Rapid Sequence Research Articles

Evaluation of NGS DNA barcoding for biosecurity diagnostic applications: case study from banana freckle incursion in Australia

AbstractMolecular diagnostics in combination with morphological identification is the method of choice for several cryptic microbial plant pathogens. For some diagnostic applications, traditional sequencing techniques can be time consuming, making them ill-suited for biosecurity incursion responses, where accurate results are needed in real time. More rapid next generation sequencing tools must be tested and compared with traditional methods to assess their utility in biosecurity applications. Here utilizing 95 samples infected with fungal pathogen Phyllosticta cavendishii, from a recent incursion in Australia, we compare species identification success using Internal Transcribed Spacer (ITS) gene barcode on conventional Sanger and Oxford Nanopore MinION sequencing platforms. For Sanger sequencing, the average pairwise identity percentage score between generated consensus sequences and P. cavendishii sequence from holotype material on NCBI database was 99.9% ± SE 0.0 whereas for MinION sequencing the average pairwise identity percentage was 99.1% ± SE 0.1. Relatively larger consensus sequences (mean 486 bp ± SE 2.4) were generated by Sanger sequencing compared to MinION sequencing (mean 435 bp ± SE 4.6). Our results confirm that both sequencing methods can reliably identify P. cavendishii. MinION sequencing, provided quicker results compared to Sanger sequencing and demonstrated diagnostic competence, with the added advantage of being portable, for front-line “point of incursion” biosecurity applications.

Role of next generation sequencing in diagnosis and management of critically ill children with suspected monogenic disorder.

Next generation sequencing based diagnosis has emerged as a promising tool for evaluating critically ill neonates and children. However, there is limited data on its utility in developing countries. We assessed its diagnostic rate and clinical impact on management of pediatric patients with a suspected genetic disorder requiring critical care. The study was conducted at a single tertiary hospital in Northern India. We analyzed 70 children with an illness requiring intensive care and obtained a precise molecular diagnosis in 32 of 70 probands (45.3%) using diverse sequencing techniques such as clinical exome, whole exome, and whole genome. A significant change in clinical outcome was observed in 13 of 32 (40.6%) diagnosed probands with a change in medication in 11 subjects and redirection to palliative care in two subjects. Additional benefits included specific dietary management (three cases), avoidance of a major procedure (one case) and better reproductive counseling. Dramatic therapeutic responses were observed in three cases with SCN1A, SCN2A and KCNQ2-related epileptic encephalopathy. A delayed turn-around for sequencing results was perceived as a major limiting factor in the study, as rapid and ultra-rapid sequencing was not available. Achieving a precise molecular diagnosis has great utility in managing critically ill patients with suspected genetic disorders in developing countries.

More Than a Decade of Rapid Genomic Sequencing: Where Are We Now?

More Than a Decade of Rapid Genomic Sequencing: Where Are We Now?

A novel fast hybrid capture sequencing method for high-efficiency common human coronavirus whole-genome acquisition.

Rapid and accurate sequencing of the entire viral genome, coupled with continuous monitoring of genetic changes, is crucial for understanding the epidemiology of coronaviruses. We designed a novel method called micro target hybrid capture system (MT-Capture) to enable whole-genome sequencing in a timely manner. The novel design of probes used in target binding exhibits a unique and synergistic "hand-in-hand" conjugation effect. The entire hybrid capture process is within 2.5 hours, overcoming the time-consuming and complex operation characteristics of the traditional liquid-phase hybrid capture (T-Capture) system. By designing specific probes for these coronaviruses, MT-Capture effectively enriched isolated strains and 112 clinical samples of coronaviruses with cycle threshold values below 37. Compared to multiplex PCR sequencing, it does not require frequent primer updates and has higher compatibility. MT-Capture is highly sensitive and capable of tracking variants.IMPORTANCEMT-Capture is meticulously designed to enable the efficient acquisition of the target genome of the common human coronavirus. Coronavirus is a kind of virus that people are generally susceptible to and is epidemic and infectious, and it is the virus with the longest genome among known RNA viruses. Therefore, common human coronavirus samples are selected to evaluate the accuracy and sensitivity of MT-Capture. This method utilizes innovative probe designs optimized through probe conjugation techniques, greatly shortening the time and simplifying the handwork compared with traditional hybridization capture processes. Our results demonstrate that MT-Capture surpasses multiplex PCR in terms of sensitivity, exhibiting a thousandfold increase. Moreover, MT-Capture excels in the identification of mutation sites. This method not only is used to target the coronaviruses but also may be used to diagnose other diseases, including various infectious diseases, genetic diseases, or tumors.

The Effect of Full-Dose Versus Half-Dose Ketamine for Induction During Rapid Sequence Intubation on Patient Outcomes.

Prehospital rapid sequence intubation (RSI), like inpatient RSI, is not without risk of adverse effects to the patient. The most notable of these adverse effects is postintubation hemodynamic instability. Air medical providers choose induction agents for critically ill patients who require emergent airway management, some of whom may already be hemodynamically unstable prior to RSI. Ketamine is often selected as the induction agent of choice for patients who are either unstable before RSI or have a high index of suspicion of becoming unstable in the postintubation period. Although widely considered to have a good safety profile for induction, ketamine administration has been correlated with episodes of postintubation hypotension. In this retrospective literature review, the effect of using half-dose ketamine for induction in patients who show pre-RSI instability (systolic blood pressure <90 mm Hg or body mass index >30) on postintubation hemodynamics is examined.

Delayed Sequence Intubation in Children, Why Not?

Abstract Tracheal intubation in pediatric patients is a clinical scenario that can quickly become an emergency. Complication rates can potentially reach up to 60% in rapid sequence intubation. An alternate to this is delayed sequence intubation, which may reduce potential complications—mostly hypoxemia—and can be especially useful in non-cooperative children. This technique consists of the prior airway and oxygenation optimization. This is done through sedation using agents that preserve ventilatory function and protective reflexes and continuous oxygen therapy—prior and after the anesthetic induction—using nasal prongs. The objective of this narrative review is to provide a broader perspective on delayed sequence intubation by defining the concept and indications; reviewing its safety, effectiveness, and complications; and describing the anesthetic agents and oxygen therapy techniques used in this procedure.

Using nanopore sequencing to identify bacterial infection in joint replacements: a preliminary study.

This project investigates if third-generation genomic sequencing can be used to identify the species of bacteria causing prosthetic joint infections (PJIs) at the time of revision surgery. Samples of prosthetic fluid were taken during revision surgery from patients with known PJIs. Samples from revision surgeries from non-infected patients acted as negative controls. Genomic sequencing was performed using the MinION device and the rapid sequencing kit from Oxford Nanopore Technologies. Bioinformatic analysis pipelines to identify bacteria included Basic Local Alignment Search Tool, Kraken2 and MinION Detection Software, and the results were compared with standard of care microbiological cultures. Furthermore, there was an attempt to predict antibiotic resistance using computational tools including ResFinder, AMRFinderPlus and Comprehensive Antibiotic Resistance Database. Bacteria identified using microbiological cultures were successfully identified using bioinformatic analysis pipelines. Nanopore sequencing and genomic classification could be completed in the time it takes to perform joint revision surgery (2-3h). Genomic sequencing in this study was not able to predict antibiotic resistance in this time frame, this is thought to be due to a short-read length and low read depth. It can be concluded that genomic sequencing can be useful to identify bacterial species in infected joint replacements. However, further work is required to investigate if it can be used to predict antibiotic resistance within clinically relevant timeframes.

A rapid PCR-free next-generation sequencing method for comprehensive diagnosis of chromosome disease syndromes in prenatal samples.

The aim of this study is to investigate the application performance of rapid copy number variation sequencing (rCNV-seq) technology for the detection of chromosomal abnormalities during prenatal diagnosis. Samples were collected from 424 pregnant women who were at high-risk for noninvasive prenatal screening in Kunming Maternal and Child Care Hospital from January 2018 to May 2022. rCNV-seq technique was used to detect fetal chromosome abnormalities and compare the results with that of chromosomal karyotype analysis. The Result showed that 330 (77.83%, 330/424) cases indicated chromosomal abnormalities among 424 high-risk pregnant women who underwent rCNV-seq. Moreover, 94 (22.17%, 94/424) cases were discovered to have copy number variations. Among the 330 fetuses with chromosomal abnormalities, common autosomal aneuploidy was observed in 203 cases (47.87%, 203/424) and sex chromosome aneuploidy was observed in 91 cases (21.46%, 91/424). Moreover, the abnormalities in multiple chromosomes were discovered in 33 cases (7.78%, 33/424), and the rare autosomal aneuploidy was observed in 3 cases (0.71%, 3/424). There were 63 fetuses (14.86%, 63/424) with pathogenic CNVs among the 94 fetuses with variable copy numbers. Of the 245 pregnant women who voluntarily selected G-band karyotyping, 1 fetus with copy number variation had normal karyotype results, and the remaining women were consistent with rCNV-seq. Our study revealed that rCNV-seq has higher accuracy in detecting common trisomy and can also detect chromosomal microdeletions or microduplications that cannot be detected by G-banding karyotype analysis. There is no effective treatment for chromosomal diseases, so it is particularly important to prevent chromosomal diseases through genetic counseling and prenatal diagnosis of chromosomal diseases.

Practice Guidelines for Monitoring Neuromuscular Blockade-Elements to Change to Increase the Quality of Anesthesiological Procedures and How to Improve the Acceleromyographic Method.

Neuromuscular blocking agents are a crucial pharmacological element of general anesthesia. Decades of observations and scientific studies have resulted in the identification of many risks associated with the uncontrolled use of neuromuscular blocking agents during general anesthesia or an incomplete reversal of neuromuscular blockade in the postoperative period. Residual relaxation and acute postoperative respiratory depression are the most serious consequences. Cyclic recommendations have been developed by anesthesiology societies from many European countries as well as from the United States and New Zealand. The newest recommendations from the American Society of Anesthesiologists and the European Society of Anesthesiology were published in 2023. These publications contain very detailed recommendations for monitoring the dosage of skeletal muscle relaxants in the different stages of anesthesia-induction, maintenance and recovery, and the postoperative period. Additionally, there are recommendations for various special situations (for example, rapid sequence induction) and patient populations (for example, those with organ failure, obesity, etc.). The guidelines also refer to pharmacological drugs for reversing the neuromuscular transmission blockade. Despite the development of several editions of recommendations for monitoring neuromuscular blockade, observational and survey data indicate that their practical implementation is very limited. The aim of this review was to present the professional, technical, and technological factors that limit the implementation of these recommendations in order to improve the implementation of the guidelines and increase the quality of anesthesiological procedures and perioperative safety.

Rapid multiple protein sequence search by parallel and heterogeneous computation.

Protein sequence database search and multiple sequence alignment (MSA) generation is a fundamental task in many bioinformatics analyses. As the data volume of sequences continues to grow rapidly, there is an increasing need for efficient and scalable multiple sequence query algorithms for super-large databases without expensive time and computational costs. We introduce Chorus, a novel protein sequence query system that leverages parallel model and heterogeneous computation architecture to enable users to query thousands of protein sequences concurrently against large protein databases on a desktop workstation. Chorus achieves over 100x speedup over BLASTP without sacrificing sensitivity. We demonstrate the utility of Chorus through a case study of analyzing a ∼1.5TB large-scale metagenomic datasets for novel CRISPR-Cas protein discovery within 30 minutes. Chorus is open-source and its code repository is available at https://github.com/Bio-Acc/Chorus. Supplementary data are available at Bioinformatics online.

Genome Sequencing of Idiopathic Speech Delay

Genetic investigations of people with speech and language disorders can provide windows into key aspects of human biology. Most genomic research into impaired speech development has so far focused on childhood apraxia of speech (CAS), a rare neurodevelopmental disorder characterized by difficulties with coordinating rapid fine motor sequences that underlie proficient speech. In 2001, pathogenic variants of FOXP2 provided the first molecular genetic accounts of CAS aetiology. Since then, disruptions in several other genes have been implicated in CAS, with a substantial proportion of cases being explained by high-penetrance variants. However, the genetic architecture underlying other speech-related disorders remains less well understood. Thus, in the present study, we used systematic DNA sequencing methods to investigate idiopathic speech delay, as characterized by delayed speech development in the absence of a motor speech diagnosis (such as CAS), a language/reading disorder, or intellectual disability. We performed genome sequencing in a cohort of 23 children with a rigorous diagnosis of idiopathic speech delay. For roughly half of the sample (ten probands), sufficient DNA was also available for genome sequencing in both parents, allowing discovery of de novo variants. In the thirteen singleton probands, we focused on identifying loss-of-function and likely damaging missense variants in genes intolerant to such mutations. We found that one speech delay proband carried a pathogenic frameshift deletion in SETD1A, a gene previously implicated in a broader variable monogenic syndrome characterized by global developmental problems including delayed speech and/or language development, mild intellectual disability, facial dysmorphisms, and behavioural and psychiatric symptoms. Of note, pathogenic SETD1A variants have been independently reported in children with CAS in two separate studies. In other probands in our speech delay cohort, likely pathogenic missense variants were identified affecting highly conserved amino acids in key functional domains of SPTBN1 and ARF3. Overall, this study expands the phenotype spectrum associated with pathogenic SETD1A variants, to also include idiopathic speech delay without CAS or intellectual disability, and suggests additional novel potential candidate genes that may harbour high-penetrance variants that can disrupt speech development.

Comparison of Magnesium Pre-treatment With Two Different Doses of Rocuronium in Rapid Sequence Intubation: A Randomized Controlled Trial

Comparison of Magnesium Pre-treatment With Two Different Doses of Rocuronium in Rapid Sequence Intubation: A Randomized Controlled Trial

Genetic aetiologies of acute liver failure.

Acute liver failure (ALF) is a rare, rapidly evolving, clinical syndrome with devastating consequences where definitive treatment is by emergency liver transplantation. Establishing a diagnosis can be challenging and, historically, the cause of ALF was unidentified in up to half of children. However, recent technological and clinical advances in genomic medicine have led to an increasing proportion being diagnosed with monogenic aetiologies of ALF. The conditions encountered include a diverse group of inherited metabolic disorders each with prognostic and treatment implications. Often these disorders are clinically indistinguishable and may even mimic disorders of immune regulation or red cell disorders. Rapid genomic sequencing for children with ALF is, therefore, a key component in the diagnostic work up today. This review focuses on the monogenic aetiologies of ALF.

The current trend of emergency airway management: a clinical review.

Airway management is a fundamental and intricate process that involves a sequence of integrated tasks. Situations requiring emergency airway management may occur in emergency department, intensive care units, and various other spaces. Emergency airway management can face a variety of challenges during preparation, intubation, and post-intubation, and may result in significant complications for the patients. Therefore, many countries are establishing step-by-step systemization and detailed guidelines, and updating the content based on the latest research. This clinical review introduces the current trends in emergency airway management, such as emergency airway management algorithms, comparison of video and direct laryngoscopy, rapid sequence intubation, pediatric airway management, pre-hospital airway management, surgical airway management, and airway management education.

Delayed presentation of traumatic hepatothorax: An anesthetic and surgical perspective

: Traumatic diaphragmatic hernia is an uncommon sequela of blunt and penetrating trauma. It is estimated that 0.8–1.6% of patients with blunt trauma develop diaphragmatic rupture. However, the diagnosis of this condition is often overlooked, with right-sided hernias missed more frequently than left-sided hernias. Early diagnosis and repair are essential to prevent future complications such as strangulation and ischemia of intra-abdominal organs, lung collapse, and cardiovascular demise.: A 17-year-old male (180 cm; 86 kg) with no comorbidities presented to the emergency department (ED) following a high-velocity, unrestricted, rear-ended motor vehicle crash (MVC). The patient was intubated and hemodynamically stabilized in the ED, with planned surgical exploration of the abdomen. Six days following the accident, the patient was extubated; he became increasingly hypoxic and tachypneic over the next day. A CT of the chest and pelvis was ordered, confirming the diagnosis of a post-traumatic right-sided diaphragmatic hernia. An exploratory laparotomy was scheduled. Difficulties with ventilation were encountered due to suspected lung compression by the liver and movement of the endotracheal tube into the right main lung bronchus. : Traumatic diaphragmatic hernia should be considered in all patients presenting with abdominal trauma and should be suspected if respiratory distress arises during the patient’s clinical course. The recommended anesthetic approach is rapid sequence intubation with a cardiovascular-stable agent like etomidate and gastric decompression. Difficulties with intubation and ventilation should be anticipated due to the presence of the visceral organs within the thoracic cavity.

'Something that helped the whole picture': Experiences of parents offered rapid prenatal exome sequencing in routine clinical care in the English National Health Service.

In October 2020, rapid prenatal exome sequencing (pES) was introduced into routine National Health Service (NHS) care in England. This study aimed to explore parent experiences and their information and support needs from the perspective of parents offered pES and of health professionals involved in its delivery. In this qualitative study, semi-structured interviews were conducted with 42 women and 6 male partners and 63 fetal medicine and genetic health professionals. Interviews were transcribed verbatim and analysed using thematic analysis. Overall views about pES were positive and parents were grateful to be offered the test. Highlighted benefits of pES included the value of the additional information for pregnancy management and planning for future pregnancies. An anxious wait for results was common, often associated with the need to make decisions near to 24weeks in pregnancy when there are legal restrictions for late termination. Descriptions of dealing with uncertainty were also common, even when results had been returned. Many parents described pES results as informing decision-making around whether or not to terminate pregnancy. Some professionals were concerned that a non-informative result could be overly reassuring and highlighted that careful counselling was needed to ensure parents have a good understanding of what the result means for their pregnancy. Emotional support from professionals was valued; however, some parents felt that post-test support was lacking. Parents and professionals welcomed the introduction of pES. Results inform parents' decision-making around the termination of pregnancy. When there are no diagnostic findings or uncertain findings from pES, personalised counselling that considers scans and other tests are crucial. Directing parents to reliable online sources of information and providing emotional support throughout could improve their experiences of care.

Characterizing Large-Scale Human Circuit Development with In Vivo Neuroimaging.

Large-scale coordinated patterns of neural activity are crucial for the integration of information in the human brain and to enable complex and flexible human behavior across the life span. Through recent advances in noninvasive functional magnetic resonance imaging (fMRI) methods, it is now possible to study this activity and how it emerges in the living fetal brain across the second half of human gestation. This work has demonstrated that functional activity in the fetal brain has several features in keeping with highly organized networks of activity, which are undergoing a highly programmed and rapid sequence of development before birth, in which long-range connections emerge and core features of the mature functional connectome (such as hub regions and a gradient organization) are established. In this review, the findings of these studies are summarized, their relationship to the known changes in developmental neurobiology is considered, and considerations for future work in the context of limitations to the fMRI approach are presented.

Utility of Fast MRIs in Pediatric Elbow Injuries

BackgroundPediatric elbow trauma presents a diagnostic challenge for clinicians. Radiographs may be insufficient in assessing the radiocapitellar alignment and advanced imaging or invasive procedures such as an intraoperative arthrogram may be indicated. The purpose of this study is to describe the application and utility of fast magnetic resonance imaging (MRI) in the diagnosis and management of traumatic elbow injuries in pediatric patients. MethodsWe reviewed pediatric patients who underwent a fast MRI of the elbow between 2014-2022. A fast MRI was defined as an MRI using rapid sequences, accelerated conventional sequences, or fewer conventional sequences than a standard exam. Imaging for chronic or non-traumatic injuries was excluded. We identified patient demographics, MRI scan times, and clinical course. ResultsTwenty-three patients (8 females and 15 males) with fast elbow MRI scans were identified. The mean age was 5 years (range 2-12 years, SD 2 years). All patients had radiographs and four had CT scans prior to MRI. The average scanning time was 14 minutes (SD 6 minutes). Only one patient required anesthesia. Twenty of the 23 patients had a fracture identified on elbow radiographs and 8 were Monteggia fracture-dislocations. Nine of these patients demonstrated subluxation of the radiocapitellar joint requiring operative management in 8 patients, and one additional patient underwent surgery for displaced radial neck fracture. Three patients sustained an elbow dislocation without fracture identified on radiographs; of these, one had persistent radiocapitellar subluxation on MRI requiring surgical management. A total of 13 patients (56.5%) did not require operative management based on fast MRI findings. Outcome data was available for 22 patients with mean follow up was 23.6 months. One patient had residual pain and 8 had some range of motion limitation at final follow-up. All patients had reduced elbow joint on follow-up radiographs. ConclusionsFast MRIs can assist with the clinical evaluation of acute elbow injury in pediatric patients with equivocal radiographic findings and particularly in those with possible radiocapitellar subluxation/dislocation. This novel imaging technique has the potential to change clinical management, decrease the need for unnecessary procedures, and prevent complications of missed injuries.

Machine learning based prediction of image quality in prostate MRI using rapid localizer images.

Diagnostic performance of prostate MRI depends on high-quality imaging. Prostate MRI quality is inversely proportional to the amount of rectal gas and distention. Early detection of poor-quality MRI may enable intervention to remove gas or exam rescheduling, saving time. We developed a machine learning based quality prediction of yet-to-be acquired MRI images solely based on MRI rapid localizer sequence, which can be acquired in a few seconds. The dataset consists of 213 (147 for training and 64 for testing) prostate sagittal T2-weighted (T2W) MRI localizer images and rectal content, manually labeled by an expert radiologist. Each MRI localizer contains seven two-dimensional (2D) slices of the patient, accompanied by manual segmentations of rectum for each slice. Cascaded and end-to-end deep learning models were used to predict the quality of yet-to-be T2W, DWI, and apparent diffusion coefficient (ADC) MRI images. Predictions were compared to quality scores determined by the experts using area under the receiver operator characteristic curve and intra-class correlation coefficient. In the test set of 64 patients, optimal versus suboptimal exams occurred in 95.3% (61/64) versus 4.7% (3/64) for T2W, 90.6% (58/64) versus 9.4% (6/64) for DWI, and 89.1% (57/64) versus 10.9% (7/64) for ADC. The best performing segmentation model was 2D U-Net with ResNet-34 encoder and ImageNet weights. The best performing classifier was the radiomics based classifier. A radiomics based classifier applied to localizer images achieves accurate diagnosis of subsequent image quality for T2W, DWI, and ADC prostate MRI sequences.

Tracing the disc: The novel qualitative morphometric MRI based disc degeneration classification system.

This study aimed to develop a classification system for lumbar disc degeneration using routine magnetic resonance images (MRIs) that is easily applicable and unaffected by existing classifications' limitations, and to compare its reliability, reproducibility, and discriminative power to the widely used Pfirrmann classification. Five features were graded. This new classification system has eight grades, with at least one of these five features altering each grade. The T2-weighted sagittal images were acquired using a rapid spin-echo sequence with a repetition time of 2680 to 4900 milliseconds, an echo time of 100 to 109 milliseconds, and an echo train length of 17. Slice thick was 4 mm and the display field of view was 32 × 32 cm. The new classification system used five features: signal intensity, disc height, disc boundary regularity, and nucleus annulus separation. Increased signal intensity, decreased height, decreased regularity, and decreased nucleus-annulus separation indicated degeneration. Four raters classified 400 discs from 80 patients using the Pfirrmann and Novel systems. Statistical analyses were conducted to investigate reliability and correlation. The overall ICC and kappa values were found to be higher in the novel classification. (0.988 indicating excellent agreement for ICC and 0.76/0.94 indicating good-very good agreement for kappa). The Kendall tau c value, which shows the correlation between the two classifications and indicates the validity of the new classification, was 0.872, which is very strong. Through the use of cross-tabulations, the discriminatory power of the two newly added classification criteria was determined. This study demonstrates the intra-rater and inter-rater reliability of an easy-to-use, discriminative novel morphometric MRI based classification system for lumbar disc degeneration. The differentiation of grades based on five distinct criteria may generate novel hypotheses regarding treatment selection and response monitoring, as well as new insights into the study of disc degeneration.