A molecular beacon design for a colorimetric loop-mediated isothermal amplification assay.

In this study, a molecular beacon (MB) was designed for colorimetric loop-mediated isothermal amplification (cLAMP). The length of complementary bases on the MB, guanine and cytosine content (GC content), and hybridization sites of complementary bases were investigated as key factors affecting the design of the MB. We designed MBs consisting of 10, 15, and 20 complementary bases located at both ends of the HRPzyme. In the case of the long dumbbell DNA structure amplified from the hlyA gene of Listeria monocytogenes, possessing a flat region (F1c-B1) of 61 base pairs (bp), an MB was designed to intercalate into the flat region between the F1c and B1 regions of the LAMP amplicons. In the case of the short dumbbell DNA structure amplified from the bcfD gene of Salmonella species possessing a flat region (F1c-B1) length of 6 bp, another MB was designed to intercalate into the LoopF or LoopB regions of the LAMP amplicons. The results revealed that the hybridization site of the MB on the LAMP amplicons was not crucial in designing the MB, but the GC content was an important factor. The highest hybridization efficiencies for LAMP amplicons were obtained from hlyA gene-specific and bcfD gene-specific MBs containing 20- and 15-base complementary sequences, respectively, which exhibited the highest GC content. Therefore, designing MBs with a high GC content is an effective solution to overcome the low hybridization efficiency of cLAMP assays. The results obtained can be used as primary data for designing MBs to improve cLAMP accessibility.

A Highly Sensitive Indirect Enzyme-Linked Immunosorbent Assay (ELISA) Based on a Monoclonal Antibody Specific to Thermal Stable-Soluble Protein in Pork Fat for the Rapid Detection of Pork Fat Adulterated in Heat-Processed Beef Meatballs.

Processed foods containing pork fat tissue to improve flavor and gain economic benefit may cause severe issues for Muslims, Jews, and vegetarians. This study aimed to develop an indirect enzyme-linked immunosorbent assay (iELISA) based on a monoclonal antibody specific to thermal stable-soluble protein in pork fat tissue and apply it to detect pork fat tissue in heat-processed (autoclave, steam, roast, and fry) beef meatballs. To develop a sensitive iELISA, the optimal sample pre-cooking time, coating conditions, primary and secondary dilution time, and various buffer systems were tested. The change in the iELISA sensitivity with different 96-well microtiter microplates was confirmed. The detection limit of iELISA performed with an appropriate microplate was 0.015% (w/w) pork fat in raw and heat-treated beef. No cross-reactions to other meats or fats were shown. These results mean that the iELISA can be used as an analytical method to detect trace amounts of pork fat mixed in beef.

Correction: Evaluation of class participation in non-face-to-face CPR training for medical students.

[This corrects the article DOI: 10.1371/journal.pone.0278273.].

Detection of a Thermal Stable-Soluble Protein (TSSP) as a Marker of Peanut Adulteration Using a Highly Sensitive Indirect Enzyme-Linked Immunosorbent Assay based on Monoclonal Antibodies.

Food allergy represents a severe problem for many societies, including sensitive populations, academies, health authorities, and the food industry. Peanut allergy occupies a special place in the food allergy spectrum. To prevent consumption by consumers suffering from a peanut allergy, a rapid and sensitive detection method is essential to identify unintended peanut adulteration in processed foods. In this study, we produced four monoclonal antibodies (MAbs; RO 3A1-12, PB 4C12-10, PB 5F9-23, and PB 6G4-30) specific to thermo-stable and soluble proteins (TSSPs) of peanut and developed an enzyme-linked immunosorbent assay (ELISA) based on the MAbs. Among them, PB 5F9-23 MAb was firmly bound to Ara h 1, and other MAbs strongly reacted to Ara h 3 in the Western blot analysis. An antibody cocktail solution of the MAbs was used to enhance the sensitivity of an indirect ELISA, and the limit of detection of the indirect ELISA based on the antibody cocktail solution was 1 ng/ml and improved compared to the indirect ELISA based on the single MAb (11 ng/ml). The cross-reaction analysis revealed the high specificity of developed MAbs to peanut TSSPs without cross-reaction to other food allergens, including nuts. Subsequently, analyzing processed foods by indirect ELISA, all foods labeled as containing peanuts in the product description were confirmed to be positive. The results indicate that the developed antibodies exhibit high specificity and sensitivity to peanuts and can be used as bio-receptors in immunoassays or biosensors to detect intentional or unintentional adulteration of peanuts in processed foods, particularly heat-processed foods.

Rapid detection of Shiga-toxin-producing Escherichia coli O157:H7 based on a colorimetric loop-mediated isothermal amplification (cLAMP) assay using a molecular beacon paired with HRPzyme.

Contamination by Escherichia coli O157:H7 is considered a threat in the livestock and food industries. Therefore, it is necessary to develop methods for the convenient and rapid detection of Shiga-toxin-producing E. coli O157:H7. This study aimed to develop a colorimetric loop-mediated isothermal amplification (cLAMP) assay using a molecular beacon to rapidly detect E. coli O157:H7. Primers and a molecular beacon were designed for targeting the Shiga-toxin-producing virulence genes (stx1 and stx2) as molecular markers. Additionally, Bst polymerase concentration and amplification conditions for bacterial detection were optimized. The sensitivity and specificity of the assay were also investigated and validated on artificially tainted (100-104 CFU/g) Korean beef samples. The cLAMP assay could detect 1 × 101 CFU/g at 65 °C for both genes, and the assay was confirmed to be specific for E. coli O157:H7. The cLAMP takes about an hour and does not require expensive devices (e.g., thermal cycler and detector). Hence, the cLAMP assay proposed herein can be used in the meat industry as a fast and simple way to detect E. coli O157:H7.

Ultrasensitive monoclonal antibodies specific to thermal stable-soluble proteins of buckwheat.

Buckwheat is considered a severe food allergen, and its adulteration and mislabeling cause serious health risks. For protecting consumers suffering from buckwheat allergy, a high-sensitivity detection method is necessary to accurately identify intentional or unintentional adulteration of buckwheat in processed foods. The study revealed that buckwheat contains a significant amount of thermally stable-soluble proteins (TSSPs), which keep antigenicity even after heat treatment. Therefore, we used TSSPs to produce three monoclonal antibodies (MAbs) specific to buckwheat. A MAbs cocktail solution was subjected to enhance the sensitivity of an indirect enzyme-linked immunosorbent assay (iELISA), and the LOD was 1 ng/mL. The MAbs cocktail solution based-iELISA can successfully detect buckwheat adulterated in processed foods. The results suggested that the TSSPs in buckwheat can be used as suitable immunogens, and MAbs produced can be used as bioreceptor to develop immunoassays and biosensors for detecting buckwheat in food facilities and processed foods.

Analyzing willingness for extracorporeal cardiopulmonary resuscitation in refractory ventricular fibrillation.

Extracorporeal cardiopulmonary resuscitation (ECPR) for refractory ventricular fibrillation/ventricular tachycardia in out-of-hospital cardiac arrest has recently been recommended for selected patients with favorable prognostic features. We aimed to identify factors affecting the willingness of emergency physicians to implement extracorporeal cardiopulmonary resuscitation (ECPR). We conducted a factorial survey with nine experimental vignettes by combining three different scene time intervals and transportation time intervals. Emergency physicians reported willingness to implement ECPR (1-100 points). Respondent characteristics that could affect the willingness were studied. Multilevel analysis of vignettes and respondent factors was conducted using a mixed-effects regression model. We obtained 486 vignette responses from 54 emergency physicians. In the case of longer scene time intervals, there was a significant difference in the willingness scores at 9 and 12 min transportation time intervals. When the pre-hospital time interval was > 40 min, emergency physicians demonstrated lower willingness to implement ECPR. Clinical experience of 15-19 years showed a significant favorable effect on willingness to implement extracorporeal membrane oxygenation (ECMO). However, the mean willingness scores of EPs for ECMO implementation were more than 75 across all vignettes. In ECPR, the prehospital time interval is an important factor, and the willingness of emergency physicians to implement ECMO could be mutually affected by scene time intervals, transportation time intervals, and total prehospital time.

Evaluation of class participation in non-face-to-face CPR training for medical students.

BACKGROUND: Cardiopulmonary resuscitation (CPR) education requires that learners practice key skills to promote mastery. Our aim in this study was to evaluate differences in post-education performance and class participation during CPR training between face-to- face (FF) and non-face-to-face (NFF) learning formats. METHODS: This was a randomized controlled study of third-year medical students from two university hospital, allocated to either the FF or NFF format for CPR education. The learning scenario addressed single-person CPR, consisting of chest compression only, and excluded breathing. The Kahoot! application was used for NFF. Between-group comparisons for class participation and CPR skills were based on video recordings. RESULTS: Seventy students participated in our study, with 35 randomly allocated to the FF and NFF groups. There were no between-group differences in terms of age, sex, previous basic life support training, and willingness and confidence in performing CPR. Compared to the FF group, the NFF group demonstrated significant differences during CPR, including fewer calling for assistance and using of defibrillator (p = 0.006), as well as fewer checking for breathing (p = 0.007), and fewer counting during chest compression (p = 0.006). Additionally, < 30% of learners in the NFF group completed rhythm analysis after the last defibrillator shock delivery and resumed immediate chest compression (p < 0.001). All students in both groups passed the post-training assessment. CONCLUSION: Class participation in NFF learning was lower than that in FF learning. Although the post-education evaluation in the NFF group was not inferior, efforts on promoting active participation in NFF learning are required.

Colorimetric detection of norovirus by helicase-dependent amplification method based on specific primers integrated with HRPzyme.

Noroviruses (NoVs) are the most common causes of epidemic gastroenteritis, responsible for at least 50% of all gastroenteritis outbreaks worldwide and significant causes of foodborne illness. In the USA, approximately 21 million illnesses attributable to NoVs have annually occurred. Therefore, there is a great demand to develop a rapid, low-cost, and accurate detection method for NoVs. This study first reported colorimetric helicase-dependent amplification (HDA) methods based on specific primers integrated with HRPzyme for the rapid and sensitive detection of NoV GI and GII. The colorimetric HDA methods exhibited a detection limit of 10 copies mL-1 of each NoV GI and GII and were confirmed to be specific to each NoV GI and GII. The period required to complete the HDA method was 2 h, including a step of RNA extraction and cDNA synthesis without expensive instruments such as a thermal cycler and detector. The cutoff value of the method for the oyster artificially inoculated with a known amount of NoV was all 102 copies g-1 for NoV GI and GII. Therefore, the HDA method developed in this study can be useful tool for the on-site detection of NoVs in food samples.

Unintended beneficial effects of COVID-19 on influenza-associated emergency department use in Korea.

BACKGROUND: Non-pharmaceutical interventions, including hand hygiene, wearing masks, and cough etiquette, and public health measures such as social distancing, used to prevent the spread of coronavirus disease 2019 (COVID-19), could reduce the incidence rate of respiratory viral infections such as influenza. We evaluated the effect of COVID-19 on the incidence of influenza in Korea. METHODS: This retrospective study included all patients who visited five urban emergency departments (EDs) during the influenza epidemic seasons of 2017-18, 2018-19, and 2019-20. Influenza was defined as ICD-10 codes J09, J10, and J11, determined from ED discharge records. The weekly incidence rates of influenza per 1000 ED visits during the 2019-20 season, when COVID-19 became a pandemic, were compared with those of 2017-18 and 2018-19. The actual incidence rate of the 2019-20 season was compared with the predicted value using a generalized estimation equation model based on 2017-18 and 2018-19 data. RESULTS: The weekly influenza incidence rate decreased from 101.6 to 56.6 between week 4 and week 5 in 2020 when the first COVID-19 patient was diagnosed and public health measures were implemented. The weekly incidence rate during week 10 and week 22 of the 2019-20 season decreased most steeply compared to 2017-18 and 2018-19. The actual influenza incidence rate observed in the 2019-20 season was lower than the rate predicted in the 2017-18 and 2018-19 seasons starting from week 7 when a COVID-19 outbreak occurred in Korea. CONCLUSIONS: The implementation of non-pharmaceutical interventions and public health measures for the COVID-19 epidemic effectively reduced the transmission of influenza and associated ED use in Korea. Implementing appropriate public health measures could reduce outbreaks and lessen the burden of influenza during future influenza epidemics.

Effect of Designating Emergency Medical Centers for Critical Care on Emergency Medical Service Systems during the COVID-19 Pandemic: A Retrospective Observational Study.

During the coronavirus disease 2019 (COVID-19) pandemic, prehospital times were delayed for patients who needed to arrive at the hospital in a timely manner to receive treatment. To address this, in March 2020, the Korean government designated emergency medical centers for critical care (EMC-CC). This study retrospectively analyzed whether this intervention effectively reduced ambulance diversion (AD) and shortened prehospital times using emergency medical service records from 219,763 patients from the Gyeonggi Province, collected between 1 January and 31 December 2020. We included non-traumatic patients aged 18 years or older. We used interrupted time series analysis to investigate the intervention effects on the daily AD rate and compared prehospital times before and after the intervention. Following the intervention, the proportion of patients transported 30-35 km and 50 km or more was 13.8% and 5.7%, respectively, indicating an increased distance compared to before the intervention. Although the change in the AD rate was insignificant, the daily AD rate significantly decreased after the intervention. Prehospital times significantly increased after the intervention in all patients (p < 0.001) and by disease group; all prehospital times except for the scene time of cardiac arrest patients increased. In order to achieve optimal treatment times for critically ill patients in a situation that pushes the limits of the medical system, such as the COVID-19 pandemic, even regional distribution of EMC-CC may be necessary, and priority should be given to the allocation of care for patients with mild symptoms.

Monitoring of characteristics of the patients visiting an emergency center in Cameroon through the development of hospital patient database.

Most countries in Sub-Saharan Africa have struggled to utilize health information technology and thus lack in accurate patient data. This paper describes the method of collecting patient data and patient characteristics in an emergency centre in Yaoundé, Cameroon. We developed an Epi InfoTM-based data entry form to collect data of the patients who visited the Centre des Urgences de Yaoundé (CURY) from January 2016 to June 2018. Demographic, clinical symptoms, treatments and outcome data were collected. Additional data on the patients with multiple trauma, chest pain, sepsis/septic shock, and stroke were also collected. During the study period, a total of 18,875 patients' data were collected (44.5% women, median age of 36). Of the total patients, 2.4% had chest pain, 2.7% had stroke, 1.9% had sepsis/septic shock, and 1.6% had multiple trauma. About 6.0% patients received operation and majority of patients were discharged either normally (48.2%) or with continuity of care (26.3%). About 5.0% of patients were transferred to other hospital and 5.2% of patients were dead. This study serves to broaden understanding of the emergency patients in Yaoundé, Cameroon.

An Ultrasensitive Silicon-Based Electrolyte-Gated Transistor for the Detection of Peanut Allergens.

The highly sensitive detection of peanut allergens (PAs) using silicon-based electrolyte-gated transistors (Si-EGTs) was demonstrated. The Si-EGT was made using a top-down technique. The fabricated Si-EGT showed excellent intrinsic electrical characteristics, including a low threshold voltage of 0.7 V, low subthreshold swing of <70 mV/dec, and low gate leakage of <10 pA. Surface functionalization and immobilization of antibodies were performed for the selective detection of PAs. The voltage-related sensitivity (SV) showed a constant behavior from the subthreshold regime to the linear regime. The current-related sensitivity (SI) was high in the subthreshold regime and then significantly decreased as the drain current increased. The limit of detection (LOD) was calculated to be as low as 25 pg/mL based on SI characteristics, which is the lowest value reported to date in the literature for various sensor methodologies. The Si-EGT showed selective detection of PA through a non-specific control test. These results confirm that Si-EGT is a high-sensitivity and low-power biosensor for PA detection.

Colonization of Listeria monocytogenes in potting soils as affected by bacterial community composition, storage temperature, and natural amendment.

This study aimed to characterize the bacterial community of commercial potting soils with or without Listeria monocytogenes inoculation at 5-35 °C using 16S metagenomic sequencing and evaluate the effect of natural amendments on the reduction L. monocytogenes in non-sterile potting soils. An increase in the expected operational taxonomic units of each sample with or without L. monocytogenes was proportional to the increasing storage temperatures after 5 days. Biodiversity was distinct among all potting soils for Shannon and inverse Simpson indices, with the highest diversity being observed in a soil sample stored at 35 °C for 5 days with L. monocytogenes. An increase in richness and diversity of soil bacterial community structure positively correlated with less survival of the invading L. monocytogenes. Particularly, garlic extract was demonstrated as a promising soil-amendment substrate, reducing L. monocytogenes by ≥ 4.50 log CFU/g in potting soils stored at 35 °C. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10068-021-00925-9.

Management challenges at end-of-life in a patient with agitated delirium and benzodiazepine withdrawal at comprehensive cancer care center.

Most people have some idea of how they want to live their life; however, an estimated two-thirds of Americans have not completed their advance directives. This becomes an issue when up to 90% of patients develop delirium during their final days of life, at which point we depend on advance directives or surrogate decision-makers. Here, we present a case of terminal delirium in a patient with advanced cancer and a history of alprazolam abuse who had not discussed his end-of-life wishes with the medical team or with his estranged family. Treatment was provided to address reversible causes of delirium, including correcting electrolyte imbalances, urinary retention, and administered antibiotics for purulent otitis media. Hyperactive delirium was managed aggressively with intravenous neuroleptics and benzodiazepine, while keeping a balance between somnolence and control of agitative symptoms. Without knowing the patient's wishes, the family continued to struggle with decision making. However, with multidisciplinary team approach patients and caregivers' symptoms were better managed. Family then requested us to transfer him to a local hospice facility. The patient eventually passed away peacefully surrounded by his family members. This case highlights the importance of advance care planning, addressing emotional distress in estranged family members regarding symptom burden, and developing the appropriate treatment regimen for a delirious patient with a history of benzodiazepine abuse. Our case serves as a reminder of the support, guidance, and impact that inpatient palliative care teams can offer to both the patient and caregivers.

The Effect of Transport Time Interval on Neurological Recovery after Out-of-Hospital Cardiac Arrest in Patients without a Prehospital Return of Spontaneous Circulation.

BACKGROUND: Longer transport adversely affects outcomes in out-of-hospital cardiac arrest (OHCA) patients who do not return to spontaneous circulation (ROSC). The aim of this study was to determine the association between the transport time interval (TTI) and neurological outcomes in OHCA patients without ROSC. METHODS: We analyzed adult OHCA patients with presumed cardiac etiology and without prehospital ROSC from 2012 to 2015. The study population was divided into 2 groups according to STI (short STI [1-5 minutes] and long STI [≥ 6 minutes]). The primary exposure was TTI, which was categorized as short (1-5 minutes), intermediate (6-10 minutes), or long (≥ 11 minutes). The primary outcome was a good neurological recovery at discharge. Multiple logistic regression analysis was used in each STI group. RESULTS: Among 57,822 patients, 23,043 (40%), 20,985 (36%), and 13,794 (24%) were classified as short, intermediate, and long TTI group. A good neurological recovery occurred in 1.0%, 0.6%, and 0.3% of the patients in the short, intermediate and long TTI group, respectively. Among 12,652 patients with short STI, a good neurological recovery occurred in 2.2%, 1.0%, and 0.4% of the patients in the short, intermediate and long TTI group, respectively. Among 45,570 patients with long STI, a good neurological recovery occurred in 0.7%, 0.5%, and 0.3% of the patients in the short, intermediate and long TTI group, respectively. When short TTI was used as a reference, the adjusted odds ratios (AOR) of TTI for good neurological recovery was different between short STI group and long STI group (AOR [95% confidence interval, 0.46 [0.32-0.67] vs. 0.72 [0.59-0.89], respectively, for intermediate TTI and 0.31 [0.17-0.55] vs. 0.49 [0.37-0.65], respectively, for long TTI). CONCLUSION: A longer TTI adversely affected the likelihood of a good neurological recovery in OHCA patients without prehospital ROSC. This negative effect was more prominent in short STI group.

Factors Associated with the Transfer Decision in Resuscitated Patients with Out-of-Hospital Cardiac Arrest Presenting to a Hospital with Limited Targeted Temperature Management Capability in Korea.

This study aimed to identify factors associated with the decision to transfer resuscitated patients with out-of-hospital cardiac arrest (OHCA) who were initially examined at hospitals with limited targeted temperature management capability (HLTC) in Korea. We included adult patients with OHCA who were initially transported to HLTC from 2012 to 2015. We limited the study population with OHCA who were admitted or transferred. Potential factors associated with the transfer decision were categorized as patient- or hospital-level characteristics. The primary outcome was emergency department disposition: transfer out versus admission to HLTC. A stepwise multivariable logistic regression analysis was conducted to identify factors associated with the outcome. Of the 64,696 patients with OHCA, 37,705 (58.3%) were initially transported to HLTC. Approximately 44.7% of patients were transferred out. In terms of patient-level factors, age, socioeconomic status, and initial electrocardiogram rhythm were associated with the transfer decision. Hospital factors, such as percutaneous coronary intervention capability of HLTC (adjusted odds ratio = 0.16; 95% confidence interval = 0.14-0.19) and location of the metropolitan or urban hospital, were negatively associated with the transfer decision. Several hospital and patient factors were associated with the decision to transfer out patients with OHCA who were initially examined at HLTC.

The effect of team-based CPR on outcomes in out of hospital cardiac arrest patients: A meta-analysis.

OBJECTIVES: The objective of this systematic review and meta-analysis was to determine the effects of team cardiopulmonary resuscitation (CPR) on outcomes of patients with out-of-hospital cardiac arrest (OHCA). METHODS: A systematic literature review was performed using PubMed, EMBASE, and the Cochrane database to identify relevant articles for this meta-analysis. All studies that described the implementation of team CPR performed by emergency medical services for OHCA patients with presumed cardiac etiology were included in this study. Outcomes included return of spontaneous circulation (ROSC), survival to hospital discharge, and good neurological recovery. RESULTS: A total of 2504 studies were reviewed. After excluding studies according to exclusion criteria, 4 studies with 15,455 OHCA patients were included in this study. The odds of survival and neurologic recovery for patients who received team CPR were higher than those for patients who did not (survival odds ratio [OR]: 1.68; 95% confidence interval [CI]: 1.48-1.91; neurologic recovery OR: 1.52; 95% CI: 1.31-1.77). There was no significant difference in the odds of ROSC between the two patient groups (OR: 1.59; 95% CI: 0.76-3.33). CONCLUSIONS: In this meta-analysis, team CPR improved the outcomes of OHCA patients, consistently increasing their odds of survival to discharge and neurologic recovery.

Preventive effects of motorcycle helmets on intracranial injury and mortality from severe road traffic injuries.

INTRODUCTION: Road traffic injuries caused by motorcycle crashes are one of the major public health burdens leading to high mortality, functional disability, and high medical costs. The helmet is crucial protective equipment for motorcyclists. This study aimed to measure the protective effect of motorcycle helmets on clinical outcomes and to compare the effects of high- and low-speed motorcycle crashes. METHODS: A cross-sectional observational study was conducted using a nationwide registry of severe trauma patients treated by emergency medical services (EMS) providers in Korea. The study population consisted of severe trauma patients injured in motorcycle crashes between January and December 2013. The primary and secondary outcomes were intracranial injury and in-hospital mortality. We calculated adjusted odds ratios (AORs) of helmet use and motorcycle speeds for study outcomes after adjusting for potential confounders. RESULTS: Among 495 eligible patients, 105 (21.2%) patients were wearing helmets at the time of the crash, and 256 (51.7%) patients had intracranial injuries. The helmeted group was less likely to have an intracranial injury compared with the un-helmeted group (41.0% vs. 54.6%, AOR: 0.53 (0.33-0.84)). However, there was no significant difference in in-hospital mortality between the two groups (16.2% vs. 16.9%, AOR: 0.91 (0.49-1.69)). In the interaction analysis, there was a significant preventive effect of motorcycle helmet use on intracranial injury when the speed of the motorcycle was <30km/h (AOR: 0.50 (0.27-0.91)). CONCLUSION: Wearing helmets for severe trauma patients in motorcycle crashes reduced intracranial injuries. The preventive effect on intracranial injury was significant in low-speed motorcycle crashes.