Blood culture-free ultra-rapid antimicrobial susceptibility testing.

Treatment assessment and patient outcome for sepsis depend predominantly on the timely administration of appropriate antibiotics1-3. However, the clinical protocols used to stratify and select patient-specific optimal therapy are extremely slow4. In particular, the major hurdle in performing rapid antimicrobial susceptibility testing (AST) remains in the lengthy blood culture procedure, which has long been considered unavoidable due to the limited number of pathogens present in the patient's blood. Here we describe an ultra-rapid AST method that bypasses the need for traditional blood culture, thereby demonstrating potential to reduce the turnaround time of reporting drug susceptibility profiles by more than 40-60 h compared with hospital AST workflows. Introducing a synthetic beta-2-glycoprotein I peptide, a broad range of microbial pathogens are selectively recovered from whole blood, subjected to species identification or instantly proliferated and phenotypically evaluated for various drug conditions using a low-inoculum AST chip. The platform was clinically evaluated by the enrolment of 190 hospitalized patients suspected of having infection, achieving 100% match in species identification. Among the eight positive cases, six clinical isolates were retrospectively tested for AST showing an overall categorical agreement of 94.90% with an average theoretical turnaround time of 13 ± 2.53 h starting from initial blood processing.

Prospective evaluation of a rapid antimicrobial susceptibility test (QMAC-dRAST) for selecting optimal targeted antibiotics in positive blood culture.

OBJECTIVES: MALDI-TOF MS has been successfully used for empirical antibiotic selection. However, limited data are available regarding the usefulness of MALDI-TOF MS in common resistant organisms compared with rapid antimicrobial susceptibility testing (AST). We prospectively evaluated the usefulness of rapid AST, compared with MALDI-TOF MS, for optimal antibiotic selection by infectious disease (ID) physicians in patients with bacteraemia including polymicrobial infection. METHODS: Three hundred and fifty-nine patients with positive blood culture were included for analysis. ID physicians prospectively decided on antibiotic regimens with consensus at each timepoint of receiving results of Gram stain, MALDI-TOF MS and rapid AST, the last of which was performed using QMAC-dRAST. RESULTS: ID physicians with MALDI-TOF MS results chose optimal targeted antibiotics in 255 (71.0%) cases, with appropriate antibiotic selection in 303 (84.4%) cases. The proportion of optimal targeted antibiotic selection and appropriate antibiotic selection was significantly lower for resistant strains than for susceptible strains [62.5% versus 79.2% (P < 0.001) and 68.2% versus 100% (P < 0.001), respectively]. QMAC-dRAST results led to optimal antibiotic treatment in 95 (91.3%) of the 104 cases receiving non-optimal targeted antibiotics. Optimal targeted treatments based on QMAC-dRAST results were possible in 322 (98.2%) of the 328 cases with monobacterial infection and in 345 (96.1%) of the 359 cases with monobacterial and polymicrobial infection. CONCLUSIONS: MALDI-TOF MS has a high chance of failure in guiding ID physicians to optimal antibiotics, especially against resistant organisms. With increasingly common resistant organisms, rapid AST is needed to identify optimal targeted antibiotics early in bacteraemia.

Fine-tuned grayscale optofluidic maskless lithography for three-dimensional freeform shape microstructure fabrication.

This article presents free-floating three-dimensional (3D) microstructure fabrication in a microfluidic channel using direct fine-tuned grayscale image lithography. The image is designed as a freeform shape and is composed of gray shades as light-absorbing features. Gray shade levels are modulated through multiple reflections of light in a digital micromirror device (DMD) to produce different height formations. Whereas conventional photolithography has several limitations in producing grayscale colors on photomask features, our method focuses on a maskless, single-shot process for fabrication of freeform 3D micro-scale shapes. The fine-tuned gray image is designed using an 8-bit grayscale color; thus, each pixel is capable of displaying 256 gray shades. The pattern of the UV light reflecting on the DMD is transferred to a photocurable resin flowing through a microfluidic channel. Here, we demonstrate diverse free-floating 3D microstructure fabrication using fine-tuned grayscale image lithography. Additionally, we produce polymeric microstructures with locally embedded gray encoding patterns, such as grayscale-encoded microtags. This functional microstructure can be applied to a biophysical detection system combined with 3D microstructures. This method would be suitable for fabricating 3D microstructures that have a specific morphology to be used for particular biological or medical applications.

Fabrication of membrane-type microvalves in rectangular microfluidic channels via seal photopolymerization.

Rectangular fluidic channels have rarely been used in microfluidic devices which use PDMS membrane-type microvalves, since the rectangular channel shape does not perfectly match the round shape of the membrane deformation. We present a polymer sealing method to fabricate PDMS membrane-type microvalves for rectangular microchannels. After fabricating the microfluidic device, photocurable oligomer is introduced into the fluidic channel and gas pressure is applied to the pneumatic channel to deform the membrane. The polymer seal is then locally polymerized by photolithography producing a structure matching the shape of the deformed membrane curvature. We compare the flow leakage between the membrane-type microvalve with and without a polymer seal. We also demonstrate a micropump and droplet generator using this embedded polymer membrane-type microvalve in a rectangular microfluidic channel. This polymeric seal technique enables the use of easily fabricated rectangular channel membrane microvalves with all the functionality of their curved channel counterparts with negligible flow leakage.

Hierarchical shape-by-shape assembly of microparticles for micrometer-scale viral delivery of two different genes.

Understanding tissue engineering using a bottom-up approach has been hindered by technical limitations because no platform can demonstrate the controlled formation of a heterogeneous population of cells in microscale. Here, we demonstrate hierarchical shape-by-shape assembly of virus-laden particles into larger ones to transfect two different genes on the seeded cells. We show that smaller daughter particles with different sizes and shapes can be assembled into the matching indentations of larger parent particles with different sizes and shapes. Then, we transfected a population of cells with two different gene-transfecting viruses, each of which was laden on the parent or daughter particles.

Direct rapid antibiotic susceptibility test (dRAST) for blood culture and its potential usefulness in clinical practice.

PURPOSE: The direct rapid antibiotic susceptibility test (dRAST), based on analysing changes in bacterial micro-colonies under antibiotic conditions, detects antibiotic resistance within 6 h of direct smear examination results. This study aimed to assess the accuracy of dRAST and evaluate its potential usefulness for improving selection of appropriate antibiotic in real clinical practice settings. METHODOLOGY: We evaluated the accuracy of dRAST by comparing the antibiotic treatments that should have been administered based on dRAST results and the broth microdilution (BMD) test and its potential usefulness via simulation. RESULT: For 49/52 (94.2 %) patients with Gram-positive bacteraemia and 66/67 (98.5 %) patients with Gram-negative bacteraemia, antibiotics indicated by dRAST results were the same as those indicated by the BMD test. Among 34 patients with ineffective and suboptimal treatment, 19 (55.9 %) of patients could have received optimal treatment 1 to 2 days earlier with dRAST results. Among 33 patients given unnecessary broad-spectrum antibiotics, 1 to 2 days earlier de-escalation could have been possible for 27 (81.8 %) patients based on dRAST results. CONCLUSION: The introduction of dRAST could increase the use of optimal antibiotics and reduce unnecessary broad-spectrum antibiotic use in the early period of bacteraemia.

An encoded viral micropatch for multiplex cell-based assays through localized gene delivery.

The increasing number of potential drug targets and compounds has led to the development of high-throughput cell-based assays. Simultaneous processing of multiple targets in the same experiment based on localized target gene expression is a very efficient strategy for this purpose. To address this need, we present an adenoviral vector-immobilized microparticle with two-dimensional (2D) shape-encoding properties that allows localized patch-like gene delivery to monolayer-cultured cells. This format conveniently achieves multiplexed gene delivery compatible with both high-throughput cellular assays and fluorescence high-content imaging instruments. A multiplex G protein-coupled receptor (GPCR) internalization assay was developed to demonstrate the compatibility of this system with high-throughput image-based cellular assays.

Direct, rapid antimicrobial susceptibility test from positive blood cultures based on microscopic imaging analysis.

For the timely treatment of patients with infections in bloodstream and cerebrospinal fluid, a rapid antimicrobial susceptibility test (AST) is urgently needed. Here, we describe a direct and rapid antimicrobial susceptibility testing (dRAST) system, which can determine the antimicrobial susceptibility of bacteria from a positive blood culture bottle (PBCB) in six hours. The positive blood culture sample is directly mixed with agarose and inoculated into a micropatterned plastic microchip with lyophilized antibiotic agents. Using microscopic detection of bacterial colony formation in agarose, the total time to result from a PBCB for dRAST was only six hours for a wide range of bacterial concentrations in PBCBs. The results from the dRAST system were consistent with the results from a standard AST, broth microdilution test. In tests of clinical isolates (n = 206) composed of 16 Gram-negative species and seven Gram-positive species, the dRAST system was accurate compared to the standard broth microdilution test, with rates of 91.11% (2613/2868) categorical agreement, 6.69% (192/2868) minor error, 2.72% (50/1837) major error and 1.45% (13/896) very major error. Thus, the dRAST system can be used to rapidly identify appropriate antimicrobial agents for the treatment of blood stream infection (BSI) and antibiotic-resistant strain infections.

Biomimetic microfingerprints for anti-counterfeiting strategies.

An unclonable, fingerprint-mimicking anti-counterfeiting strategy is presented that encrypts polymeric particles with randomly generated silica film wrinkles. The generated wrinkle codes are as highly unique as human fingerprints and are technically irreproducible. Superior to previous physical unclonable functions, codes are tunable on demand and generable on various geometries. Reliable authentication of real-world products that have these microfingerprints is demonstrated using optical decoding methods.

Lithographically encoded polymer microtaggant using high-capacity and error-correctable QR code for anti-counterfeiting of drugs.

A QR-coded microtaggant for the anti-counterfeiting of drugs is proposed that can provide high capacity and error-correction capability. It is fabricated lithographically in a microfluidic channel with special consideration of the island patterns in the QR Code. The microtaggant is incorporated in the drug capsule ("on-dose authentication") and can be read by a simple smartphone QR Code reader application when removed from the capsule and washed free of drug.