Genetic and phenotypic profiling of single living circulating tumor cells from patients with microfluidics.

Accurate prediction of the efficacy of immunotherapy for cancer patients through the characterization of both genetic and phenotypic heterogeneity in individual patient cells holds great promise in informing targeted treatments, and ultimately in improving care pathways and clinical outcomes. Here, we describe the nanoplatform for interrogating living cell host-gene and (micro-)environment (NICHE) relationships, that integrates micro- and nanofluidics to enable highly efficient capture of circulating tumor cells (CTCs) from blood samples. The platform uses a unique nanopore-enhanced electrodelivery system that efficiently and rapidly integrates stable multichannel fluorescence probes into living CTCs for in situ quantification of target gene expression, while on-chip coculturing of CTCs with immune cells allows for the real-time correlative quantification of their phenotypic heterogeneities in response to immune checkpoint inhibitors (ICI). The NICHE microfluidic device provides a unique ability to perform both gene expression and phenotypic analysis on the same single cells in situ, allowing us to generate a predictive index for screening patients who could benefit from ICI. This index, which simultaneously integrates the heterogeneity of single cellular responses for both gene expression and phenotype, was validated by clinically tracing 80 non-small cell lung cancer patients, demonstrating significantly higher AUC (area under the curve) (0.906) than current clinical reference for immunotherapy prediction.

An isothermal CRISPR- based lateral flow assay for detection of Neisseria meningitidis.

BACKGROUND: Neisseria meningitidis can cause life-threatening meningococcal meningitis and meningococcemia. Old standard microbiological results from CSF/blood cultures are time consuming. This study aimed to combine the sensitivity of loop-mediated isothermal nucleic acid amplification (LAMP) with the specificity of CRISPR/Cas12a cleavage to demonstrate a reliable diagnostic assay for rapid detection of N. meningitidis. METHODS: A total of n = 139 samples were collected from patients with suspected meningococcal disease and were used for evaluation. The extracted DNA was subjected to qualitative real-time PCR, targeting capsular transporter gene (ctrA) of N. meningitidis. LAMP-specific primer pairs, also targeting the ctrA, were designed and the LAMP products were subjected to CRISPR/Cas12 cleavage reaction. the readout was on a lateral flow strip. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of LAMP-CRISPR/Cas was compared with real-time PCR assays. The limit of detection (LOD) was established with serial dilutions of the target N. meningitidis DNA and calculated by Probit regression analysis. RESULTS: Six LAMP assay-specific primers were developed targeting the ctrA gene of N. meningitidis, which is conserved in all meningococcal serogroups. The LAMP primers did not amplify DNA from other bacterial DNA tested, showing 100% specificity. The use of 0.4 M betaine increased the sensitivity and stability of the reaction. LAMP-CRISPR/Cas detected meningococcal serogroups (B, C, W). The assay showed no cross-reactivity and was specific for N. meningitidis. The LOD was 74 (95% CI: 47-311) N. meningitidis copies. The LAMP-CRISPR/Cas performed well compared to the gold standard. In the 139 samples from suspected patients, the sensitivity and specificity of the test were 91% and 99% respectively. CONCLUSION: This developed and optimized method can complement for the available gold standard for the timely diagnosis of meningococcal meningitis and meningococcemia.

Single-Cell Sequencing Reveals Functional Alterations in Tuberculosis.

Despite its importance, the functional heterogeneity surrounding the dynamics of interactions between mycobacterium tuberculosis and human immune cells in determining host immune strength and tuberculosis (TB) outcomes, remains far from understood. This work now describes the development of a new technological platform to elucidate the immune function differences in individuals with TB, integrating single-cell RNA sequencing and cell surface antibody sequencing to provide both genomic and phenotypic information from the same samples. Single-cell analysis of 23 990 peripheral blood mononuclear cells from a new cohort of primary TB patients and healthy controls enables to not only show four distinct immune phenotypes (TB, myeloid, and natural killer (NK) cells), but also determine the dynamic changes in cell population abundance, gene expression, developmental trajectory, transcriptomic regulation, and cell-cell signaling. In doing so, TB-related changes in immune cell functions demonstrate that the immune response is mediated through host T cells, myeloid cells, and NK cells, with TB patients showing decreased naive, cytotoxicity, and memory functions of T cells, rather than their immunoregulatory function. The platform also has the potential to identify new targets for immunotherapeutic treatment strategies to restore T cells from dysfunctional or exhausted states.

Alignment-free construction of double emulsion droplet generation devices incorporating surface wettability contrast.

Although polydimethylsiloxane (PDMS) is a versatile and easy-to-use material for microfluidics, its inherent hydrophobicity often necessitates specific hydrophilic treatment to fabricate microchip architectures for generating double emulsions. These additional processing steps frequently lead to increased complexity, potentially creating barriers to the wider use of promising microfluidic techniques. Here we describe an alignment-free spatial hydrophilic PDMS patterning technique to produce devices for the creation of double emulsions using combinations of PDMS and PDMS/surfactant bilayers. The technique enables us to achieve selective patterning and alignment-free bonding, producing reliable and reproducible water-in-oil-in-water W/O/W droplet emulsions. Our method involves processing devices in a vertical orientation, with the wetting transition contrast being achieved simply by imaging whilst adjusting the PDMS pouring speed (using a mobile phone, for example). We successfully obtain hydrophilic surfaces without distinguishable hydrophobic recovery using a range of surfactant concentrations. Droplet emulsions were produced with low coefficients of variation aligned with those generated with other, more complex, techniques (e.g. 3.8% and 3.1% for the inner and outer diameters, respectively). As a further example, the methods were also demonstrated for liposome production. In future we anticipate that the technique may be applied to other fields, including e.g. reagent delivery, DNA amplification, and encapsulated cell studies.

LoCKAmp: lab-on-PCB technology for <3 minute virus genetic detection.

The recent COVID-19 outbreak highlighted the need for lab-on-chip diagnostic technology fit for real-life deployment in the field. Existing bottlenecks in multistep analytical microsystem integration and upscalable, standardized fabrication techniques delayed the large-scale deployment of lab-on-chip solutions during the outbreak, throughout a global diagnostic test shortage. This study presents a technology that has the potential to address these issues by redeploying and repurposing the ubiquitous printed circuit board (PCB) technology and manufacturing infrastructure. We demonstrate the first commercially manufactured, miniaturised lab-on-PCB device for loop-mediated isothermal amplification (LAMP) genetic detection of SARS-CoV-2. The system incorporates a mass-manufactured, continuous-flow PCB chip with ultra-low cost fluorescent detection circuitry, rendering it the only continuous-flow µLAMP platform with off-the-shelf optical detection components. Ultrafast, SARS-CoV-2 RNA amplification in wastewater samples was demonstrated within 2 min analysis, at concentrations as low as 17 gc µL-1. We further demonstrate our device operation by detecting SARS-CoV-2 in 20 human nasopharyngeal swab samples, without the need for any RNA extraction or purification. This renders the presented miniaturised nucleic-acid amplification-based diagnostic test the fastest reported SARS-CoV-2 genetic detection platform, in a practical implementation suitable for deployment in the field. This technology can be readily extended to the detection of alternative pathogens or genetic targets for a very broad range of applications and matrices. LoCKAmp lab-on-PCB chips are currently mass-manufactured in a commercial, ISO-compliant PCB factory, at a small-scale production cost of £2.50 per chip. Thus, with this work, we demonstrate a high technology-readiness-level lab-on-chip-based genetic detection system, successfully benchmarked against standard analytical techniques both for wastewater and nasopharyngeal swab SARS-CoV-2 detection.

Phage-Inducible Chromosomal Islands as a Diagnostic Platform to Capture and Detect Bacterial Pathogens.

Phage-inducible chromosomal islands (PICIs) are a family of phage satellites that hijack phage components to facilitate their mobility and spread. Recently, these genetic constructs are repurposed as antibacterial drones, enabling a new toolbox for unorthodox applications in biotechnology. To illustrate a new suite of functions, the authors have developed a user-friendly diagnostic system, based upon PICI transduction to selectively enrich bacteria, allowing the detection and sequential recovery of Escherichia coli and Staphylococcus aureus. The system enables high transfer rates and sensitivities in comparison with phages, with detection down to ≈50 CFU mL-1 . In contrast to conventional detection strategies, which often rely on nucleic acid molecular assays, and cannot differentiate between dead and live organisms, this approach enables visual sensing of viable pathogens only, through the expression of a reporter gene encoded in the PICI. The approach extends diagnostic sensing mechanisms beyond cell-free synthetic biology strategies, enabling new synthetic biology/biosensing toolkits.

Sequence terminus dependent PCR for site-specific mutation and modification detection.

The detection of changes in nucleic acid sequences at specific sites remains a critical challenge in epigenetics, diagnostics and therapeutics. To date, such assays often require extensive time, expertise and infrastructure for their implementation, limiting their application in clinical settings. Here we demonstrate a generalizable method, named Specific Terminal Mediated Polymerase Chain Reaction (STEM-PCR) for the detection of DNA modifications at specific sites, in a similar way as DNA sequencing techniques, but using simple and widely accessible PCR-based workflows. We apply the technique to both for site-specific methylation and co-methylation analysis, importantly using a bisulfite-free process - so providing an ease of sample processing coupled with a sensitivity 20-fold better than current gold-standard techniques. To demonstrate the clinical applicability through the detection of single base mutations with high sensitivity and no-cross reaction with the wild-type background, we show the bisulfite-free detection of SEPTIN9 and SFRP2 gene methylation in patients (as key biomarkers in the prognosis and diagnosis of tumours).

Meniscal and Articular Cartilage Predictors of Outcome After Revision ACL Reconstruction: A 6-Year Follow-up Cohort Study.

BACKGROUND: Meniscal and chondral damage is common in the patient undergoing revision anterior cruciate ligament (ACL) reconstruction. PURPOSE: To determine if meniscal and/or articular cartilage pathology at the time of revision ACL surgery significantly influences a patient's outcome at 6-year follow-up. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: Patients undergoing revision ACL reconstruction were prospectively enrolled between 2006 and 2011. Data collection included baseline demographics, surgical technique, pathology, treatment, and scores from 4 validated patient-reported outcome instruments: International Knee Documentation Committee (IKDC), Knee injury and Osteoarthritis Outcome Score (KOOS), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), and Marx Activity Rating Scale. Patients were followed up at 6 years and asked to complete the identical set of outcome instruments. Regression analysis assessed the meniscal and articular cartilage pathology risk factors for clinical outcomes 6 years after revision ACL reconstruction. RESULTS: An overall 1234 patients were enrolled (716 males, 58%; median age, 26 years). Surgeons reported the pathology at the time of revision surgery in the medial meniscus (45%), lateral meniscus (36%), medial femoral condyle (43%), lateral femoral condyle (29%), medial tibial plateau (11%), lateral tibial plateau (17%), patella (30%), and trochlea (21%). Six-year follow-up was obtained on 79% of the sample (980/1234). Meniscal pathology and articular cartilage pathology (medial femoral condyle, lateral femoral condyle, lateral tibial plateau, trochlea, and patella) were significant drivers of poorer patient-reported outcomes at 6 years (IKDC, KOOS, WOMAC, and Marx). The most consistent factors driving outcomes were having a medial meniscal excision (either before or at the time of revision surgery) and patellofemoral articular cartilage pathology. Six-year Marx activity levels were negatively affected by having either a repair/excision of the medial meniscus (odds ratio range, 1.45-1.72; P≤ .04) or grade 3-4 patellar chondrosis (odds ratio, 1.72; P = .04). Meniscal pathology occurring before the index revision surgery negatively affected scores on all KOOS subscales except for sports/recreation (P < .05). Articular cartilage pathology significantly impaired all KOOS subscale scores (P < .05). Lower baseline outcome scores, higher body mass index, being a smoker, and incurring subsequent surgery all significantly increased the odds of reporting poorer clinical outcomes at 6 years. CONCLUSION: Meniscal and chondral pathology at the time of revision ACL reconstruction has continued significant detrimental effects on patient-reported outcomes at 6 years after revision surgery.

Mobile Health Access and Utilisation in Uganda: Knowledge, Attitudes and Perceptions of Health and Veterinary Workers.

Background: Despite its strong growth in many parts of the world, mobile health access is still limited in low- and middle-income countries. Among the many factors restricting implementation are the lack of information security, insufficient evidence base, low sensitization, and user acceptance. Limited evidence has been obtained on current practices, perceptions, and user acceptability in such settings. The aim of this study was therefore to evaluate the knowledge, attitude, and perceptions on mobile health use among health workers and veterinary officers in Uganda. Materials and Methods: A cross-section study was carried out, targeting health practitioners in both hospitals and veterinary laboratories/clinics. A structured questionnaire was used to collect data from the Central, Eastern, Northern, and Western representative regions. Interviews with selected health workers were also conducted as well as a focused group discussion. Results: Of the 120 health practitioners that were targeted, a total of 80 health workers and 7 veterinary practitioners participated in the study of which 46% were men and 54% women. Majority of the health workers had encountered m-health but had never used it, whereas the 15 practitioners who had used it before the survey did not use it for disease diagnosis in hospitals but used it for ordering medicine online, for patient consultations with the doctors, result interpretation, tracking women menstrual cycles, tuberculosis assessment. Discussion and Conclusion: Participants expressed significant interest in mobile health as it addresses key challenges including challenges with management of patient data, and long patient queues, which would ultimately improve service delivery. However, there is some skepticism about access as many rural facilities lack access to smartphones and stable internet.

Changes in Oxygenation Levels During Moderate Altitude Simulation (Hypoxia-Induced): A Pilot Study Investigating the Impact of Skin Pigmentation in Pulse Oximetry.

The current COVID-19 pandemic has shown us that the pulse oximeter is a key medical device for monitoring blood-oxygen levels non-invasively in patients with chronic or acute illness. It has also emphasised limitations in accuracy for individuals with darker skin pigmentation, calling for new methods to provide better measurements. The aim of our study is to identify the impact of skin pigmentation on pulse oximeter measurements. We also explored the benefits of a multi-wavelength approach with an induced change of arterial oxygen saturation. A total of 20 healthy volunteers were recruited. We used time domain diffuse reflectance spectroscopy (TDDRS) from a broad band light source, collecting spectra from the index finger along with three different pulse oximeters used simultaneously for monitoring purposes. Five acute hypoxic events were induced by administering 11% FiO2, produced by a Hypoxico altitude training system, for 120 sec through a face mask with a one-way valve. Our multi-wavelength approach revealed a correlation between the signature of skin pigmentation and the dynamic range of oxygen saturation measurements. Principal component analysis (PCA) showed separation between a range of different pigmented volunteers (PC1 = 56.00%) and oxygen saturation (PC2 = 22.99%). This emphasises the need to take into account skin pigmentation in oximeter measurements. This preliminary study serves to validate the need to better understand the impact of skin pigmentation absorption on optical readings in pulse oximeters. Multi-wavelength approaches have the potential to enable robust and accurate measurements across diverse populations.

Tracking Molecular Diffusion across Biomaterials' Interfaces Using Stimulated Raman Scattering.

The determination of molecular diffusion across biomaterial interfaces, including those involving hydrogels and tissues remains important, underpinning the understanding of a broad range of processes including, for example, drug delivery. Current techniques using Raman spectroscopy have previously been established as a method to quantify diffusion coefficients, although when using spontaneous Raman spectroscopy, the signal can be weak and dominated by interferences such as background fluorescence (including biological autofluoresence). To overcome these issues, we demonstrate the use of the stimulated Raman scattering technique to obtain measurements in soft tissue samples that have good signal-to-noise ratios and are largely free from fluorescence interference. As a model illustration of a small metabolite/drug molecule being transported through tissue, we use deuterated (d7-) glucose and monitor the Raman C-D band in a spectroscopic region free from other Raman bands. The results show that although mass transport follows a diffusion process characterized by Fick's laws within hydrogel matrices, more complex mechanisms appear within tissues.

Descriptive Characteristics and Outcomes of Patients Undergoing Revision Anterior Cruciate Ligament Reconstruction With and Without Tunnel Bone Grafting.

BACKGROUND: Lytic or malpositioned tunnels may require bone grafting during revision anterior cruciate ligament reconstruction (rACLR) surgery. Patient characteristics and effects of grafting on outcomes after rACLR are not well described. PURPOSE: To describe preoperative characteristics, intraoperative findings, and 2-year outcomes for patients with rACLR undergoing bone grafting procedures compared with patients with rACLR without grafting. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: A total of 1234 patients who underwent rACLR were prospectively enrolled between 2006 and 2011. Baseline revision and 2-year characteristics, surgical technique, pathology, treatment, and patient-reported outcome instruments (International Knee Documentation Committee [IKDC], Knee injury and Osteoarthritis Outcome Score [KOOS], Western Ontario and McMaster Universities Osteoarthritis Index, and Marx Activity Rating Scale [Marx]) were collected, as well as subsequent surgery information, if applicable. The chi-square and analysis of variance tests were used to compare group characteristics. RESULTS: A total of 159 patients (13%) underwent tunnel grafting-64 (5%) patients underwent 1-stage and 95 (8%) underwent 2-stage grafting. Grafting was isolated to the femur in 31 (2.5%) patients, the tibia in 40 (3%) patients, and combined in 88 patients (7%). Baseline KOOS Quality of Life (QoL) and Marx activity scores were significantly lower in the 2-stage group compared with the no bone grafting group (P≤ .001). Patients who required 2-stage grafting had more previous ACLRs (P < .001) and were less likely to have received a bone-patellar tendon-bone or a soft tissue autograft at primary ACLR procedure (P≤ .021) compared with the no bone grafting group. For current rACLR, patients undergoing either 1-stage or 2-stage bone grafting were more likely to receive a bone-patellar tendon-bone allograft (P≤ .008) and less likely to receive a soft tissue autograft (P≤ .003) compared with the no bone grafting group. At 2-year follow-up of 1052 (85%) patients, we found inferior outcomes in the 2-stage bone grafting group (IKDC score = 68; KOOS QoL score = 44; KOOS Sport/Recreation score = 65; and Marx activity score = 3) compared with the no bone grafting group (IKDC score = 77; KOOS QoL score = 63; KOOS Sport/Recreation score = 75; and Marx activity score = 7) (P≤ .01). The 1-stage bone graft group did not significantly differ compared with the no bone grafting group. CONCLUSION: Tunnel bone grafting was performed in 13% of our rACLR cohort, with 8% undergoing 2-stage surgery. Patients treated with 2-stage grafting had inferior baseline and 2-year patient-reported outcomes and activity levels compared with patients not undergoing bone grafting. Patients treated with 1-stage grafting had similar baseline and 2-year patient-reported outcomes and activity levels compared with patients not undergoing bone grafting.

Returning to Activity After Anterior Cruciate Ligament Revision Surgery: An Analysis of the Multicenter Anterior Cruciate Ligament Revision Study (MARS) Cohort at 2 Years Postoperative.

BACKGROUND: Patients with anterior cruciate ligament (ACL) revision report lower outcome scores on validated knee questionnaires postoperatively compared to cohorts with primary ACL reconstruction. In a previously active population, it is unclear if patient-reported outcomes (PROs) are associated with a return to activity (RTA) or vary by sports participation level (higher level vs. recreational athletes). HYPOTHESES: Individual RTA would be associated with improved outcomes (ie, decreased knee symptoms, pain, function) as measured using validated PROs. Recreational participants would report lower PROs compared with higher level athletes and be less likely to RTA. STUDY DESIGN: Cohort study; Level of evidence, 2. METHODS: There were 862 patients who underwent a revision ACL reconstruction (rACLR) and self-reported physical activity at any level preoperatively. Those who did not RTA reported no activity 2 years after revision. Baseline data included patient characteristics, surgical history and characteristics, and PROs: International Knee Documentation Committee questionnaire, Marx Activity Rating Scale, Knee injury and Osteoarthritis Outcome Score, and the Western Ontario and McMaster Universities Osteoarthritis Index. A binary indicator was used to identify patients with same/better PROs versus worse outcomes compared with baseline, quantifying the magnitude of change in each direction, respectively. Multivariable regression models were used to evaluate risk factors for not returning to activity, the association of 2-year PROs after rACLR surgery by RTA status, and whether each PRO and RTA status differed by participation level. RESULTS: At 2 years postoperatively, approximately 15% did not RTA, with current smokers (adjusted odds ratio [aOR] = 3.3; P = .001), female patients (aOR = 2.9; P < .001), recreational participants (aOR = 2.0; P = .016), and those with a previous medial meniscal excision (aOR = 1.9; P = .013) having higher odds of not returning. In multivariate models, not returning to activity was significantly associated with having worse PROs at 2 years; however, no clinically meaningful differences in PROs at 2 years were seen between participation levels. CONCLUSION: Recreational-level participants were twice as likely to not RTA compared with those participating at higher levels. Within a previously active cohort, no RTA was a significant predictor of lower PROs after rACLR. However, among patients who did RTA after rACLR, approximately 20% reported lower outcome scores. Most patients with rACLR who were active at baseline improved over time; however, patients who reported worse outcomes at 2 years had a clinically meaningful decline across all PROs.

Reprogrammed tracrRNAs enable repurposing of RNAs as crRNAs and sequence-specific RNA biosensors.

In type II CRISPR systems, the guide RNA (gRNA) comprises a CRISPR RNA (crRNA) and a hybridized trans-acting CRISPR RNA (tracrRNA), both being essential in guided DNA targeting functions. Although tracrRNAs are diverse in sequence and structure across type II CRISPR systems, the programmability of crRNA-tracrRNA hybridization for Cas9 is not fully understood. Here, we reveal the programmability of crRNA-tracrRNA hybridization for Streptococcus pyogenes Cas9, and in doing so, redefine the capabilities of Cas9 proteins and the sources of crRNAs, providing new biosensing applications for type II CRISPR systems. By reprogramming the crRNA-tracrRNA hybridized sequence, we show that engineered crRNA-tracrRNA interactions can not only enable the design of orthogonal cellular computing devices but also facilitate the hijacking of endogenous small RNAs/mRNAs as crRNAs. We subsequently describe how these re-engineered gRNA pairings can be implemented as RNA sensors, capable of monitoring the transcriptional activity of various environment-responsive genomic genes, or detecting SARS-CoV-2 RNA in vitro, as an Atypical gRNA-activated Transcription Halting Alarm (AGATHA) biosensor.

Programmable design of isothermal nucleic acid diagnostic assays through abstraction-based models.

Accelerating the design of nucleic acid amplification methods remains a critical challenge in the development of molecular tools to identify biomarkers to diagnose both infectious and non-communicable diseases. Many of the principles that underpin these mechanisms are often complex and can require iterative optimisation. Here we focus on creating a generalisable isothermal nucleic acid amplification methodology, describing the systematic implementation of abstraction-based models for the algorithmic design and application of assays. We demonstrate the simplicity, ease and flexibility of our approach using a software tool that provides amplification schemes de novo, based upon a user-input target sequence. The abstraction of reaction network predicts multiple reaction pathways across different strategies, facilitating assay optimisation for specific applications, including the ready design of multiplexed tests for short nucleic acid sequence miRNAs or for difficult pathogenic targets, such as highly mutating viruses.

Multiplex, Real-Time, Point-of-care RT-LAMP for SARS-CoV-2 Detection Using the HFman Probe.

Viral evolution impacts diagnostic test performance through the emergence of variants with sequences affecting the efficiency of primer binding. Such variants that evade detection by nucleic acid-based tests are subject to selective pressure, enabling them to spread more efficiently. Here, we report a variant-tolerant diagnostic test for SARS-CoV-2 using a loop-mediated isothermal nucleic acid-based amplification (LAMP) assay containing high-fidelity DNA polymerase and a high-fidelity DNA polymerase-medicated probe (HFman probe). In addition to demonstrating a high tolerance to variable SARS-CoV-2 viral sequences, the mechanism also overcomes frequently observed limitations of LAMP assays arising from non-specific amplification within multiplexed reactions performed in a single "pot". Results showed excellent clinical performance (sensitivity 94.5%, specificity 100%, n = 190) when compared directly to a commercial gold standard reverse transcription quantitative polymerase chain reaction assay for the extracted RNA from nasopharyngeal samples and the capability of detecting a wide range of sequences containing at least alpha and delta variants. To further validate the test with no sample processing, directly from nasopharyngeal swabs, we also detected SARS-CoV-2 in positive clinical samples (n = 49), opening up the possibility for the assay's use in decentralized testing.

High-Speed Diagnosis of Bacterial Pathogens at the Single Cell Level by Raman Microspectroscopy with Machine Learning Filters and Denoising Autoencoders.

Accurate and rapid identification of infectious bacteria is important in medicine. Raman microspectroscopy holds great promise in performing label-free identification at the single-cell level. However, due to the naturally weak Raman signal, it is a challenge to build extensive databases and achieve both accurate and fast identification. Here, we used signal-to-noise ratio (SNR) as a standard indicator for Raman data quality and performed bacterial identification using 11, 141 single-cell Raman spectra from nine bacterial strains. Subsequently, using two machine learning methods, a simple filter, and a neural network-based denoising autoencoder (DAE), we demonstrated 92% (simple filter using 1 s/cell spectra) and 84% (DAE using 0.1 s/cell spectra) identification accuracy. Our machine learning-aided Raman analysis paves the way for high-speed Raman microspectroscopic clinical diagnostics.

Paper microfluidic implementation of loop mediated isothermal amplification for early diagnosis of hepatitis C virus.

The early diagnosis of active hepatitis C virus (HCV) infection remains a significant barrier to the treatment of the disease and to preventing the associated significant morbidity and mortality seen, worldwide. Current testing is delayed due to the high cost, long turnaround times and high expertise needed in centralised diagnostic laboratories. Here we demonstrate a user-friendly, low-cost pan-genotypic assay, based upon reverse transcriptase loop mediated isothermal amplification (RT-LAMP). We developed a prototype device for point-of-care use, comprising a LAMP amplification chamber and lateral flow nucleic acid detection strips, giving a visually-read, user-friendly result in <40 min. The developed assay fulfils the current guidelines recommended by World Health Organisation and is manufactured at minimal cost using simple, portable equipment. Further development of the diagnostic test will facilitate linkage between disease diagnosis and treatment, greatly improving patient care pathways and reducing loss to follow-up, so assisting in the global elimination strategy.

Association Between Graft Choice and 6-Year Outcomes of Revision Anterior Cruciate Ligament Reconstruction in the MARS Cohort.

BACKGROUND: Although graft choice may be limited in the revision setting based on previously used grafts, most surgeons believe that graft choice for anterior cruciate ligament (ACL) reconstruction is an important factor related to outcome. HYPOTHESIS: In the ACL revision setting, there would be no difference between autograft and allograft in rerupture rate and patient-reported outcomes (PROs) at 6-year follow-up. STUDY DESIGN: Cohort study; Level of evidence, 2. METHODS: Patients who had revision surgery were identified and prospectively enrolled in this cohort study by 83 surgeons over 52 sites. Data collected included baseline characteristics, surgical technique and pathology, and a series of validated PRO measures. Patients were followed up at 6 years and asked to complete the identical set of PRO instruments. Incidence of additional surgery and reoperation because of graft failure were also recorded. Multivariable regression models were used to determine the predictors (risk factors) of PROs, graft rerupture, and reoperation at 6 years after revision surgery. RESULTS: A total of 1234 patients including 716 (58%) men were enrolled. A total of 325 (26%) underwent revision using a bone-patellar tendon-bone (BTB) autograft; 251 (20%), soft tissue autograft; 289 (23%), BTB allograft; 302 (25%), soft tissue allograft; and 67 (5%), other graft. Questionnaires and telephone follow-up for subsequent surgery information were obtained for 809 (66%) patients, while telephone follow-up was only obtained for an additional 128 patients for the total follow-up on 949 (77%) patients. Graft choice was a significant predictor of 6-year Marx Activity Rating Scale scores (P = .024). Specifically, patients who received a BTB autograft for revision reconstruction had higher activity levels than did patients who received a BTB allograft (odds ratio [OR], 1.92; 95% CI, 1.25-2.94). Graft rerupture was reported in 5.8% (55/949) of patients by their 6-year follow-up: 3.5% (16/455) of patients with autografts and 8.4% (37/441) of patients with allografts. Use of a BTB autograft for revision resulted in patients being 4.2 times less likely to sustain a subsequent graft rupture than if a BTB allograft were utilized (P = .011; 95% CI, 1.56-11.27). No significant differences were found in graft rerupture rates between BTB autograft and soft tissue autografts (P = .87) or between BTB autografts and soft tissue allografts (P = .36). Use of an autograft was found to be a significant predictor of having fewer reoperations within 6 years compared with using an allograft (P = .010; OR, 0.56; 95% CI, 0.36-0.87). CONCLUSION: BTB and soft tissue autografts had a decreased risk in graft rerupture compared with BTB allografts. BTB autografts were associated with higher activity level than were BTB allografts at 6 years after revision reconstruction. Surgeons and patients should consider this information when choosing a graft for revision ACL reconstruction.