Membrane-Based In-Gel Loop-Mediated Isothermal Amplification (mgLAMP) System for SARS-CoV-2 Quantification in Environmental Waters.

Since the COVID-19 pandemic is expected to become endemic, quantification of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in ambient waters is critical for environmental surveillance and for early detection of outbreaks. Herein, we report the development of a membrane-based in-gel loop-mediated isothermal amplification (mgLAMP) system that is designed for the rapid point-of-use quantification of SARS-CoV-2 particles in environmental waters. The mgLAMP system integrates the viral concentration, in-assay viral lysis, and on-membrane hydrogel-based RT-LAMP quantification using enhanced fluorescence detection with a target-specific probe. With a sample-to-result time of less than 1 h, mgLAMP successfully detected SARS-CoV-2 below 0.96 copies/mL in Milli-Q water. In surface water, the lowest detected SARS-CoV-2 concentration was 93 copies/mL for mgLAMP, while the reverse transcription quantitative polymerase chain reaction (RT-qPCR) with optimal pretreatment was inhibited at 930 copies/mL. A 3D-printed portable device is designed to integrate heated incubation and fluorescence illumination for the simultaneous analysis of nine mgLAMP assays. Smartphone-based imaging and machine learning-based image processing are used for the interpretation of results. In this report, we demonstrate that mgLAMP is a promising method for large-scale environmental surveillance of SARS-CoV-2 without the need for specialized equipment, highly trained personnel, and labor-intensive procedures.

Aminophylline infusion for status asthmaticus in the pediatric critical care unit setting is independently associated with increased length of stay and time for symptom improvement.

INTRODUCTION: The role of aminophylline in the treatment of severe acute asthma in the pediatric critical care unit (PCCU) is not clear. We sought to examine the association of aminophylline treatment with PCCU length of stay and time to symptom improvement. MATERIAL AND METHODS: Patients with severe acute asthma who were admitted to our PCCU and received aminophylline infusion were retrospectively compared with similar patients who did not receive aminophylline. The primary outcome measure was functional length of stay (i.e. time to which patients could be transferred to a general pediatric ward bed). A secondary outcome was time to symptom improvement. RESULTS: Adjusted functional length of stay was longer for subjects who received aminophylline (n = 49) than for the patients who did not (n = 47) (hazard ratio 0.396, p < 0.001), as well as the time for symptom improvement (hazard ratio 0.359, p < 0.001). In the group of subjects receiving aminophylline, those with a serum theophylline level ≥ 10 mcg/ml (therapeutic) (n = 31) had longer functional length of stay (hazard ratio 0.457, p = 0.0225) and time to symptom improvement (hazard ratio 0.403, p = 0.0085) than those with levels < 10 mcg/ml (sub-therapeutic) (n = 18). CONCLUSIONS: The addition of aminophylline to therapy with corticosteroids and inhaled ß-agonists was associated with statistically and clinically significant increases in functional length of stay and time to symptom improvement in the PCCU. This potential morbidity supports the National Asthma Education and Prevention Program guideline proscribing aminophylline use in acute asthma.

Changes in Provider Perceptions and Practices Regarding Dosing Units for Oral Liquid Medications.

OBJECTIVE: A 2015 survey of primary care providers (PCPs) found that while many believed that milliliter (mL)-only dosing was safest for oral liquid medications, few would use mL alone in dosing instructions. Since 2015, many recommendations have promoted "mL-only" dosing. In 2019, a follow-up survey was conducted to assess if PCP perceptions and practices have changed. METHODS: Pediatricians, family medicine physicians, nurse practitioners, and internists participating in the 2015 and 2019 DocStyles cross-sectional, web-based surveys were asked about their perceptions and practices regarding dosing units for oral liquid medications. RESULTS: In 2019, among 1392 respondents, the proportion of PCPs who reported they believed using mL-only is the safest dosing instruction ranged from 55.1% of internists to 80.8% of pediatricians. While fewer PCPs believed patients/caregivers prefer dosing instructions in mL-only (23.9% of nurse practitioners to 48.4% of pediatricians), more held this belief in 2019 compared to 2015; pediatricians had the greatest absolute increase (+14.4%) and family medicine physicians had the smallest increase (+1.3%). While 61.6% of pediatricians reported they would use mL-only dosing, only 36.0% of internists, 36.6% of nurse practitioners, and 42.5% of family medicine physicians reported they would do so. After controlling for age, gender, region, and specialty, 2019 PCP survey participants were more likely to report that they would use mL-only dosing compared to 2015 participants (adjusted odds ratio 1.51, 95% confidence interval 1.29-1.77). CONCLUSIONS: Broader educational efforts may be necessary to reach nonpediatricians, to encourage prescribing and communication with patients/caregivers using mL-only dosing.

Results of the BfR MEAL Study: The food type has a stronger impact on calcium, potassium and phosphorus levels than factors such as seasonality, regionality and type of production.

The BfR MEAL Study aims to provide representative levels of chemical substances in foods consumed by the population in Germany for dietary exposure assessment. Calcium, potassium and phosphorus (Ca, K, P) are essential to obtain physiological functions in humans. Levels were investigated in 356 foods. Foods were purchased representatively, prepared as typically consumed and pooled before analysis. High mean levels were found in milk, dairy products, legumes, nuts, oilseeds and spices as well as chia seeds (Ca, K, P), chewing gum (Ca) and cocoa powder (K). Different levels comparing organically and conventionally produced foods were determined among others in cereal cracker (puffed), olives and tofu. Higher K levels were found in fried compared to boiled potatoes. Similar P levels were mainly found in regionally and seasonally sampled foods. These data provide a substantially improved basis to address dietary exposure assessment of the population in Germany for Ca, K and P.

Partial warfarin reversal prior to hip fracture surgical intervention in geriatric trauma patients effects on blood loss and transfusions.

BACKGROUND: Warfarin reversal is typically sought prior to surgery for geriatric hip fractures; however, patients often proceed to surgery with partial warfarin reversal. The effect of partial reversal (defined as having an international normalized ratio [INR] > 1.5) remains unclear. METHODS: This was a retrospective cohort study. Geriatric patients (≥65 y/o) admitted to six level I trauma centers from 01/2014-01/2018 with isolated hip fractures requiring surgery who were taking warfarin pre-injury were included. Warfarin reversal methods included: vitamin K, factor VIIa, (a)PCC, fresh frozen plasma (FFP), and the "wait and watch" method. An INR of ≤ 1.5 defined complete reversal. The primary outcome was the volume of blood loss during surgery; other outcomes included packed red blood cell (pRBC) and FFP transfusions, and time to surgery. RESULTS: There were 135 patients, 44% partially reversed and 56% completely reversed. The median volume of blood loss was 100 mL for both those completely and partially reversed, p = 0.72. There was no difference in the proportion of patients with blood loss by study arm, 95% vs. 95%, p > 0.99. Twenty-five percent of those completely reversed and 39% of those partially reversed had pRBCs transfused, p = 0.08. Of those completely reversed 5% received an FFP transfusion compared to 14% of those partially reversed, p = 0.09. There were no statistically significant differences observed for the volume of pRBC or FFP transfused, or for time to surgery. CONCLUSIONS: Partial reversal may be safe for blood loss and blood product transfusions for geriatric patients with isolated hip fractures. Complete warfarin reversal may not be necessary prior to hip fracture surgery, especially for mildly elevated INRs.

FEV1 decline in relation to blood eosinophils and neutrophils in a population-based asthma cohort.

BACKGROUND: The relationship between lung function decline and eosinophils and neutrophils has important therapeutic implications among asthmatics, but it has rarely been studied in large cohort studies. OBJECTIVE: The aim is to study the relationship between blood eosinophils and neutrophils and FEV1 decline in a long-term follow-up of a population-based adult asthma cohort. METHODS: In 2012-2014, an adult asthma cohort was invited to a follow-up including spirometry, blood sampling, and structured interviews, and n = 892 participated (55% women, mean age 59 y, 32-92 y). Blood eosinophils, neutrophils and FEV 1 decline were analyzed both as continuous variables and divided into categories with different cut-offs. Regression models adjusted for smoking, exposure to vapors, gas, dust, or fumes (VGDF), use of inhaled and oral corticosteroids, and other possible confounders were utilized to analyze the relationship between eosinophils and neutrophils at follow-up and FEV1 decline. RESULTS: The mean follow-up time was 18 years, and the mean FEV 1 decline was 27 ml/year. The annual FEV1 decline was related to higher levels of both blood eosinophils and neutrophils at follow-up, but only the association with eosinophils remained when adjusted for confounders. Further, the association between FEV1 decline and eosinophils was stronger among those using ICS. With EOS <0.3 × 109/L as reference, a more rapid decline in FEV1 was independently related to EOS ≥0.4 × 109/L in adjusted analyses. CONCLUSIONS AND CLINICAL RELEVANCE: Besides emphasizing the importance of smoking cessation and reduction of other harmful exposures, our real-world results indicate that there is an independent relationship between blood eosinophils and FEV1 decline among adults with asthma.

Hepatitis B Virus Infection of Normal Hepatic Stem/Progenitor Cells.

BACKGROUND/AIMS: Whether the hepatitis B virus (HBV) infects normal hepatic stem/progenitor cells (NSCs) and if so, whether such infections play a role in the pathogenesis of HBV-induced chronic liver disease (CLD) and/or hepatocellular carcinoma (HCC) remains to be determined. The objectives of this study were to determine whether HBV infects NSCs and whether such infections alter NSC activity in a manner likely to contribute to the development of CLD and/or HCC. METHODS: Liver biopsies from five hepatitis B surface antigen (HBsAg) positive patients were co-stained for HBcAg and HBx and the stem cell markers EpCAM, Oct-4 and Nanog. In addition, primary NSCs derived from healthy human livers were exposed to HBV contaminated serum in vitro. Supernatant and/or cellular HBsAg, HBcAg and HBV-DNA expression were documented over the subsequent 30 days of culture. Pro- and anti-inflammatory cytokine expression, membrane potential differences (PDs), proliferative and telomerase activities of HBV-infected NSCs were also documented. RESULTS: Markers of HBV infection were present within the NSC population of all five biopsy specimens. In vitro, HBV markers appeared within three days of exposure, peaked in expression after 10-15 days and remained positive thereafter for the duration of cell viability. There were no consistent changes in HBV-infected NSC pro- or anti-inflammatory cytokine expression, membrane PDs, proliferative or telomerase activities. CONCLUSIONS: Although the results of this study need to be confirmed, they suggest that HBV infects human NSCs but in the short term, do not alter those NSC features or activities associated with CLD and/or HCC.

Extension of the molecular analysis to the promoter region of the iduronate 2-sulfatase gene reveals genomic alterations in mucopolysaccharidosis type II patients with normal coding sequence.

Hunter disease or mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal disorder caused by the deficit of the enzyme iduronate-2-sulfatase (IDS), involved in the catabolism of the glycosaminoglycans heparan and dermatan sulfate. Our aim was to search for molecular defects in the promoter region of the IDS gene in patients with previous biochemical diagnosis of MPS II and after we sequenced the whole IDS coding region and the exon/intron boundaries without detecting any pathogenic mutations. Screening of the promoter region of four patients detected in two of them a 178 bp deletion and in the other two a single nucleotide substitution 818 bp upstream of the coding region. The latter had never been described before in MPS II patients and it turned out to be a polymorphism. Our experience suggests that MPS II patients with no mutations detected in the IDS coding region should be screened in the promoter region of the gene. Findings will hopefully help to clarify the relationship between genotype and phenotype and will be useful for the correct molecular diagnosis of Hunter patients and the identification of female carriers, the latter particularly important for genetic counseling.