Comprehensive identification of pathogenic microbes and antimicrobial resistance genes in food products using nanopore sequencing-based metagenomics.

Foodborne pathogens, particularly antimicrobial-resistant (AMR) bacteria, remain a significant threat to global health. Given the limitations of conventional culture-based approaches, which are limited in scope and time-consuming, metagenomic sequencing of food products emerges as a promising solution. This method provides a fast and comprehensive way to detect the presence of pathogenic microbes and antimicrobial resistance genes (ARGs). Notably, nanopore long-read sequencing provides more accurate bacterial taxonomic classification in comparison to short-read sequencing. Here, we revealed the impact of food types and attributes (origin, retail place, and food processing methods) on microbial communities and the AMR profile using nanopore metagenomic sequencing. We analyzed a total of 260 food products, including raw meat, sashimi, and ready-to-eat (RTE) vegetables. Clostridium botulinum, Acinetobacter baumannii, and Vibrio parahaemolyticus were identified as the top three foodborne pathogens in raw meat and sashimi. Importantly, even with low pathogen abundance, higher percentages of samples containing carbapenem and cephalosporin resistance genes were identified in chicken and RTE vegetables, respectively. In parallel, our results demonstrated that fresh, peeled, and minced foods exhibited higher levels of pathogenic bacteria. In conclusion, this comprehensive study offers invaluable data that can contribute to food safety assessments and serve as a basis for quality indicators.

Optimizing a mentorship program from the perspective of academic medicine leadership - a qualitative study.

BACKGROUND: Effective mentorship is an important contributor to academic success. Given the critical role of leadership in fostering mentorship, this study sought to explore the perspectives of departmental leadership regarding 1) current departmental mentorship processes; and 2) crucial components of a mentorship program that would enhance the effectiveness of mentorship. METHODS: Department Division Directors (DDDs), Vice-Chairs, and Mentorship Facilitators from the Department of Medicine at the University of Toronto Temerty Faculty of Medicine were interviewed between April and December 2021 using a semi-structured guide. Interviews were audio-recorded and transcribed verbatim, then coded. Analysis occurred in 2 steps: 1) codes were organized to identify emergent themes; then 2) the Social Ecological Model (SEM) was applied to interpret the findings. RESULTS: Nineteen interviews (14 DDDs, 3 Vice-Chairs, and 2 Mentorship Facilitator) were completed. Analysis revealed three themes: (1) a culture of mentorship permeated the department as evidenced by rigorous mentorship processes, divisional mentorship innovations, and faculty that were keen to mentor; (2) barriers to the establishment of effective mentoring relationships existed at 3 levels: departmental, interpersonal (mentee-mentor relationships), and mentee; and (3) strengthening the culture of mentorship could entail scaling up pre-existing mentorship processes and promoting faculty engagement. Application of SEM highlighted critical program features and determined that two components of interventions (creating tools to measure mentorship outcomes and systems for mentor recognition) were potential enablers of success. CONCLUSIONS: Establishing 'mentorship outcome measures' can incentivize and maintain relationships. By tangibly delineating departmental expectations for mentorship and creating systems that recognize mentors, these measures can contribute to a culture of mentorship.

Long-Read Sequencing with Hierarchical Clustering for Antiretroviral Resistance Profiling of Mixed Human Immunodeficiency Virus Quasispecies.

BACKGROUND: HIV infections often develop drug resistance mutations (DRMs), which can increase the risk of virological failure. However, it has been difficult to determine if minor mutations occur in the same genome or in different virions using Sanger sequencing and short-read sequencing methods. Oxford Nanopore Technologies (ONT) sequencing may improve antiretroviral resistance profiling by allowing for long-read clustering. METHODS: A new ONT sequencing-based method for profiling DRMs in HIV quasispecies was developed and validated. The method used hierarchical clustering of long amplicons that cover regions associated with different types of antiretroviral drugs. A gradient series of an HIV plasmid and 2 plasma samples was prepared to validate the clustering performance. The ONT results were compared to those obtained with Sanger sequencing and Illumina sequencing in 77 HIV-positive plasma samples to evaluate the diagnostic performance. RESULTS: In the validation study, the abundance of detected quasispecies was concordant with the predicted result with the R2 of > 0.99. During the diagnostic evaluation, 59/77 samples were successfully sequenced for DRMs. Among 18 failed samples, 17 were below the limit of detection of 303.9 copies/µL. Based on the receiver operating characteristic analysis, the ONT workflow achieved an F1 score of 0.96 with a cutoff of 0.4 variant allele frequency. Four cases were found to have quasispecies with DRMs, in which 2 harbored quasispecies with more than one class of DRMs. Treatment modifications were recommended for these cases. CONCLUSIONS: Long-read sequencing coupled with hierarchical clustering could differentiate the quasispecies resistance profiles in HIV-infected samples, providing a clearer picture for medical care.

The precedence effect in spatial hearing manifests in cortical neural population responses.

BACKGROUND: To localize sound sources accurately in a reverberant environment, human binaural hearing strongly favors analyzing the initial wave front of sounds. Behavioral studies of this "precedence effect" have so far largely been confined to human subjects, limiting the scope of complementary physiological approaches. Similarly, physiological studies have mostly looked at neural responses in the inferior colliculus, the main relay point between the inner ear and the auditory cortex, or used modeling of cochlear auditory transduction in an attempt to identify likely underlying mechanisms. Studies capable of providing a direct comparison of neural coding and behavioral measures of sound localization under the precedence effect are lacking. RESULTS: We adapted a "temporal weighting function" paradigm previously developed to quantify the precedence effect in human for use in laboratory rats. The animals learned to lateralize click trains in which each click in the train had a different interaural time difference. Computing the "perceptual weight" of each click in the train revealed a strong onset bias, very similar to that reported for humans. Follow-on electrocorticographic recording experiments revealed that onset weighting of interaural time differences is a robust feature of the cortical population response, but interestingly, it often fails to manifest at individual cortical recording sites. CONCLUSION: While previous studies suggested that the precedence effect may be caused by early processing mechanisms in the cochlea or inhibitory circuitry in the brainstem and midbrain, our results indicate that the precedence effect is not fully developed at the level of individual recording sites in the auditory cortex, but robust and consistent precedence effects are observable only in the auditory cortex at the level of cortical population responses. This indicates that the precedence effect emerges at later cortical processing stages and is a significantly "higher order" feature than has hitherto been assumed.

Rate of Refracture After Removal of Hardware in Pediatric Femur Fractures.

PURPOSE: Both flexible intramedullary nails (FINs) and plate osteosynthesis are commonly used for the treatment of femoral shaft fractures in pediatric patients. The purpose of this study is to determine the refracture rate after hardware removal in pediatric femur fractures. METHODS: This is a retrospective cohort study that utilized the Pediatric Health Information System database to determine the number of pediatric patients ages 4 to 10 who underwent surgical fixation of a femur fracture and subsequent hardware removal between the years 2015 and 2019. All patients had a minimum of a 2-year follow-up to assess for refracture. Patients with metabolic bone disease, neuromuscular conditions, bone fragility disorders, nutritional deficiencies, and pathologic fractures were excluded. RESULTS: Of the total, 2805 pediatric patients with 2881 femoral shaft fractures who underwent FIN (48.4%), plate fixation (36.1%), splinting/casting (14.9%), or external fixation (0.6%) were included. The mean age of patients with index fracture was 7.2 years (SD, 2.1) and 69% were males. Eight hundred eighty patients (60%) in the FIN group had their hardware removed compared with 693 patients (68%) in the plate fixation group ( P = 0.07), at an average of 287 ± 191 days versus 320 ± 203 days ( P = 0.03). Refracture occurred in 13 patients (1.5%) who had their hardware retained and in 21 patients (1.4%) who had their hardware removed ( P = 0.732). Among 65% of patients who underwent hardware removal, refracture occurred in 7 patients with FIN (0.8%) and 14 patients with plate fixation (2.2%) ( P = 0.04). Refracture occurred within 365 days from hardware removal in 1 patient with FIN (0.1%) and 7 patients with plate fixation (1%) ( P = 0.01). In logistic regression, patients with FIN fixation had lower odds of refracture after hardware removal compared with plate fixation (adjusted odds ratio: 0.39; 95% CI: 0.15-0.97). Age and payor status did not reach statistical significance in multivariate analysis. CONCLUSIONS: The rate of refracture after hardware removal for pediatric patients with a femoral shaft fracture was similar between patients with hardware retained and removal. However, patients with FIN had a lower rate of refracture AFTER hardware removal compared with plate fixation. This information can be helpful for advising families regarding the risks of refracture after hardware removal. LEVEL OF EVIDENCE: Level IV-retrospective cohort study.

The Clinical Utility of Two High-Throughput 16S rRNA Gene Sequencing Workflows for Taxonomic Assignment of Unidentifiable Bacterial Pathogens in Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Bacterial pathogens that cannot be identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) are occasionally encountered in clinical laboratories. The 16S rRNA gene is often used for sequence-based analysis to identify these bacterial species. Nevertheless, traditional Sanger sequencing is laborious, time-consuming, and low throughput. Here, we compared two commercially available 16S rRNA gene sequencing tests that are based on Illumina and Nanopore sequencing technologies, respectively, in their ability to identify the species of 172 clinical isolates that failed to be identified by MALDI-TOF MS. Sequencing data were analyzed by the respective built-in programs (MiSeq Reporter software of Illumina and Epi2me of Nanopore) and BLAST+ (v2.11.0). Their agreement with Sanger sequencing on species-level identification was determined. Discrepancies were resolved by whole-genome sequencing. The diagnostic accuracy of each workflow was determined using the composite sequencing result as the reference standard. Despite the high base-calling accuracy of Illumina sequencing, we demonstrated that the Nanopore workflow had a higher taxonomic resolution at the species level. Using built-in analysis algorithms, the concordance of Sanger 16S with the Illumina and Nanopore workflows was 33.14% and 87.79%, respectively. The agreement was 65.70% and 83.14%, respectively, when BLAST+ was used for analysis. Compared with the reference standard, the diagnostic accuracy of Nanopore 16S was 96.36%, which was identical to that of Sanger 16S and better than that of Illumina 16S (69.07%). The turnaround time of the Illumina workflow and the Nanopore workflow was 78 h and 8.25 h, respectively. The per-sample cost of the Illumina and Nanopore workflows was US$28.5 and US$17.7, respectively.

Human Papillomavirus Vaccination Trends and Disparities in the United States: Who Is Getting Left Behind?

BACKGROUND: United States guidelines recommend human papillomavirus (HPV) vaccination for males and females up to 26, with more recent extended coverage for those 27 to 45 years based on discussion with patients' clinician. This study seeks to assess trends and disparities of vaccination in the United States based on demographic characteristics. METHODS: Data were obtained from the National Health and Nutrition Examination Survey between 2007 and 2016. χ2 Analyses were used for statistical methods. RESULTS: Of 12,844 participants (median age, 22 years; range, 9-36 years), 2711 (21.3%) initiated HPV vaccination, of which 1358 (56.3%) completed the 3-dose vaccination series. Vaccination rates were higher in females compared with males (24.6% vs 13.0%; P < 0.001) and in Whites compared to Mexican Americans (22.6% vs 19.4%; P = 0.02). The uninsured had lower vaccination rates than private insurance and Medicaid (12.5% vs 22.4% vs 28.5%; P < 0.001). We divided the 10 year study into five separate periods (2007-2008, 2009-2010, 2011-2012, 2013-2014, and 2015-2016) to analyze trends. Vaccine initiation increased from 19.6% to 49.6% for 14-19-year olds (P < 0.001), 10.4% to 35.5% for females (P < 0.001), and 8.5% to 32.9% for Blacks (P < 0.001). Although on trend analyses, the vaccination rates with the highest proportional increase were found in those: older than 25 to 29 years (4.56-fold), Mexican Americans (4.56 fold), below high school education (2.32 fold), and low income group (2.90 fold) over time. CONCLUSIONS: The HPV vaccination rates in Mexican Americans increased nearly 5-fold over the last 10 years. However, their vaccination rates continue to lag behind Whites and Blacks.

Racial disparities in high-risk uterine cancer histologic subtypes: A United States Cancer Statistics study.

OBJECTIVE: Black women with uterine cancer on average have worse survival outcomes compared to White women, in part due to higher rates of aggressive, non-endometrioid subtypes. However, analyses of incidence trends by specific high-risk subtypes are lacking, including those with hysterectomy and active pregnancy correction. The objective of our study was to evaluate racial disparities in age-adjusted incidence of non-endometrioid uterine cancer in 720,984 patients. METHODS: Data were obtained from United States Cancer Statistics using SEER*Stat. We used the Behavioral Risk Factor Surveillance System to correct for hysterectomy and active pregnancy. Age-adjusted, corrected incidence of uterine cancer from 2001 to 2016 and annual percent change (APC) were calculated using Joinpoint regression. RESULTS: Of 720,984 patients, 560,131 (77.7%) were White, 72,328 (10.0%) were Black, 56,239 (7.8%) were Hispanic, and 22,963 (3.2%) were Asian/Pacific Islander. Age-adjusted incidence of uterine cancer increased from 40.8 (per 100,000) in 2001 to 42.9 in 2016 (APC = 0.5, p < 0.001). Black women had the highest overall incidence at 49.5 (APC = 2.3, p < 0.001). The incidence of non-endometrioid subtypes was higher in Black compared to White women, with the most pronounced differences seen in serous carcinoma (9.1 vs. 3.0), carcinosarcoma (6.1 vs. 1.8), and leiomyosarcoma (1.3 vs. 0.6). In particular, Black women aged 70-74 with serous carcinoma had the highest incidence (61.3) and the highest APC (7.3, p < 0.001). CONCLUSIONS: Black women have a two to four-fold higher incidence of high-risk uterine cancer subtypes, particularly serous carcinoma, carcinosarcoma, and leiomyosarcoma, compared to White women after correcting for hysterectomy and active pregnancy.

Five-Strand Versus Four-Strand Hamstring Autografts in Anterior Cruciate Ligament Reconstruction-A Prospective Randomized Controlled Study.

PURPOSE: To compare the clinical outcomes of the routine use of 5-strand hamstring grafts (where possible) with those of 4-strand grafts in primary anterior cruciate ligament (ACL) reconstruction. METHODS: A total of 64 patients were enrolled in a prospective randomized controlled study comparing the use of 5-strand and 4-strand semitendinosus-gracilis autografts in single bundle ACL reconstruction (n = 32 in each group). Four participants in each group were lost to follow-up and were excluded from the outcome analysis. The outcomes of 28 patients in the 5-strand group and 28 patients in the 4-strand group were analyzed. The diameters of all grafts were measured intraoperatively. Patients were assessed postoperatively at 2 years with objective assessments (anterior knee laxity using the KT-2000 arthrometer, Lachman test, pivot-shift test, hop test) and patient-reported outcome scores (Lysholm knee score, Knee Injury and Osteoarthritis Outcome Score, International Knee Documentation Committee subjective knee score, SF-36 physical and mental components, Tegner activity scale). Postoperative graft ruptures were also noted. RESULTS: There were improvements in all outcome measures postoperatively regardless of the number of graft strands. When we compared the study and control groups, there were no significant differences in all subjective and objective outcome measures except the Knee Injury and Osteoarthritis Outcome Score symptoms score (5-strand group 93.3 ± 9.2 vs 4-strand group 86.2 ± 14.7, P = .04). The KT-2000 side-to-side difference was 2.79 ± 2.11 mm in the 5-strand group and 2.54 ± 1.75 mm in the 4-strand group (P = .63). The 5-strand study group had 2 graft ruptures at 1 year, whereas the 4-strand control group had one partial graft rupture at 6 months. CONCLUSIONS: At 2-year follow-up, the routine use of the 5-strand hamstring tendon autograft was not superior to that of the quadrupled or 4-strand graft in primary ACL reconstruction. LEVEL OF EVIDENCE: Level I, prospective randomized controlled trial.

Epidemiology of patients with anterior cruciate ligament injuries undergoing reconstruction surgery in a multi-ethnic Asian population.

We conducted an epidemiological study to identify the demographics and pre-operative characteristics of patients undergoing primary anterior cruciate ligament (ACL) reconstruction in a multi-ethnic Asian population. A retrospective study was performed on 696 patients who registered in our ACL reconstruction registry from January 2013 to August 2016. Important inter-ethnic differences were found. Indian patients were significantly older compared to Malay and Chinese patients. Malay and Indian patients were more likely to be male, overweight, and obese compared to Chinese patients. Soccer was the predominant sport involved, although ethnic predominance in specific sports exists. Novel findings from this study include increased prevalence of concomitant chondral injuries in the Chinese population compared to Malays and Indians, and significantly higher rates of concomitant intra-articular injuries in soccer and basketball compared to netball. Identification of these high-risk subgroups would serve useful in guiding the formulation of prevention strategies within our multi-ethnic community.

Microsecond sensitivity to envelope interaural time differences in rats.

Currently, there is controversy around whether rats can use interaural time differences (ITDs) to localize sound. Here, naturalistic pulse train stimuli were used to evaluate the rat's sensitivity to onset and ongoing ITDs using a two-alternative forced choice sound lateralization task. Pulse rates between 50 Hz and 4.8 kHz with rectangular or Hanning windows were delivered with ITDs between ±175 µs over a near-field acoustic setup. Similar to other mammals, rats performed with 75% accuracy at ∼50 µs ITD, demonstrating that rats are highly sensitive to envelope ITDs.

The presence of concomitant intra-articular injuries and bone bruise does not affect pre-operative knee pain and symptoms in patients undergoing anterior cruciate ligament reconstruction.

PURPOSE: Increased knee pain at the time of anterior cruciate ligament (ACL) reconstruction may predict increased pain post-operatively, a prolonged recovery and a more difficult rehabilitation. The main objective of our study was to identify preoperative factors, such as concomitant intra-articular injuries and bone bruises, that may be associated with increased knee pain and symptoms in patients undergoing ACL reconstruction. METHODS: Patient data was queried from our institution's prospectively maintained ACL reconstruction registry. Two-hundred and seventy patients who underwent primary ACL reconstruction within 3 months of injury were included in the study. Predictors such as demographic characteristics (age, body mass index and gender) and injury characteristics (mechanism of injury, meniscal injury, chondral injury and bone bruise) were recorded. The association between the pre-operative knee injury and Osteoarthritis Outcome Score (KOOS) pain and symptom subscales and the Short Form-36 (SF-36) bodily pain subscale, and the predictors were assessed using logistic regression for categorical variables and linear regression for continuous variables. RESULTS: The mean age of our patient group was 25.4 years with 211 out of 270 (78%) being males. Bone bruise was present in 243 patients (90%), meniscal injury in 165 (61%) patients and chondral injury in 40 (15%) patients. The presence of bone bruise, meniscal injury or chondral injury was not significantly associated with worse preoperative KOOS pain and symptom and SF-36 bodily pain scores. Other factors that were not associated were demographic characteristics (age, BMI and gender) and mechanism of injury. CONCLUSION: The presence of bone bruise and concomitant intra-articular injuries does not affect pre-operative knee pain and symptoms in patients undergoing ACL reconstruction within 3 months of injury. This knowledge would aid the surgeon in pre-operative counselling, and prognostication of post-operative pain and rehabilitation after ACL reconstruction.

Anterior cruciate ligament reconstruction performed within 12 months of the index injury is associated with a lower rate of medial meniscus tears.

PURPOSE: To verify the correlation of time to surgery with the prevalence of concomitant intra-articular injuries detected on arthroscopy during anterior cruciate ligament (ACL) reconstruction. METHODS: The medical records of 653 patients who underwent ACL reconstruction surgery were retrospectively analyzed. Univariate and multivariate logistic regression analysis was performed to identify factors that were associated with the presence of at least one intra-articular injury, medial meniscus tears, lateral meniscus tears and chondral injuries at the time of surgery. Further univariate analysis was conducted to determine the earliest time-point for surgery, after which the rate of concomitant injuries was significantly higher. RESULTS: Longer time to surgery (OR 1.019 95% CI 1.010, 1.028, p = 0.000), male sex (OR 1.695 95% CI 1.074, 2.675 p = 0.023), and higher BMI (OR 1.050 95% CI 1.006, 1.097 p = 0.025) were correlated with a higher prevalence of medial meniscus tears. There was an increased prevalence of medial meniscus tears when surgery was carried out more than 12 months after the index injury (OR 2.274 95% CI 1.469, 3.522, p = 0.000). The correlation between longer time to surgery and chondral injuries approached statistical significance (OR 1.006 95% CI 0.999, 1.012, p = 0.073). However, a longer time to surgery was not associated with an increased prevalence of lateral meniscus tears (OR 1.003 95% CI 0.998, 1.009, p = n.s.). CONCLUSIONS: Longer time to surgery is associated with an increased prevalence of medial meniscus tears in ACL reconstruction. Surgery performed within 12 months of the index injury reduces the prevalence of medial meniscus tears. Prioritizing males and overweight patients for counselling and early intervention can be considered. LEVEL OF EVIDENCE: Therapeutic level III retrospective cohort study.

Two year outcomes of minimally invasive hallux valgus surgery.

BACKGROUND: We report our experience with the Minimally Invasive Chevron Akin (MICA) technique for correcting hallux valgus, and evaluate its effectiveness and associated complications. METHODS: Case series of 13 feet with mild to moderate symptomatic hallux valgus treated surgically from July 2013 to December 2014, with at least 48-months follow-up. Patients were assessed pre-operatively and post-operatively with radiographical measurements (Hallux Valgus Angle (HVA) and Intermetatarsal Angle (IMA)) and clinical scores (American Orthopaedic Foot and Ankle Society (AOFAS), 36-Item Short Form Health Survery (SF-36), Visual Analog Scale (VAS)). RESULTS: Mean HVA and IMA decreased from 30.4° and 13.9°-10.9° and 10.2° respectively (p<0.05). The mean AOFAS score improved from an average of 59.0-93.7 (p<0.05). All patients reported a VAS score of 0 post-operatively, and the 4 SF-36 domains improved significantly (p<0.05). CONCLUSIONS: The MICA technique is a safe and effective method in the surgical correction of mild to moderate hallux valgus deformity, and continued use is justified.

A study on the chiral inversion of mandelic acid in humans.

Mandelic acid is a chiral metabolite of the industrial pollutant styrene and is used in chemical skin peels, as a urinary antiseptic and as a component of other medicines. In humans, S-mandelic acid undergoes rapid chiral inversion to R-mandelic acid by an undefined pathway but it has been proposed to proceed via the acyl-CoA esters, S- and R-2-hydroxy-2-phenylacetyl-CoA, in an analogous pathway to that for Ibuprofen. This study investigates chiral inversion of mandelic acid using purified human recombinant enzymes known to be involved in the Ibuprofen chiral inversion pathway. Both S- and R-2-hydroxy-2-phenylacetyl-CoA were hydrolysed to mandelic acid by human acyl-CoA thioesterase-1 and -2 (ACOT1 and ACOT2), consistent with a possible role in the chiral inversion pathway. However, human α-methylacyl-CoA racemase (AMACR; P504S) was not able to catalyse exchange of the α-proton of S- and R-2-hydroxy-2-phenylacetyl-CoA, a requirement for chiral inversion. Both S- and R-2-phenylpropanoyl-CoA were epimerised by AMACR, showing that it is the presence of the hydroxy group that prevents epimerisation of R- and S-2-hydroxy-2-phenylacetyl-CoAs. The results show that it is unlikely that 2-hydroxy-2-phenylacetyl-CoA is an intermediate in the chiral inversion of mandelic acid, and that the chiral inversion of mandelic acid is via a different pathway to that of Ibuprofen and related drugs.

A dataset on two decades of Japan's energy investments in Southeast Asian countries, 2000-2020.

Between 2000 and 2020, Japan played a crucial role in transforming Southeast Asia's energy sector, contributing significantly to local, national, and regional development. This period saw substantial Japanese investments in various energy sources, including renewable energy initiatives and continued use of fossil fuels, while emphasising capacity development. Our research involved an extensive and systematic inventory of secondary resources, with meticulous validation and fact-checking of critical project data. The resulting dataset provides funding amounts and details channelled through Japanese government-backed institutions and private corporations. This dataset could enhance our understanding of Japan-supported energy infrastructure and soft-skill capacity-building projects, allowing us to analyse further how these investments were aligned with the broader economic and sustainability objectives of Southeast Asian countries. Covering investment types, funding sources, and project locations, this valuable resource is useful for scholars across disciplines. Asian studies researchers can gain insights into Japan's strategic involvement in the region, while energy studies specialists can gain a nuanced understanding of the evolving energy finance landscape. Energy policy experts can also use this data to analyse the implications of Japan's contributions to Southeast Asia's energy transition.

Good short-term results following arthroscopic deepening trochleoplasty combined with medial patellofemoral ligament reconstruction: surgical technique and outcomes.

PURPOSE: To report short-term results and clinical outcomes of arthroscopic deepening trochleoplasty combined with medial patellofemoral ligament (MPFL) reconstruction utilizing standard arthroscopic instruments in patients of recurrent patellar dislocation and trochlear dysplasia. METHODS: This is a case series of 13 patients between the ages of 14 and 20 years who presented with recurrent patellar dislocation and severe trochlear dysplasia (Dejour grade D). They were treated surgically using an arthroscopic technique from February 2017 to January 2019 and were followed for 18 months. Patients were assessed preoperatively and postoperatively (at 6, 12, and 18 months) with clinical scores (Tegner Activity Score, Lysholm Knee Score, and Kujala Score). RESULTS: There were 69.2% females, and the mean age was 16.4 â€‹± â€‹2.0 years. There were statistically significant improvements in the mean Lysholm and Kujala scores when comparing pre-operative and post-operative scores at every follow-up landmark (p â€‹< â€‹0.05). Comparing the preoperative and 18-month postoperative scores-the Lysholm score improved from 68.2 â€‹± â€‹10.3 to 98.7 â€‹± â€‹2.1 (p â€‹< â€‹0.001), and the Kujala score improved from 50.3 â€‹± â€‹12.0 to 95.4 â€‹± â€‹4.8 (p â€‹< â€‹0.001). Five patients were able to achieve premorbid Tegner activity levels at 12 months, with an additional 5 patients achieving the same premorbid Tegner activity at the 18-month mark. The remaining 3 patients were able to attain >90% of their activity level at 18 months' follow-up. No complications were observed during the follow-up period. CONCLUSIONS: This proposed arthroscopic deepening trochleoplasty technique combined with MPFL reconstruction has demonstrated excellent and reproducible early clinical outcomes. LEVEL OF EVIDENCE: IV.

Genetic mechanisms of co-emergence of INH-resistant Mycobacterium tuberculosis strains during the standard course of antituberculosis therapy.

The incidence of isoniazid (INH) resistant Mycobacterium tuberculosis is increasing globally. This study aimed to identify the molecular mechanisms behind the development of INH resistance in M. tuberculosis strains collected from the same patients during the standard course of treatment. Three M. tuberculosis strains were collected from a patient before and during antituberculosis (anti-TB) therapy. The strains were characterized using phenotypic drug susceptibility tests, Mycobacterial Interspersed Repeated Unit-Variable-Number Tandem Repeats (MIRU-VNTR), and whole-genome sequencing (WGS) to identify mutations associated with INH resistance. To validate the role of the novel mutations in INH resistance, the mutated katG genes were electroporated into a KatG-deleted M. tuberculosis strain (GA03). Three-dimensional structures of mutated KatG were modeled to predict their impact on INH binding. The pre-treatment strain was susceptible to INH. However, two INH-resistant strains were isolated from the patient after anti-TB therapy. MIRU-VNTR and WGS revealed that the three strains were clonally identical. A missense mutation (P232L) and a nonsense mutation (Q461Stop) were identified in the katG of the two post-treatment strains, respectively. Transformation experiments showed that katG of the pre-treatment strain restored INH susceptibility in GA03, whereas the mutated katG genes from the post-treatment strains rendered negative catalase activity and INH resistance. The protein model indicated that P232L reduced INH-KatG binding affinity while Q461Stop truncated gene transcription. Our results showed that the two katG mutations, P232L and Q461Stop, accounted for the co-emergence of INH-resistant clones during anti-TB therapy. The inclusion of these mutations in the design of molecular assays could increase the diagnostic performance.IMPORTANCEThe evolution of drug-resistant strains of Mycobacterium tuberculosis within the lung lesions of a patient has a detrimental impact on treatment outcomes. This is particularly concerning for isoniazid (INH), which is the most potent first-line antimycobacterial drug. However, the precise genetic factors responsible for drug resistance in patients have not been fully elucidated, with approximately 15% of INH-resistant strains harboring unknown genetic factors. This raises concerns about the emergence of drug-resistant clones within patients, further contributing to the global epidemic of resistance. In this study, we revealed the presence of two novel katG mutations, which emerged independently due to the stress exerted by antituberculosis (anti-TB) treatment on a parental strain. Importantly, we experimentally demonstrated the functional significance of both mutations in conferring resistance to INH. Overall, this research sheds light on the genetic mechanisms underlying the evolution of INH resistance within patients and provides valuable insights for improving diagnostic performance by targeting specific mutations.

Identification of microbial community in the urban environment: The concordance between conventional culture and nanopore 16S rRNA sequencing.

Introduction: Microbes in the built environment have been implicated as a source of infectious diseases. Bacterial culture is the standard method for assessing the risk of exposure to pathogens in urban environments, but this method only accounts for <1% of the diversity of bacteria. Recently, full-length 16S rRNA gene analysis using nanopore sequencing has been applied for microbial evaluations, resulting in a rise in the development of long-read taxonomic tools for species-level classification. Regarding their comparative performance, there is, however, a lack of information. Methods: Here, we aim to analyze the concordance of the microbial community in the urban environment inferred by multiple taxonomic classifiers, including ARGpore2, Emu, Kraken2/Bracken and NanoCLUST, using our 16S-nanopore dataset generated by MegaBLAST, as well as assess their abilities to identify culturable species based on the conventional culture results. Results: According to our results, NanoCLUST was preferred for 16S microbial profiling because it had a high concordance of dominant species and a similar microbial profile to MegaBLAST, whereas Kraken2/Bracken, which had similar clustering results as NanoCLUST, was also desirable. Second, for culturable species identification, Emu with the highest accuracy (81.2%) and F1 score (29%) for the detection of culturable species was suggested. Discussion: In addition to generating datasets in complex communities for future benchmarking studies, our comprehensive evaluation of the taxonomic classifiers offers recommendations for ongoing microbial community research, particularly for complex communities using nanopore 16S rRNA sequencing.

The clinical utility of Nanopore 16S rRNA gene sequencing for direct bacterial identification in normally sterile body fluids.

The prolonged incubation period of traditional culture methods leads to a delay in diagnosing invasive infections. Nanopore 16S rRNA gene sequencing (Nanopore 16S) offers a potential rapid diagnostic approach for directly identifying bacteria in infected body fluids. To evaluate the clinical utility of Nanopore 16S, we conducted a study involving the collection and sequencing of 128 monomicrobial samples, 65 polymicrobial samples, and 20 culture-negative body fluids. To minimize classification bias, taxonomic classification was performed using 3 analysis pipelines: Epi2me, Emu, and NanoCLUST. The result was compared to the culture references. The limit of detection of Nanopore 16S was also determined using simulated bacteremic blood samples. Among the three classifiers, Emu demonstrated the highest concordance with the culture results. It correctly identified the taxon of 125 (97.7%) of the 128 monomicrobial samples, compared to 109 (85.2%) for Epi2me and 102 (79.7%) for NanoCLUST. For the 230 cultured species in the 65 polymicrobial samples, Emu correctly identified 188 (81.7%) cultured species, compared to 174 (75.7%) for Epi2me and 125 (54.3%) for NanoCLUST. Through ROC analysis on the monomicrobial samples, we determined a threshold of relative abundance at 0.058 for distinguishing potential pathogens from background in Nanopore 16S. Applying this threshold resulted in the identification of 107 (83.6%), 117 (91.4%), and 114 (91.2%) correctly detected samples for Epi2me, Emu, and NanoCLUST, respectively, in the monomicrobial samples. Nanopore 16S coupled with Epi2me could provide preliminary results within 6 h. However, the ROC analysis of polymicrobial samples exhibited a random-like performance, making it difficult to establish a threshold. The overall limit of detection for Nanopore 16S was found to be about 90 CFU/ml.