteria in human joint fluids.
- Analysis of nine clinical samples showed negative results for all culture-negative cases, confirming reduced false positives.
- EMA pre-treatment was identified as crucial for accurate live bacterial detection, particularly for culture-positive samples.
Conclusions:
- The developed microfluidic system offers a rapid (within 55 minutes) and effective tool for diagnosing PJI by detecting live bacteria.
- The EMA-based assay integrated into the microfluidic platform significantly enhances diagnostic accuracy and reduces false-positive results.
- This technology holds potential for broader clinical applications in infectious disease diagnostics beyond PJI.
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