Performance of QuantaMatrix Microfluidic Agarose Channel system integrated with mycobacteria growth indicator tube liquid culture.

The QuantaMatrix Microfluidic Agarose Channel (QMAC) system was used for rapid drug susceptibility testing (DST). Here, we performed DST using QMAC integrated with the mycobacteria growth indicator tube (MGIT) liquid culture employing a specially designed cross agarose channel for the tuberculosis chip. MGIT-, QMAC-, and Löwenstein-Jensen (LJ)-DSTs were performed using 13 drugs. The protocol for QMAC-DST was optimized using the inoculum obtained after the disaggregation of Mycobacterium tuberculosis clumps in MGIT culture. The completion times of QMAC-DST and MGIT-DST were analyzed, and the results of all three DSTs were compared. Discrepant results were analyzed using line probe assays and DNA sequencing. Nontuberculous mycobacteria were distinguished using the ρ-nitrobenzoic acid inhibition test. The overall agreement rate of QMAT-DST and LJ-DST was 97.0% and that of QMAT-DST and MGIT-DST was 86.3%. An average turnaround time for DST was 5.4 days, which was considerably less than the time required for MGIT-DST. The overall time required to obtain DST results using QMAC-DST integrated with MGIT culture was an average of 18.6 days: 13.2 days for culture and identification and 5.4 days for DST. Hence, QMAC-DST integrated with liquid culture can be used to perform DSTs with short turnaround times and effective detection. KEY POINTS: • QMAC system can simultaneously perform phenotypic DST with 13 anti-TB drugs and PNB. • An optimized DST protocol led to a marked decrease in clumping in MGIT culture. • QMAC system integrated with MGIT liquid culture system reduced the turnaround time.

A high-throughput cell culture system based on capillary and centrifugal actions for rapid antimicrobial susceptibility testing.

Antibiotic resistance is a global threat to modern society. Rapid determination of suitable antibiotics that inhibit bacterial growth can effectively reduce antibiotic resistance and improve clinical treatment. The conventional methods of antimicrobial susceptibility testing (AST) depend on optical density measurements, which require long-time incubation. Various kinds of rapid AST systems which utilize various technologies from the field of lab on a chip have promised a great reduction in measurement time, but cannot achieve high-throughput, user-friendly testing due to the complexity of the testing system. Here, we introduce a capillary and centrifuge-based rapid AST system that reduces the time of loading the sample and culture media while achieving a high-throughput testing capacity. The capability of the proposed system is validated in a systematic analysis that includes sample loading characteristics and AST trials with standard strains. The proposed system provides a useful tool for drug testing in cell-culture systems with user-friendly and high-throughput analysis.

Clinical Validation of the QMAC-DST System for Testing the Drug Susceptibility of Mycobacterium tuberculosis to First- and Second-Line Drugs.

There is a high demand for novel approaches to counter the various challenges of conventional drug susceptibility testing (DST) for tuberculosis, the most prevalent infectious disease with significant global mortality. The QMAC-DST system was recently developed for rapid DST using image technology to track the growth of single cells of Mycobacterium tuberculosis (MTB). The purpose of this study was to clinically validate the QMAC-DST system compared to conventional DST. In total, 178 MTB isolates recovered from clinical specimens in Asan Medical Center in 2016 were tested by both QMAC-DST and absolute concentration methods using Lowenstein-Jensen media (LJ-DST). Among the isolates, 156 were subjected to DST using BACTEC MGIT 960 SIRE kits (BD, Sparks, MD, United States) (MGIT-DST). The susceptibility/resistance results obtained by QMAC-DST were read against 13 drugs after 7 days of incubation and compared with those of LJ-DST. Based on the gold standard LJ-DST, the agreement rates of QMAC-DST for all drugs were 97.8%, 97.9%, and 97.8% among susceptible, resistant, and total isolates, respectively, while the overall agreement of MGIT-DST tested for 156 isolates against first-line drugs was 95.5%. QMAC-DST showed the highest major error of 6.4% for rifampin, however, it could be corrected by a revised threshold of growth since false-resistant isolates showed grew only half than the true-resistant isolates. The rapid and accurate performance of QMAC-DST warrants ideal phenotypic DST for a wide range of first-line and second-line drugs.

A rapid culture system uninfluenced by an inoculum effect increases reliability and convenience for drug susceptibility testing of Mycobacterium tuberculosis.

The Disc Agarose Channel (DAC) system utilizes microfluidics and imaging technologies and is fully automated and capable of tracking single cell growth to produce Mycobacterium tuberculosis (MTB) drug susceptibility testing (DST) results within 3~7 days. In particular, this system can be easily used to perform DSTs without the fastidious preparation of the inoculum of MTB cells. Inoculum effect is one of the major problems that causes DST errors. The DAC system was not influenced by the inoculum effect and produced reliable DST results. In this system, the minimum inhibitory concentration (MIC) values of the first-line drugs were consistent regardless of inoculum sizes ranging from ~103 to ~108 CFU/mL. The consistent MIC results enabled us to determine the critical concentrations for 12 anti-tuberculosis drugs. Based on the determined critical concentrations, further DSTs were performed with 254 MTB clinical isolates without measuring an inoculum size. There were high agreement rates (96.3%) between the DAC system and the absolute concentration method using Löwenstein-Jensen medium. According to these results, the DAC system is the first DST system that is not affected by the inoculum effect. It can thus increase reliability and convenience for DST of MTB. We expect that this system will be a potential substitute for conventional DST systems.

Clinical Evaluation of QMAC-dRAST for Direct and Rapid Antimicrobial Susceptibility Test with Gram-Positive Cocci from Positive Blood Culture Bottles / 대한임상미생물학회지

BACKGROUND: Timely intervention in the treatment of bloodstream infection is important for prescription of appropriate antimicrobials. With prompt determination of the antimicrobial susceptibility of a causative agent, rapid antimicrobial susceptibility test (AST) can help select the appropriate antimicrobial therapy. This clinical study is for evaluation of the clinical performance of the QMAC-dRAST for rapid AST directly from positive blood culture (PBC)s with Gram-positive cocci. METHODS: A total of 115 PBC samples with Gram-positive organisms (76 Staphylococcus spp. and 39 Enterococcus spp.) were evaluated by the QMAC-dRAST system, and their pure culture isolates were evaluated by the MicroScan WalkAway (Beckman Coulter, USA) as the comparative AST system. Thirteen antimicrobial agents were included, and the agreement and discrepancy rates of the QMAC-dRAST system (Quantamatrix Inc., Republic of Korea) compared to the MicroScan WalkAway were calculated. To resolve discrepancies, the broth microdilution method was performed. RESULTS: The QMAC-dRAST system exhibited a categorical agreement rate of 94.9% (1,126/1,187) and an essential agreement rate of 98.3% (1,167/1,187). The QMAC-dRAST system yielded very major (false-susceptible) errors at 1.0% (5/485), major (false-resistant) errors at 1.3% (9/693), and minor errors at 4.0% (47/1,187) compared to the MicroScan WalkAway. The QMAC-dRAST system significantly eliminated 30 hours of total turnaround time by combination of direct inoculation of PBC and an image-based approach. CONCLUSION: The results of the QMAC-dRAST system were highly accurate. Thereby, the QMAC-dRAST may provide essential information to accelerate therapeutic decisions for earlier and adequate antibiotic treatment and patient management in clinical settings.

Correction: Clinical Evaluation of QMAC-dRAST for Direct and Rapid Antimicrobial Susceptibility Test with Gram-Positive Cocci from Positive Blood Culture Bottles / 대한임상미생물학회지

There was an error in the article, the names of manufacturers and countries of equipments in the Korean abstract were reversed.

Rapid drug susceptibility test of Mycobacterium tuberculosis using microscopic time-lapse imaging in an agarose matrix.

Tuberculosis (TB) is a major global health problem, and multi-drug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) are spreading throughout the world. However, conventional drug susceptibility test (DST) methods, which rely on the detection of the colony formation on a solid medium, require 1-2 months to the result. A rapid and accurate DST is necessary to identify patients with drug-resistant TB and treat them with appropriate drugs. Here, we used microscopic imaging of Mycobacterium tuberculosis (MTB) immobilized in an agarose matrix for a rapid DST. The agarose matrix, which was molded in a microfluidic chip, was inoculated with MTB, and TB drugs in liquid culture medium diffused throughout the agarose to reach the MTB immobilized in the agarose matrix. After the responses of MTB to drugs were tracked with an automated microscopic system, an image-processing program automatically determined the susceptibility and resistance of MTB to specific doses of TB drugs. The automatic DST system was able to assess the drug susceptibility of various drug-resistant clinical TB strains within 9 days with an accuracy comparable to that of conventional method. Our rapid DST method based on microscopic time-lapse imaging greatly reduces the time required for a DST and can be used to rapidly and accurately treat TB patients.

New Cyst Nematode, Heterodera sojae n. sp. (Nematoda: Heteroderidae) from Soybean in Korea.

A new soybean cyst nematode Heterodera sojae n. sp. was found from the roots of soybean plants in Korea. Cysts of H. sojae n. sp. appeared more round, shining, and darker than that of H. glycines. Morphologically, H. sojae n. sp. differed from H. glycines by fenestra length (23.5-54.2 µm vs. 30-70 µm), vulval silt length (9.0-24.4 µm vs. 43-60 µm), tail length of J2 (54.3-74.8 µm vs. 40-61 µm), and hyaline part of J2 (32.6-46.3 µm vs. 20-30 µm). It is distinguished from H. elachista by larger cyst (513.4-778.3 µm × 343.4-567.1 µm vs. 350-560 µm × 250-450 µm) and longer stylet length of J2 (23.8-25.3 µm vs. 17-19 µm). Molecular analysis of rRNA large subunit (LSU) D2-D3 segments and ITS gene sequence shows that H. sojae n. sp. is more close to rice cyst nematode H. elachista than H. glycines. Heterodera sojae n. sp. was widely distributed in Korea. It was found from soybean fields of all three provinces sampled.

A rapid antimicrobial susceptibility test based on single-cell morphological analysis.

A rapid antibiotic susceptibility test (AST) is desperately needed in clinical settings for fast and appropriate antibiotic administration. Traditional ASTs, which rely on cell culture, are not suitable for urgent cases of bacterial infection and antibiotic resistance owing to their relatively long test times. We describe a novel AST called single-cell morphological analysis (SCMA) that can determine antimicrobial susceptibility by automatically analyzing and categorizing morphological changes in single bacterial cells under various antimicrobial conditions. The SCMA was tested with four Clinical and Laboratory Standards Institute standard bacterial strains and 189 clinical samples, including extended-spectrum ß-lactamase-positive Escherichia coli and Klebsiella pneumoniae, imipenem-resistant Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococci from hospitals. The results were compared with the gold standard broth microdilution test. The SCMA results were obtained in less than 4 hours, with 91.5% categorical agreement and 6.51% minor, 2.56% major, and 1.49% very major discrepancies. Thus, SCMA provides rapid and accurate antimicrobial susceptibility data that satisfy the recommended performance of the U.S. Food and Drug Administration.

Potentiometric multichannel cytometer microchip for high-throughput microdispersion analysis.

The parallelization of microfluidic cytometry is expected to lead to considerably enhanced throughput enabling point-of-care diagnosis. In this article, the development of a microfluidic potentiometric multichannel cytometer is presented. Parallelized microfluidic channels sharing a fluid path inevitably suffer from interchannel signal crosstalk that results from electrical coupling within the microfluidic channel network. By employing three planar electrodes within a single detection channel, we electrically decoupled each channel unit, thereby enabling parallel analysis by using a single cytometer microchip with multiple microfluidic channels. The triple-electrode configuration is validated by analyzing the size and concentration of polystyrene microbeads (diameters: 1.99, 2.58, 3, and 3.68 µm; concentration range: ∼2 × 10(5) mL(-1) to ∼1 × 10(7) mL(-1)) and bacterial microdispersion samples (Bacillus subtilis, concentration range: ∼4 × 10(5) CFU mL(-1) to ∼3 × 10(6) CFU mL(-1)). Crosstalk-free parallelized analysis is then demonstrated using a 16-channel potentiometric cytometer (maximum cross-correlation coefficients |r|: < 0.13 in all channel combinations). A detection throughput of ∼48,000 s(-1) was achieved; the throughout can be easily increased with the degree of parallelism of a single microchip without additional technical complexities. Therefore, this methodology should enable high-throughput and low-cost cytometry.

Optofluidic in situ maskless lithography of charge selective nanoporous hydrogel for DNA preconcentration.

An optofluidic maskless photopolymerization process was developed for in situ negatively charged nanoporous hydrogel [poly-AMPS (2-acrylamido-2-methyl-1-propanesulfonic acid)] fabrication. The optofluidic maskless lithography system, which combines a high power UV source and digital mirror device, enables fast polymerization of arbitrary shaped hydrogels in a microfluidic device. The poly-AMPS hydrogel structures were positioned near the intersections of two microchannels, and were used as a cation-selective filter for biological sample preconcentration. Preconcentration dynamics as well as the fabricated polymer shape were analyzed in three-dimensions using fluorescein sample and a confocal microscope. Finally, single-stranded DNA preconcentration was demonstrated for polymerase chain reaction-free signal enhancement.