Rapid Detection of Uropathogens Using an Integrated Multiplex Digital Nucleic Acid Detection Assay Powered by a Digital-to-Droplet Microfluidic Device.

The digital nucleic acid detection assay features the capability of absolute quantitation without the need for calibration, thereby facilitating the rapid identification of pathogens. Although several integrated digital nucleic acid detection techniques have been developed, there are still constraints in terms of automation and analysis throughput. To tackle these challenges, this study presents a digital-to-droplet microfluidic device comprising a digital microfluidics (DMF) module at the bottom and a droplet microfluidics module at the top. Following sample introduction, the extraction of nucleic acid and the dispensation of nucleic acid elution for mixing with the multiple amplification reagents are carried out in the DMF module. Subsequently, the reaction droplets are transported to the sample inlet of the droplet microfluidic module via a liquid outlet, and then droplet generation in four parallel units within the droplet microfluidics module is actuated by negative pressure generated by a syringe vacuum. The digital-to-droplet microfluidic device was employed to execute an integrated multiplex digital droplet nucleic acid detection assay (imDDNA) incorporating loop-mediated isothermal amplification (LAMP). This assay was specifically designed to enable simultaneous detection of four uropathogens, namely, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Enterococcus faecalis. The entire process of the imDDNA is completed within 75 min, with a detection range spanning 5 orders of magnitude (9.43 × 10-2.86 × 104 copies µL-1). The imDDNA was employed for the detection of batched clinical specimens, showing a consistency of 91.1% when compared with that of the conventional method. The imDDNA exhibits simplicity in operation and accuracy in quantification, thus offering potential advantages in achieving rapid pathogen detection.

[Priligy combined with behavioral therapy and psychological counseling for primary premature ejaculation].

OBJECTIVE: To observe the clinical effect of priligy (dapoxetine hydrochloride) combined with behavioral therapy and psychological counseling in the treatment of primary premature ejaculation (PPE). METHODS: A total of 202 PPE patients diagnosed from 2017 to 2018 were randomized into a control (n = 100) and an experimental group (n = 102), the former treated with oral priligy at 30 mg 1－3 hours before anticipated sexual activity, and the latter by the same medication combined with 30-minute behavioral therapy and psychological counseling once a month for two times. The therapeutic effects were evaluated according to the Premature Ejaculation Profile (PEP) scores of the patients at 1 and 2 months of treatment. RESULTS: After 1 month of treatment, both groups of the patients showed significant improvement, as compared with the baseline, in the PEP scores on personal distress related to ejaculation (P < 0 05), interpersonal difficulty related to ejaculation (P < 0.05) and satisfaction with sexual intercourse (P < 0.05) but not on perceived control over ejaculation (P > 0.05). At 2 months, however, the patients' scores on all the four PEP items were dramatically improved, even more significantly in the experimental than in the control group, as on perceived control over ejaculation (2.73 ± 0.95 vs 2.22 ± 0.68, P < 0.05), personal distress related to ejaculation (2.97 ± 1.07 vs 2.57 ± 0.69, P < 0.05), interpersonal difficulty related to ejaculation (3.19 ± 1.03 vs 2.77 ± 0.69, P < 0 05) and satisfaction with sexual intercourse (2.85 ± 0.99 vs 2.35 ± 0.63, P < 0.05). There was no statistically significant difference in the incidence rate of adverse events between the experimental and control groups (21.6% vs 20.0%, P > 0.05), and all the symptoms were relieved within 24 hours. CONCLUSIONS: Priligy combined with behavioral therapy and psychological counseling is more effective than priligy alone in improving the sexual function of PPE patients, raise their interest in sexual life and increase the intimacy between the partners, and can even achieve clinical cure in some patients.

Hybrid operation to revascularize long-segment occluded internal carotid artery prevent further ischemic events.

PURPOSE: The effects and complications of hybrid procedure (combined carotid endarterectomy and carotid stenting) to revascularize chronic long-segment occlusion of internal carotid artery (ICA) are currently unknown and the purpose of this study. METHODS: Sixty-five patients with long-segment occlusion of ICA were prospectively enrolled and divided into two groups of revascularization with hybrid operation (n = 30) and medication group (n = 35), and clinical and angiographic data were analyzed. RESULTS: The duration from symptom onset to revascularization ranged 17-120 days (mean 40.5 ± 5.0) in the hybrid operation, with a success revascularization rate of 100%. All patients had thrombi extracted with the clot length ranging 5-8 cm (mean 6.3 ± 0.9). The thrombolysis in cerebral infarction grade (TICI) was significantly (P < 0.0001) greater immediately after (median 2,) than before recanalization (0). Periprocedural complications included recurrent laryngeal nerve injury in one patient and intracranial hemorrhage in another (6.7%), but no severe neurological deficits occurred. The symptoms were significantly (P < 0.0001) improved after compared with before operation, with the modified Rankin score of 2.5 ± 0.6 at 3 months postoperation which was significantly (P < 0.0001) improved compared with before revascularization (3.4 ± 0.6). Follow-up angiography revealed patent ICA in all patients with hybrid operation. In the medication alone group, no significant (P > 0.05) improvement was observed with the mRS score of 3.5 ± 0.8 at admission and 3.4 ± 0.7 at 3 months, which was significantly (P < 0.001) greater than in the hybrid operation. CONCLUSION: Hybrid operation may be safe and effective in revascularizing long-segment occlusion of internal carotid artery for prevention of further ischemic events.

Outcomes of middle cerebral artery angioplasty and stenting with Wingspan at a high-volume center.

INTRODUCTION: This study was to investigate the periprocedural stroke rates, safety, and long-term effect of Wingspan stenting for symptomatic severe stenosis of the middle cerebral artery (MCA) at a high-volume center. METHODS: Between July 2007 and April 2013, 196 consecutive patients with severe MCA atherosclerotic stenosis (≥70%) who were treated with Wingspan stenting were retrospectively studied. All patients had arterial stenosis-related temporary ischemic attack or strokes. The demographic data, cerebral angiography, technical success rate, periprocedural complications, and clinical and imaging follow-up were analyzed. RESULTS: The successful stenting rate was 98.0%, and the stenosis rate was improved from pre-stenting (80.6 ± 8.3 %) to post-stenting (15.5 ± 6.8%). The 30-day periprocedural stroke or death rate was 7.1%, with a disabling or fatal rate of 2.6%. The perioprocedural stroke rate was significantly (P < 0.01) greater in the early learning stage (16.0%) than in the later technical maturation stage (4.1%). The total periprocedural ischemic and perforator stroke rates were greater in patients with the most stenosis in the distal MCA 1/3 segment (6.8 and 5.7%, respectively) than in the proximal and middle 2/3 segments (0.9 and 0%, respectively). The ipsilateral stroke or death rate beyond 30 days (6-69 months, mean 30 ± 16) was 4.8%, with the 1- and 2-year cumulative stroke rates of 9.6 and 12.1%, respectively. Imaging follow-up 6-69 months (mean 10.9 ± 8.5) revealed restenosis in 21 cases (20.4 %). CONCLUSION: Intracranial stenting of MCA stenoses may have the potential of better clinical outcomes if patients are properly selected and treated by an experienced operator at a high-volume center.

[Research on integrated application of tumor magnetic induction hyperthermia treatment planning system and modern medical information systems].

Magnetic induction hyperthermia becomes a very important tumor treatment method at present. In order to ensure a successful operation, doctors should make hyperthermia treatment planning before surgery. Based on Integration Healthcare Enterprise (IHE) framework and Digital Imaging and Communications in Medcine (DICOM) standard, we proposed and carried out a network workflow integrated with modern medical information systems for the dissemination of information in magnetic induction hyperthermia like accurate accessing patient information and radiology image data, storing processed images, sharing and verifying hyperthermia reports. The results proved that our system could not only improve the efficiency of magnetic induction hyperthermia treatment planning, but also save medical resources and reduce labor costs.

Significance of atherosclerotic plaque location in recanalizing non-acute long-segment occlusion of the internal carotid artery.

To investigate the significance of atherosclerotic plaque location in hybrid surgery comprising both endovascular recanalization approaches and carotid endarterectomy for symptomatic atherosclerotic non-acute long-segment occlusion of the internal carotid artery (ICA), 162 patients were enrolled, including 120 (74.1%) patients in the proximal plaque group and 42 (25.9%) in the distal plaque group. Surgical recanalization was performed in all patients, with successful recanalization in 119 (99.2%) patients in the proximal and 39 (92.9%) in the distal plaque group. The total successful recanalization rate was 97.5% (158/162) with a failure rate of 2.5% (4/162). Periprocedural complications occurred in 5 (4.2% or 5/120) patients in the proximal plaque group, including neck infection in two (1.7%), recurrent nerve injury in 1 (0.8%), and laryngeal edema in 2 (1.7%), and 2 (4.8%) in the distal plaque group, including femoral puncture infection in 2 (4.8%). No severe complications occurred in either group. Univariate analysis showed plaque location was a significant (P = 0.018) risk factor for successful recanalization, and multivariate analysis indicated that the plaque location remained a significant independent risk factor for recanalization success (P = 0.017). In follow-up 6-48 months after the recanalization surgery, reocclusion occurred in two (2.8%) patients in the proximal plaque group and 4 (13.3%) in the distal plaque group. In conclusion, although hybrid surgery achieves similar outcomes in patients with ICA occlusion caused by either proximal or distal atherosclerotic plaques, plaque location may be a significant risk factor for successful recanalization of symptomatic non-acute long-segment ICA occlusion.

Effects of percutaneous endovascular angioplasty for severe stenosis or occlusion of subclavian artery.

To investigate the effect and safety of percutaneous endovascular angioplasty (PEA) with optional stenting for the treatment of severe stenosis or occlusion of subclavian artery, patients with severe stenosis ≥ 70% or occlusion of subclavian artery treated with PEA were retrospectively enrolled. The clinical data were analyzed. A total of 222 patients were retrospectively enrolled, including 151 males (68.0%) and 71 females (32.0%) aged 48-86 (mean 63.9 ± 9.0) years. Forty-seven (21.2%) patients had comorbidities. Subclavian artery stenosis ≥ 70% was present in 201 (90.5%) patients and complete subclavian occlusion in 21 (9.5%) cases. Angioplasty was successfully performed in all (100%) patients. Balloon-expandable stents were used in 190 (85.6%) cases, and self-expandable stents in 20 (9.0%) cases. Only 12 (5.4%) cases were treated with balloon dilation only. Among 210 patients treated with stent angioplasty, 71 (33.8% or 71/210) cases underwent balloon pre-dilation, 139 (66.2% or 139/210) had direct deployment of balloon-expandable stents, and 2 (1.0% or 2/210) experienced balloon post-dilation. Distal embolization protection devices were used in 5 (2.3% or 5/222) cases. Periprocedural complications occurred in 3 (1.4%) patients, including aortic dissection in 2 (0.9%) cases and right middle cerebral artery embolism in 1 (0.5%). No hemorrhage occurred. Among 182 (82.0%) patients with 6-month follow-up, restenosis > 70% occurred in 1 (0.5%) patient, and among 68 (30.6%) patients with 12-month follow-up, restenosis > 70% took place in 11 (16.2%) patients. Percutaneous endovascular angioplasty can be safely and efficiently performed for the treatment of severe stenosis ≥ 70% or occlusion of subclavian artery.

Application of microcatheter shaping based on computational fluid dynamics simulation of cerebral blood flow in the intervention of posterior communicating aneurysm of the internal carotid artery.

Introduction: The present study aimed to investigate the application of the aneurysm embolization microcatheter plasticity method based on computational fluid dynamics (CFD) to simulate cerebral blood flow in the interventional treatment of posterior communicating aneurysms in the internal carotid artery and to evaluate its practicality and safety. Methods: A total of 20 patients with posterior internal carotid artery communicating aneurysms who used CFD to simulate cerebral flow lines from January 2020 to December 2022 in our hospital were analyzed. Microcatheter shaping and interventional embolization were performed according to the main cerebral flow lines, and the success rate, stability, and effect of the microcatheter being in place were analyzed. Results: Among the 20 patients, the microcatheters were all smoothly placed and the catheters were stable during the in vitro model test. In addition, the microcatheters were all smoothly placed during the operation, with a success rate of 100%. The catheter tips were stable and well-supported intraoperatively, and no catheter prolapse was registered. The aneurysm was completely embolized in 19 cases immediately after surgery, and a small amount of the aneurysm neck remained in one case. There were no intraoperative complications related to the embolization catheter operation. Conclusion: Microcatheter shaping based on CFD simulation of cerebral blood flow, with precise catheter shaping, leads to a high success rate in catheter placing, stability, and good support, and greatly reduces the difficulty of catheter shaping. This catheter-shaping method is worthy of further study and exploration.

Cascade filtration and droplet digital detection integrated microfluidic assay enables isolating culture-free phenotypic identification of carbapenem-resistant organisms.

Carbapenem-resistant organisms (CROs) are characterized by high drug resistance, rapid transmission, and high lethality. Therefore, rapid detection for CROs is essential for appropriate applying antibiotics and implementing quarantine. Droplet digital chromogenic assays (DDCA) have been accepted as an effective means for rapid microbial detection as the small droplet volumes facilitate a significant enhancement in the local concentration of chromogenic factors and, therefore, reduce the required time of the test. Nevertheless, as their dependence on the time-consuming isolation culture, the DDCA is still associated with a long turnaround time. To overcome this limitation, we develop here a microfluidic chip-based CRO phenotypic identification method that integrates cascade filtration (CF) with DDCA (CF-DDCA). After a body fluid sample is introduced to the microfluidic chip, particles with sizes >5 µm are removed out by the primary filter, and Gram (+) cocci with sizes <1 µm removed out by the secondary filter so that only Gram (-) bacilli with sizes between 1.5 and 5 µm are selectively retained. The purified Gram (-) bacilli, along with chromogenic reagents and carbapenem antibiotics, are then subjected to the DDCA. We demonstrate that the CF can remove 99.9% of the interfering microorganisms and thus eliminates the isolating culture. Benefited from the isolating culture-free DDCA, phenotypic identification of CROs can be achieved within 3.5 h. Clinical urine sample testing shows that the sensitivity and specificity of the CF-DDCA for CRO identification are all 100%, and the total coincidence rate between CF-DDCA and the conventional assay is also 100%.

Transradial access with intra-aortic catheter looping for the treatment of intracranial aneurysms.

Purpose: The study aimed to investigate the feasibility and effect of transradial access with intra-aortic catheter looping for the treatment of intracranial aneurysms. Materials and methods: This retrospective one-center study was performed on patients with intracranial aneurysms which were embolized through transradial access with intra-aortic catheter looping because of the difficulty of transfemoral access or transradial access without intra-aortic catheter looping. The imaging and clinical data were analyzed. Results: A total of 11 patients were enrolled, including seven (63.6%) male patients. Most patients were associated with one or two risk factors of atherosclerosis. There were nine aneurysms in the left internal carotid artery system and two aneurysms in the right internal carotid artery system. All 11 patients had complications with different anatomic variations or vascular diseases, which made endovascular operation via the transfemoral artery difficult or a failure. The right transradial artery approach was adopted in all patients, and the success rate of intra-aortic catheter looping was 100%. Embolization of intracranial aneurysms was successfully completed in all patients. No instability of the guide catheter was encountered. No puncture site complications or surgical-related neurological complications occurred. Conclusion: Transradial access with intra-aortic catheter looping for embolization of intracranial aneurysms is technically feasible, safe, and efficient as an important supplementary approach to the routine transfemoral access or transradial access without intra-aortic catheter looping.

Comprehensive analysis of autophagy-related gene expression profiles identified five gene biomarkers associated with immune infiltration and advanced plaques in carotid atherosclerosis.

BACKGROUND: Autophagy plays an important role in the progression of carotid atherosclerosis (CAS). This study aimed to identify hub autophagy-related genes (ATGs) associated with CAS. METHODS: GSE43292 and GSE28829 datasets of early and advanced CAS plaques were enrolled from the Gene Expression Omnibus (GEO) database. A comprehensive analysis of differentially expressed ATGs (DE-ATGs) was conducted. Functional enrichment assay was used to explore biological functions of DE-ATGs. The hub ATGs were identified by protein-protein interaction (PPI) network. Immunohistochemistry (IHC) and Real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to validate hub ATGs at the protein level and mRNA level. Correlation analysis of hub ATGs with immune cells was also conducted. In addition, a competitive endogenous RNA (ceRNA) network was constructed, and diagnostic value of hub ATGs was evaluated. RESULTS: A total of 19 DE-ATGs were identified in early and advanced CAS plaques. Functional enrichment analysis of DE-ATGs suggested that they were closely correlated to autophagy, apoptosis, and lipid regulation. Moreover, 5 hub ATGs, including TNFSF10, ITGA6, CTSD, CCL2, and CASP1, were identified and further verified by IHC. The area under the curve (AUC) values of the 5 hub ATGs were 0.818, 0.732, 0.792, 0.814, and 0.812, respectively. Competing endogenous RNA (ceRNA) networks targeting the hub ATGs were also constructed. In addition, the 5 hub ATGs were found to be closely associated with immune cell infiltration in CAS. CONCLUSION: In this study, we identified 5 hub ATGs including CASP1, CCL2, CTSD, ITGA6 and TNFSF10, which could serve as candidate diagnostic biomarkers and therapeutic targets.

Droplet Encoding-Pairing Enabled Multiplexed Digital Loop-Mediated Isothermal Amplification for Simultaneous Quantitative Detection of Multiple Pathogens.

Despite the advantages of digital nucleic acid analysis (DNAA) in terms of sensitivity, precision, and resolution, current DNAA methods commonly suffer a limitation in multiplexing capacity. To address this issue, a droplet encoding-pairing enabled DNAA multiplexing strategy is developed, wherein unique tricolor combinations are deployed to index individual primer droplets. The template droplets and primer droplets are sequentially introduced into a microfluidic chip with a calabash-shaped microwell array and are pairwise trapped and merged in the microwells. Pre-merging and post-amplification image analysis with a machine learning algorithm is used to identify, enumerate, and address the droplets. By incorporating the amplification signals with droplet encoding information, simultaneous quantitative detection of multiple targets is achieved. This strategy allows for the establishment of flexible multiplexed DNAA by simply adjusting the primer droplet library. Its flexibility is demonstrated by establishing two multiplexed (8-plex) droplet digital loop-mediated isothermal amplification (mddLAMP) assays for individually detecting lower respiratory tract infection and urinary tract infection causative pathogens. Clinical sample analysis shows that the microbial detection outcomes of the mddLAMP assays are consistent with those of the conventional assay. This DNAA multiplexing strategy can achieve flexible high-order multiplexing on demand, making it a desirable tool for high-content pathogen detection.

Transradial intra-aortic catheter looping in the angioplasty of severe intracranial symptomatic arteriosclerotic diseases.

Purpose: This study aims to investigate the effect and feasibility of intra-aortic catheter looping via transradial access in angioplasty for symptomatic intracranial severe (>70%) atherosclerotic stenosis or occlusion of large arteries (SISOLAs). Materials and methods: Patients with SISOLAs who underwent transradial endovascular angioplasty using the catheter looping technique in the ascending aorta were retrospectively enrolled. The clinical data and treatment outcomes were analyzed. Results: Fifteen patients aged 48-71 years were enrolled in this study. Left vertebrobasilar artery occlusion was present in 1 (6.7%) patient, severe left middle cerebral artery stenosis in 7 (46.7%) patients, severe left internal carotid artery (ICA) stenosis of the ophthalmic segment in 4 (26.7%) patients, severe left ICA stenosis of the cavernous segment in 2 (13.3%) patients, and severe right middle cerebral artery stenosis in 2 (13.3%) patients. The arterial stenosis ranged from 70 to 92% (mean 86%) before stenting. The looping of a guiding catheter in the ascending aorta via transradial access for angioplasty was successful in all patients (100%). The vertebral artery intracranial segment occlusion was successfully recanalized, while severe stenosis in the remaining 14 patients was successfully eliminated. After endovascular recanalization, the residual stenosis was reduced by 12-26% (median 18%). No puncture-related complications or surgical-related neurological complications occurred in these patients. In the follow-up angiography conducted on 10 (66.7%) patients after 6-25 months, no in-stent restenosis was detected. Conclusion: Intra-aortic guiding catheter looping via transradial access for endovascular angioplasty of SISOLAs is technically safe, feasible, and effective, especially when the transfemoral artery approach is difficult or impossible to undertake.

Active droplet-array microfluidics-based chemiluminescence immunoassay for point-of-care detection of procalcitonin.

The application of conventional chemiluminescence immunoassay (CLIA) in resource-limited settings is limited due to the large apparatus footprint, cumbersome operation and maintenance process, and high consumption of reagents. To address this issue, we developed an active droplet-array (ADA) microfluidics-based CLIA system, which consists of a compact microchip analyzer and microfluidic chips with preloaded reagents. The microfluidic chip contains microslit-connected microchambers, in which all the required reagents were preloaded in water-in-oil droplets. The microfluidic chip analyzer can manipulate five microfluidic chips in parallel in a single run. By interacting the microchip with magnetic, thermal, optical mechanisms programmatically, the entire workflow of CLIA can be accomplished in an automated manner. With the proposed CLIA, the detection of procalcitonin (PCT) can be completed in 12 min, with a limit of detection (LOD) of 0.044 ng mL-1 and a detection range from 0.044 to 100 ng mL-1. We found a good linear correlation between the microfluidic CLIA and the conventional electrochemiluminescence immunoassay (R2=0.98).The microfluidic CLIA has significant advantages over the conventional ELISA in detection sensitivity, dynamic range, instrument size and turnaround time, and can provide more consistent and reliable results than the lateral flow immunoassays. The compact microfluidic system can perform automated and parallelized CLIA in a short turnaround time, and thus well suited to Point-of-Care detection of disease biomarkers.

Benign testicular cavernous hemangioma presenting with acute onset: A case report.

Cavernous hemangioma is a congenital, benign vascular tumor that occurs in the deep dermis and subcutaneous tissue. Testicular cavernous hemangioma is extremely rare, mostly occurring during childhood or adolescence. Testicular cavernous hemangioma is a benign tumor that appears as a slowly growing painless mass. In rare cases, it may be associated with acute testicular infarction or torsion with acute onset. We herein report the case of a patient with an atypical presentation of testicular cavernous hemangioma, characterized by acute painful testicular enlargement triggered by minor injury. The patient underwent right radical orchiectomy, and histopathological examination confirmed the diagnosis of testicular cavernous hemangioma. Although this is a rare tumor, it should be considered in the differential diagnosis of testicular tumors.

Use of a First Large-Sized Coil Versus Conventional Coils for Embolization of Cerebral Aneurysms: Effects on Packing Density, Coil Length, and Durable Occlusion.

OBJECTIVE: To investigate effects of using a large-sized coil first on embolizing cerebral aneurysms compared with conventional coils. MATERIALS AND METHODS: Forty-six patients with 51 saccular intracranial aneurysms who underwent embolization with a large-sized coil first were enrolled as the large-sized coil group. There were 33 female and 13 male patients with a mean age of 56.9 ± 8.8 years. The treatment modality was coiling alone in 30 aneurysms and stent-assisted coiling in 21. Meanwhile, 50 patients with 53 intracranial aneurysms who were treated with conventional-sized coils were selected as the control conventional-sized coil group, including 36 female and 14 male patients with a mean age of 54.6 ± 5.8 years. The treatment modality was coiling alone in 29 aneurysms and stent-assisted coiling in 24 aneurysms. The occlusion rate, percent packing volume, total coil number and length, and follow-up occlusion rate were compared between the 2 groups. RESULTS: Significantly (P < 0.001) decreased percent packing volume (19.54% ± 6.44% vs. 27.39% ± 5.68%), decreased coil number (2.98 ± 1.09 vs. 6.38 ± 1.65), and length (26.20 ± 26.57 vs. 44.35 ± 35.88 cm) were achieved in the large versus the conventional coil group. At angiographic follow-up of 8 months, only 1 aneurysm (2.2%) recurred in the large coil group compared with 5 aneurysms recurrent (11.1%) in the conventional coil group. CONCLUSIONS: The use of a large-sized coil as the first one for embolizing cerebral aneurysms may be a better embolization strategy because it achieves similar initial occlusion rates, decreased packing density, decreased coil numbers and lengths, and reduced recurrence prevalence at follow-up.

Direct antimicrobial susceptibility testing of bloodstream infection on SlipChip.

This paper describes an integrated microfluidic SlipChip device for rapid antimicrobial susceptibility testing (AST) of bloodstream pathogens in positive blood cultures. Unlike conventional AST methods, which rely on an overnight subculture of positive blood cultures to obtain isolated colonies, this device enables direct extraction and enrichment of the bacteria from positive blood cultures by dielectrophoresis. SlipChip technology enables parallel inoculation of the extracted bacteria into nanoliter-scale broth droplets to perform multiplexed ASTs simultaneously. The nanoliter confinement in the droplets increases the effective inoculation amount of the bacteria, shortens the diffusion distance of nutrient elements and gases, and allows faster growth and proliferation rates. Entropy-based image analysis used for the characterization of bacterial susceptibility patterns eliminates the requirement for single-cell morphological analysis and fluorescence labeling. As a proof-of-concept, the susceptibility patterns of Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 6538p, and a positive blood culture containing Escherichia coli against several broad-spectrum antibiotics were determined by the SlipChip device. The on-chip AST results were well matched with those respectively reported by the broth microdilution method and a BD Phoenix Automated Microbiology System. Reliable AST results can be reported to clinicians within 3-8 h using this simple device after positive blood culture, allowing earlier proper administration of antimicrobial therapy.

Capacity of rTth polymerase to detect RNA in the presence of various inhibitors.

The full potential of the real-time reverse transcription polymerase chain reaction (RT-PCR) as a rapid and accurate diagnostic method is limited by DNA polymerase inhibitors as well as reverse transcriptase inhibitors which are ubiquitous in clinical samples. rTth polymerase has proven to be more resistant to DNA polymerase inhibitors present in clinical samples for DNA detection and also exhibits reverse transcriptase activity in the presence of Mn2+ ions. However, the capacity of rTth polymerase, which acts as DNA polymerase and reverse transcriptase, to detect RNA in the presence of various inhibitors has not been investigated in detail. Herein, the inhibitors originating from various clinical samples such as blood, urine, feces, bodily fluids, tissues and reagents used during nucleic acid extraction were employed to evaluate the capacity of rTth polymerase to detect RNA. The results show that the inhibitors have different inhibitory effects on the real-time RT-PCR reactions by rTth polymerase, and the inhibitory effects are concentration dependent. Additionally, the capacity of rTth polymerase to detect RNA in the presence of various inhibitors is better or at least comparable with its capacity to detect DNA in the presence of various inhibitors. As a consequence, RNA may be directly detected in the presence of co-purified inhibitors or even directly from crude clinical samples by rTth polymerase.

Direct enrichment of pathogens from physiological samples of high conductivity and viscosity using H-filter and positive dielectrophoresis.

The full potential of microfluidic techniques as rapid and accurate methods for the detection of disease-causing agents and foodborne pathogens is critically limited by the complex sample preparation process, which commonly comprises the enrichment of bacterial cells to detectable levels. In this manuscript, we describe a microfluidic device which integrates H-filter desalination with positive dielectrophoresis (pDEP) for direct enrichment of bacterial cells from physiological samples of high conductivity and viscosity, such as cow's milk and whole human blood. The device contained a winding channel in which electrolytes in the samples continuously diffused into deionized (DI) water (desalination), while the bacterial cells remained in the samples. The length of the main channel was optimized by numerical simulation and experimentally evaluated by the diffusion of fluorescein into DI water. The effects of another three factors on H-filter desalination were also investigated, including (a) the flow rate ratio between the sample and DI water, (b) sample viscosity, and (c) non-Newtonian fluids. After H-filter desalination, the samples were withdrawn into the dielectrophoresis chamber in which the bacterial cells were captured by pDEP. The feasibility of the device was demonstrated by the direct capture of the bacterial cells in 1× PBS buffer, cow's milk, and whole human blood after H-filter desalination, with the capture efficiencies of 70.7%, 90.0%, and 80.2%, respectively. We believe that this simple method can be easily integrated into portable microfluidic diagnosis devices for rapid and accurate detection of disease-causing agents and foodborne pathogens.

Direct DNA and RNA detection from large volumes of whole human blood.

PCR inhibitors in clinical specimens negatively affect the sensitivity of diagnostic PCR and RT-PCR or may even cause false-negative results. To overcome PCR inhibition, increase the sensitivity of the assays and simplify the detection protocols, simple methods based on quantitative nested real-time PCR and RT-PCR were developed to detect exogenous DNA and RNA directly from large volumes of whole human blood (WHB). Thermus thermophilus (Tth) polymerase is resistant to several common PCR inhibitors and exhibits reverse transcriptase activity in the presence of manganese ions. In combination with optimized concentrations of magnesium ions and manganese ions, Tth polymerase enabled efficient detection of DNA and RNA from large volumes of WHB treated with various anticoagulants. The applicability of these methods was further demonstrated by examining WHB specimens collected from different healthy individuals and those stored under a variety of conditions. The detection limit of these methods was determined by detecting exogenous DNA, RNA, and bacteria spiked in WHB. To the best of our knowledge, direct RNA detection from large volumes of WHB has not been reported. The results of the developed methods can be obtained within 4 hours, making them possible for rapid and accurate detection of disease-causing agents from WHB.