Efficient synthesis of CRISPR-Cas13a-antimicrobial capsids against MRSA facilitated by silent mutation incorporation.

In response to the escalating global threat of antimicrobial resistance, our laboratory has established a phagemid packaging system for the generation of CRISPR-Cas13a-antimicrobial capsids targeting methicillin-resistant Staphylococcus aureus (MRSA). However, a significant challenge arose during the packaging process: the unintentional production of wild-type phages alongside the antimicrobial capsids. To address this issue, the phagemid packaging system was optimized by strategically incorporated silent mutations. This approach effectively minimized contamination risks without compromising packaging efficiency. The study identified the indispensable role of phage packaging genes, particularly terL-terS, in efficient phagemid packaging. Additionally, the elimination of homologous sequences between the phagemid and wild-type phage genome was crucial in preventing wild-type phage contamination. The optimized phagemid-LSAB(mosaic) demonstrated sequence-specific killing, efficiently eliminating MRSA strains carrying target antibiotic-resistant genes. While acknowledging the need for further exploration across bacterial species and in vivo validation, this refined phagemid packaging system offers a valuable advancement in the development of CRISPR-Cas13a-based antimicrobials, shedding light on potential solutions in the ongoing battle against bacterial infections.

FRET probe for detecting two mutations in one EGFR mRNA.

Technologies for visualizing and tracking RNA are essential in molecular biology, including in disease-related fields. In this study, we propose a novel probe set (DAt-probe and T-probe) that simultaneously detects two mutations in the same RNA using fluorescence resonance energy transfer (FRET). The DAt-probe carrying the fluorophore Atto488 and the quencher Dabcyl were used to detect a cancer mutation (exon19del), and the T-probe carrying the fluorophore Tamra was used to detect drug resistance mutations (T790M) in epidermal growth factor receptor (EGFR) mRNA. These probes were designed to induce FRET when both mutations were present in the mRNA. Gel electrophoresis confirmed that the two probes could efficiently bind to the mutant mRNA. We measured the FRET ratios using wild-type and double-mutant RNAs and found a significant difference between them. Even in living cells, the FRET probe could visualize mutant RNA. As a result, we conclude that this probe set provides a method for detecting two mutations in the single EGFR mRNA via FRET.

Systems approach to improving traumatic brain injury care in Myanmar: a mixed-methods study from lived experience to discrete event simulation.

OBJECTIVES: Traumatic brain injury (TBI) is a global health problem, whose management in low-resource settings is hampered by fragile health systems and lack of access to specialist services. Improvement is complex, given the interaction of multiple people, processes and institutions. We aimed to develop a mixed-method approach to understand the TBI pathway based on the lived experience of local people, supported by quantitative methodologies and to determine potential improvement targets. DESIGN: We describe a systems approach based on narrative exploration, participatory diagramming, data collection and discrete event simulation (DES), conducted by an international research collaborative. SETTING: The study is set in the tertiary neurotrauma centre in Yangon General Hospital, Myanmar, in 2019-2020 (prior to the SARS-CoV2 pandemic). PARTICIPANTS: The qualitative work involved 40 workshop participants and 64 interviewees to explore the views of a wide range of stakeholders including staff, patients and relatives. The 1-month retrospective admission snapshot covered 85 surgical neurotrauma admissions. RESULTS: The TBI pathway was outlined, with system boundaries defined around the management of TBI once admitted to the neurosurgical unit. Retrospective data showed 18% mortality, 71% discharge to home and an 11% referral rate. DES was used to investigate the system, showing its vulnerability to small surges in patient numbers, with critical points being CT scanning and observation ward beds. This explorative model indicated that a modest expansion of observation ward beds to 30 would remove the flow-limitations and indicated possible consequences of changes. CONCLUSIONS: A systems approach to improving TBI care in resource-poor settings may be supported by simulation and informed by qualitative work to ground it in the direct experience of those involved. Narrative interviews, participatory diagramming and DES represent one possible suite of methods deliverable within an international partnership. Findings can support targeted improvement investments despite coexisting resource limitations while indicating concomitant risks.

Casemix, management, and mortality of patients rreseceiving emergency neurosurgery for traumatic brain injury in the Global Neurotrauma Outcomes Study: a prospective observational cohort study.

BACKGROUND: Traumatic brain injury (TBI) is increasingly recognised as being responsible for a substantial proportion of the global burden of disease. Neurosurgical interventions are an important aspect of care for patients with TBI, but there is little epidemiological data available on this patient population. We aimed to characterise differences in casemix, management, and mortality of patients receiving emergency neurosurgery for TBI across different levels of human development. METHODS: We did a prospective observational cohort study of consecutive patients with TBI undergoing emergency neurosurgery, in a convenience sample of hospitals identified by open invitation, through international and regional scientific societies and meetings, individual contacts, and social media. Patients receiving emergency neurosurgery for TBI in each hospital's 30-day study period were all eligible for inclusion, with the exception of patients undergoing insertion of an intracranial pressure monitor only, ventriculostomy placement only, or a procedure for drainage of a chronic subdural haematoma. The primary outcome was mortality at 14 days postoperatively (or last point of observation if the patient was discharged before this time point). Countries were stratified according to their Human Development Index (HDI)-a composite of life expectancy, education, and income measures-into very high HDI, high HDI, medium HDI, and low HDI tiers. Mixed effects logistic regression was used to examine the effect of HDI on mortality while accounting for and quantifying between-hospital and between-country variation. FINDINGS: Our study included 1635 records from 159 hospitals in 57 countries, collected between Nov 1, 2018, and Jan 31, 2020. 328 (20%) records were from countries in the very high HDI tier, 539 (33%) from countries in the high HDI tier, 614 (38%) from countries in the medium HDI tier, and 154 (9%) from countries in the low HDI tier. The median age was 35 years (IQR 24-51), with the oldest patients in the very high HDI tier (median 54 years, IQR 34-69) and the youngest in the low HDI tier (median 28 years, IQR 20-38). The most common procedures were elevation of a depressed skull fracture in the low HDI tier (69 [45%]), evacuation of a supratentorial extradural haematoma in the medium HDI tier (189 [31%]) and high HDI tier (173 [32%]), and evacuation of a supratentorial acute subdural haematoma in the very high HDI tier (155 [47%]). Median time from injury to surgery was 13 h (IQR 6-32). Overall mortality was 18% (299 of 1635). After adjustment for casemix, the odds of mortality were greater in the medium HDI tier (odds ratio [OR] 2·84, 95% CI 1·55-5·2) and high HDI tier (2·26, 1·23-4·15), but not the low HDI tier (1·66, 0·61-4·46), relative to the very high HDI tier. There was significant between-hospital variation in mortality (median OR 2·04, 95% CI 1·17-2·49). INTERPRETATION: Patients receiving emergency neurosurgery for TBI differed considerably in their admission characteristics and management across human development settings. Level of human development was associated with mortality. Substantial opportunities to improve care globally were identified, including reducing delays to surgery. Between-hospital variation in mortality suggests changes at an institutional level could influence outcome and comparative effectiveness research could identify best practices. FUNDING: National Institute for Health Research Global Health Research Group.

Management and outcomes following emergency surgery for traumatic brain injury - A multi-centre, international, prospective cohort study (the Global Neurotrauma Outcomes Study).

INTRODUCTION: Traumatic brain injury (TBI) accounts for a significant amount of death and disability worldwide and the majority of this burden affects individuals in low-and-middle income countries. Despite this, considerable geographical differences have been reported in the care of TBI patients. On this background, we aim to provide a comprehensive international picture of the epidemiological characteristics, management and outcomes of patients undergoing emergency surgery for traumatic brain injury (TBI) worldwide. METHODS AND ANALYSIS: The Global Neurotrauma Outcomes Study (GNOS) is a multi-centre, international, prospective observational cohort study. Any unit performing emergency surgery for TBI worldwide will be eligible to participate. All TBI patients who receive emergency surgery in any given consecutive 30-day period beginning between 1st of November 2018 and 31st of December 2019 in a given participating unit will be included. Data will be collected via a secure online platform in anonymised form. The primary outcome measures for the study will be 14-day mortality (or survival to hospital discharge, whichever comes first). Final day of data collection for the primary outcome measure is February 13th. Secondary outcome measures include return to theatre and surgical site infection. ETHICS AND DISSEMINATION: This project will not affect clinical practice and has been classified as clinical audit following research ethics review. Access to source data will be made available to collaborators through national or international anonymised datasets on request and after review of the scientific validity of the proposed analysis by the central study team.

Assisted Education for Specialized Medicine: A Sustainable Development Plan for Neurosurgery in Myanmar.

BACKGROUND: With only 4 active certified neurosurgeons for a population of 50 million, neurosurgical care was seriously underdeveloped in Myanmar in 2012. Together with the local neurosurgical community, Swiss Neurosurgeons International started a program of assisted education with the aim of increasing the neurosurgical capacity to 60 active neurosurgeons by 2023. METHODS: Our program included a fully sponsored fellowship at an academic hospital in Switzerland for every Myanmar neurosurgeon in training to observe contemporary neurosurgery and the organization of regular workshops, lectures, and educational surgeries in Myanmar. Halfway through the program, we performed a survey to assess its benefits and shortcomings. RESULTS: In 2019, the number of active neurosurgeons in Myanmar had increased to 20, with 50 more in training. Intracranial pressure monitoring, cervical spine surgery, transsphenoidal surgery, and intraoperative electrophysiology have become established procedures. Our survey showed an increased interest by the residents in tumor and vascular surgery and a new interest in spinal surgery. The overall rating of the fellowship was "very good," with language barriers, cultural differences, and coping with inclement weather reported as the greatest difficulties. CONCLUSIONS: Since the start of our project in 2013, a series of contemporary neurosurgical technologies and procedures have been introduced to Myanmar with the help of Swiss Neurosurgeons International. Our survey results have shown the strong points of the ongoing educational program and the weaknesses to be addressed. Midway through our 10-year project, with an expanded and improved education program, our goal of 60 active neurosurgeons in Myanmar by 2023 seems well within reach.

The First Neurosurgery Boot Camp in Southeast Asia: Evaluating Impact on Knowledge and Regional Collaboration in Yangon, Myanmar.

BACKGROUND: For the first time in Southeast Asia, a Fundamentals of Neurosurgery Boot Camp was held at the University of Medicine 1 in Yangon, Myanmar, February 24-26, 2017. The aim of this course was to teach and train fundamental skills to neurosurgery residents. METHODS: The Myanmar Neurosurgical Society, Foundation for International Education in Neurosurgery, Society for Neurological Surgeons, The University of Medicine 1 in Yangon, Myanmar, and the Henry Ford Department of Neurosurgery developed a 2-day resident training course. Day 1 activities consisted of lectures by faculty, small group case discussions, and industry-supported demonstrations of surgical techniques. Day 2 activities consisted of hands-on skill stations for common neurosurgical procedures with each station supervised by attending faculty. Written evaluations were distributed before the meeting, immediately after the meeting, and 6 months after the meeting. RESULTS: Boot camp attendees included 40 residents and 24 neurosurgical faculty from Myanmar, Cambodia, Nepal, Singapore, South Korea, Thailand, and Vietnam. There were 35 evaluations completed before the boot camp, 34 completed immediately after boot camp, and 20 completed 6 months after boot camp. Knowledge of participants improved from 62.75% before boot camp to 71.50% 6 months after boot camp (P = 0.046). CONCLUSIONS: Boot camps provide fundamental didactic and technical exposure to trainees in developed and developing countries and help standardize training in basic neurosurgical competencies, while exposing local faculty to important teaching methods. This model provides a sustainable solution to educational needs and demonstrates to local neurosurgeons how they can take ownership of the educational process.

Evaluation of a tuberculosis active case finding project in peri-urban areas, Myanmar: 2014-2016.

OBJECTIVES: We assessed the effect of an active case finding (ACF) project on tuberculosis (TB) case notification and the yields from a household and neigbourhood intervention (screening contacts of historical index TB patients diagnosed >24months ago) and a community intervention (screening attendants of health education sessions/mobile clinics). DESIGN: Cross-sectional analysis of project records, township TB registers and annual TB reports. RESULTS: In the household and neigbourhood intervention, of 56,709 people screened, 1,076 were presumptive TB and 74 patients were treated for active TB with a screening yield of 0.1% and a yield from presumptive cases of 6.9%. In the community intervention, of 162,881 people screened, 4,497 were presumptive TB and 984 were treated for active TB with a screening yield of 0.6% and yield from presumptive cases of 21.9%. Of active TB cases, 94% were new, 89% were pulmonary, 44% were bacteriologically-confirmed and 5% had HIV. Case notification rates per 100,000 in project townships increased from 142 during baseline (2011-2013) to 148 during intervention (2014-2016) periods. CONCLUSIONS: The yield from household and neigbourhood intervention was lower than community intervention. This finding highlights reconsidering the strategy of screening of contacts from historical index cases. Strategies to reach high-risk groups should be explored for future ACF interventions to increase yield of TB.

A systems approach to trauma care in Myanmar: from health partnership to academic collaboration.

Experience from a variety of disciplines suggests that improving healthcare, particularly in resource-poor environments, can benefit from a systems approach. However, putting this into practice is challenging, especially in the context of an international institutional health partnership. In this article, we outline how a systems approach to the improvement of trauma care has informed both clinical improvement and academic collaboration as part of an ongoing partnership involving Cambridge University Hospitals NHS Foundation Trust, the University of Cambridge, and Cambridge Global Health Partnerships in the UK, and Yangon General Hospital, University of Medicine 1, and the Tropical Health and Education Trust (THET) in Myanmar. Improving and researching trauma care is an exemplar of a systems problem, requiring an understanding of the relevant people, equipment, processes, institutions, and power structures that result in the delivery of care at all points of the patient's journey from injury to rehabilitation. Exploring this in the explicit context of traumatic brain injury is one of the research themes of the NIHR Global Health Research Group on Neurotrauma, allowing systems research to directly inform efforts at practical improvement.

Safe and sustainable: the extracranial approach toward frontoethmoidal meningoencephalocele repair.

OBJECTIVE Although rare, frontoethmoidal meningoencephaloceles continue to pose a challenge to neurosurgeons and plastic reconstructive surgeons. Especially when faced with limited infrastructure and resources, establishing reliable and safe surgical techniques is of paramount importance. The authors present a case series in order to evaluate a previously proposed concise approach for meningoencephalocele repair, with a focus on sustainability of internationally driven surgical efforts. METHODS Between 2001 and 2016, a total of 246 patients with frontoethmoidal meningoencephaloceles were treated using a 1-stage extracranial approach by a single surgeon in the Department of Neurosurgery of the Yangon General Hospital in Yangon, Myanmar, initially assisted by European surgeons. Outcomes and complications were evaluated. RESULTS A total of 246 patients (138 male and 108 female) were treated. Their ages ranged from 75 days to 32 years (median 8 years). The duration of follow-up ranged between 4 weeks and 16 years (median 4 months). Eighteen patients (7.3%) showed signs of increased intracranial pressure postoperatively, and early CSF rhinorrhea was observed in 27 patients (11%), with 5 (2%) of them requiring operative dural repair. In 8 patients, a decompressive lumbar puncture was performed. There were 8 postoperative deaths (3.3%) due to meningitis. In 15 patients (6.1%), recurrent herniation of brain tissue was observed; this herniation led to blindness in 1 case. The remaining patients all showed good to very good aesthetic and functional results. CONCLUSIONS A minimally invasive, purely extracranial approach to frontoethmoidal meningoencephalocele repair may serve well, especially in middle- and low-income countries. This case series points out how the frequently critiqued lack of sustainability in the field of humanitarian surgical missions, as well as the often-cited missing aftercare and dependence on foreign supporters, can be circumvented by meticulous training of local surgeons.

High treatment success rate among multidrug-resistant tuberculosis patients in Myanmar, 2012-2014: a retrospective cohort study.

Background: Since 2011, Myanmar has adopted domiciliary care for multidrug-resistant tuberculosis (MDR-TB) patients and implemented several patient-support measures such as community-based directly observed treatment, nutritional support and financial incentives for patients and providers. We assessed treatment outcomes among MDR-TB patients registered for treatment in the Yangon and Mandalay Regions of Myanmar during 2012-2014 and factors associated with unfavourable treatment outcomes. Methods: We performed a retrospective cohort study involving secondary analysis of routine programmatic data extracted from the electronic MDR-TB treatment registries. We calculated the adjusted risk ratio (aRR) and 95% confidence interval (CI). Results: Of 2185 MDR-TB patients (75% HIV tested, 14% HIV positive with 70% of them receiving antiretroviral therapy), 1746 (80%) were successfully treated (cured and treatment completed) and 20% had unfavourable outcomes (14% died, 3% lost to follow-up, 2% failure and 1% not evaluated). Compared with young patients (<25 y), patients 25-54 y of age (aRR 2.0 [95% CI 1.3 to 2.9]) and >55 y (aRR 3.2 [95% CI 2.1 to 4.8]) were more likely to have unfavourable outcomes. HIV-positive patients (especially not receiving ART; aRR 2.2 [95% CI 1.4 to 3.6]) and patients with 'unknown HIV status' (aRR 1.9 [95% CI 1.5-2.4]) had a higher risk of unfavourable outcomes compared with HIV-negative patients. Conclusions: Treatment success was high and deaths accounted for three-fourths of unfavourable outcomes. Joint care and management of MDR-TB and HIV co-infected patients should be strengthened.