Rapid isolation and diagnosis of live bacteria from human joint fluids by using an integrated microfluidic system

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.

Vancomycin-resistant gene identification from live bacteria on an integrated microfluidic system by using low temperature lysis and loop-mediated isothermal amplification

“…Then, the sample was first treated with 1 ll of 1 mg/ml EMA (Molecular Probes V R , USA) in the reaction chamber and exposed under visible light (18 W, 1200 lm) for 5 min (Figure 1(a)). According to our previous study (Chang et al (2014)), EMA can distinguish live bacteria from dead ones after 5-min of light exposure. EMA can be employed to distinguish between live and dead bacteria as it can penetrate into the broken cell walls of dead bacteria and intercalate into DNA irreversibly (Chang et al (2014);; and Liu et al (2012)).…”

“…According to our previous study (Chang et al (2014)), EMA can distinguish live bacteria from dead ones after 5-min of light exposure. EMA can be employed to distinguish between live and dead bacteria as it can penetrate into the broken cell walls of dead bacteria and intercalate into DNA irreversibly (Chang et al (2014);; and Liu et al (2012)). Then, reagents for low temperature chemical lysis (detail information for low temperature chemical lysis was described in Section II D) and 5 ll of 16S ribosomal ribonucleic acid (16S rRNA) nucleotide probecoated magnetic beads (7 Â 10 5 beads/ll) (Bergin et al (2010)) were added into the reaction chamber ( Figure 1(b)).…”

“…The dimensions of the microfluidic chip were measured to be 65 mm  88 mm  7 mm. The detail information about the working principle and performance of the normally closed micro-valves and micropumps used in the study can be found in our previous works (Weng et al (2011) and Chang et al (2014)). Please note that entire detection procedures including bio-sample and reagents loading, microfluidic chip loading, real-time fluorescence detection, and fluorescent signal analysis and diagnosis can be completed automatically on the microfluidic control and detection system by just one single touch (Figure 2(c)).…”

“…Not only does it increase medical cost and prolong the treatment period, but it also might give rise to a new class of antibiotic-resistant bacteria. Previously, our group has developed an integrated microfluidic systems for live bacteria detection by using vancomycin-coated magnetic beads for sample pre-treatment, ethidium monoazide (EMA) for live or dead bacteria distinction, and PCR or nanogold probes for gene identification (Chang et al (2014(Chang et al ( ), (2015; and Liu et al (2012)) with detection limits of about 10 2 colony formation units (CFUs) per reaction. However, these systems cannot be used to detect VRE because vancomycin-coated magnetic beads have no affinity to VRE due to the mutated peptides on the cell wall (Cetinkaya et al (2000)).…”

Advances in microfluidics in combating infectious diseases

“…Making use of mannose binding lectin (MBL) coated magnetic beads and subsequent magnetic flux concentrator, Cooper et al were able to spread captured Candida (C.) albicans fungi (99%) thinly to optimize optical imaging (Cooper et al, 2014). Wang et al argued that it is also important to differentiate live from dead bacteria to prevent unnecessary administration of antibiotics (Chang et al, 2014). The group thus designed a microfluidic system based on ethidium monoazide-based assay and PCR to probe for live bacteria from fluid isolated from periprosthetic joint infection.…”