Serological Detection for Toxoplasmosis among Patients with Covid-19 in Thi-Qar Province

Covid-19 is a viral disease that affects humans caused by a type of virus belonging to the family Coronaviridae called the SARS-CoV-2 virus. The parasitic infection associated with this disease affects the host's immune response regulation. The levels of IgG and IgM of Toxoplasma gondii in the serum of patients with COVID-19 were measured by immunoassay of the patient's sera by ELISA. Also, the level of interferon-gamma (IFN-gamma) in a covid-19 patient with or without Toxoplasmosis was evaluated. 120 samples were collected, 60 were positive for COVID-19, confirmed by clinically and radiographic examination, and 30 were in the control group. The results showed a significant difference between the infection with Covid-19 and T. gondii during the chronic phase of Toxoplasmosis compared to the negative relationship in the acute phase. The results of INF-gamma levels among Covid-19 patients were positive for all samples included in the test (30 Covid-19 patients and 30 patients COVID-19(+)/T. gondii IgG) compared to the control group. The chronic form of Toxoplasma disease, due to change in the production of this interferon, the COVID-19 infection has changed.Copyright © 2023 by Razi Vaccine & Serum Research Institute.

Molecular detection of plasmid-mediated quinolone resistance genes among clinical isolates of Klebsiella pneumoniae during Covid-19 pandemic

Plasmid-mediated quinolone resistance (PMQR) genes confer low resistance to Fluoroquinolones (FQs). This study aims to detect five PMQR genes among FQs-resistant Klebsiella pneumoniae isolated from various clinical specimens. Out of 120 K. pneumoniae isolates, 68 FQs-resistance K. pneumoniae were included in a molecular study. Standard microbiological tests were used for identification and antimicrobial susceptibility. For the detection of PMQR genes, conventional polymerase chain reaction was used. A molecular study revealed that (73.5%) of samples harbored PMQR genes, and among them, 58% were co-carriages of PMQR gene variants. Aac (6')-Ib-cr gene was predominant (47.1%) among samples, and qepA had the lowest percentage (11.8%), qnr genes were (32.4%) (29.4%) (20.6%) qnrS, qnrB, and qnrA respectively. Overall, high percentages of PMQR genes were detected, and almost all of samples were phenotypically resistant to ciprofloxacin. As well, there was a significant statistical relationship between phenotypically ESBL-producers and qnrB and qepA genes.Copyright © Abdulkareem MM et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The Evolution of SARS-CoV-2 Infection in the Pediatric Ward at St. Andrew's County Emergency Clinical Hospital Constanta

In the period April 24, 2020 - December 31, 2021 at Saint Andrew's County Emergency Clinical Hospital's Molecular Biology Laboratory were performed 2856 RT-PCR tests on childrens. This method consists of two steps: extraction and amplification. RT-PCR is the golden standard to diagnose infection with Covid-19. The tests were performed on patients of both genders and under 18 years old. Statistics show that both male and female patients were affected by Sars-Cov-2 in relatively equal proportions: in the first year (male 46% and female 54%), followed by the next year (male 48% and female 52%). The results concluding, that during the study, in 2020, 6.98% of the total number of tests came out positive, 92.32% came out negative, 0.70% inconclusive. In 2021, 4.21% of the total number of tests came out positive, 95.56% came out negative, 0.23% inconclusive. This study highlights the situation of Covid-19 cases encountered at childrens from the Pediatric section diagnosed in Constanta, Romania.Copyright © 2022 Ramona-Anca Sterian et al., published by Sciendo.

WCN23-1206 Glomerulopathies Biobank in Uruguay. Description of the first 110 patients enrolled

Introduction: The incidence of glomerular diseases varies across different countries and criteria for kidney biopsy has changed over time. In Uruguay, glomerular diseases (GD) are a frequent cause of end stage kidney disease (ESKD) and renal replacement therapy with an annual incidence of 25.0 patients per million population according to data from the Uruguayan Dialysis Registry (UDR, year 2020). Since 1970, the Uruguayan Registry of Glomerulopathies has been recording the incidence, epidemiology and evolution of patients with GP in our country. In 2018, the Glomerulopathies Biobank (GB) began to operate including all patients with a native kidney biopsy performed at the Nephrology Department of the teaching hospital Hospital de Clinicas in Montevideo, Uruguay. The purpose of the BG is to record the phenotype (clinical and paraclinical) of patients with GD diagnosed by renal biopsy and at the same time store blood, urine, renal tissue and DNA samples. The aim of this report is to communicate the first 110 patients enrolled in the BG, which started in February 2018. Method(s): The BG protocol includes the collection of patronymic data, personal history, and clinical and paraclinical features of renal pathology. Plasma, urine and cell samples are stored for subsequent DNA extraction at the time of the kidney biopsy. In our country, all renal biopsies are performed by nephrologists. The Glomerular Biobank project is funded by the Nephrology Research Fund (School of Medicine, University of the Repubic) and was approved by the Ethics Committee of the Hospital de Clinicas and the Regulatory Verification Unit of the National Institute of Donation and Transplantation. The results are presented as mean and standard deviation (SD) for the continuous variables;and qualitative variables are described with percentages. Result(s): Patient recruitment began in February 2018 and we have recruited 110 patients. The mean age at the time of biopsy was 38.3+/-16.1 (min:16;max:78) years. Regarding sex distribution, the female sex slightly predominated (55.3%). Plasma creatinine was 2.1+/-1.45 mg/dL, proteinuria was 3.1+/-3.7 gr/dL and albuminaemia was 3.2+/-1.0 mg/dL. Microhaematuria was present in 61% of patients in the sediment study. Figure 1 shows the negative impact of the COVID 19 pandemic on the incidence of patients undergoing kidney biopsy. IgA nephropathy (13,8%)was the most frequent primary glomerular disease, followed by d focal and segmental glomerulosclerosis and membranous nephropathy. Consernig the glomerulopathies secondary to a systemic disease, the most frequent diagnosis was lupus nephritis (34,5%) followed by vasculitis, amyloidosis and diabetes. Conclusion(s): Having a prospective cohort of patients with glomerular disease, including reliable data and biological samples, will allow us to perform clinical and epidemiological analyses quickly and reliably in the future. The data and aliquots of biological material are available to any local nephrologist who proposes a hypothesis and has the approval of the corresponding ethics committee. The medium-term objective is to incorporate other national reference institutions in the care of patients with glomerular diseases. The data collected by the Glomerular Biobank will be a valuable input to the process of continuous improvement, and will serve as a basis for future nephrological research of excellence. No conflict of interestCopyright © 2023

Evaluation of DNA Extraction Methods for Reliable Quantification of Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa.

Detection and quantification of DNA biomarkers relies heavily on the yield and quality of DNA obtained by extraction from different matrices. Although a large number of studies have compared the yields of different extraction methods, the repeatability and intermediate precision of these methods have been largely overlooked. In the present study, five extraction methods were evaluated, using digital PCR, to determine their efficiency in extracting DNA from three different Gram-negative bacteria in sputum samples. The performance of two automated methods (GXT NA and QuickPick genomic DNA extraction kit, using Arrow and KingFisher Duo automated systems, respectively), two manual kit-based methods (QIAamp DNA mini kit; DNeasy UltraClean microbial kit), and one manual non-kit method (CTAB), was assessed. While GXT NA extraction kit and the CTAB method have the highest DNA yield, they did not meet the strict criteria for repeatability, intermediate precision, and measurement uncertainty for all three studied bacteria. However, due to limited clinical samples, a compromise is necessary, and the GXT NA extraction kit was found to be the method of choice. The study also showed that dPCR allowed for accurate determination of extraction method repeatability, which can help standardize molecular diagnostic approaches. Additionally, the determination of absolute copy numbers facilitated the calculation of measurement uncertainty, which was found to be influenced by the DNA extraction method used.

Virucidal efficacy of household dishwashers

Not only since SARS-CoV-2, have transmission routes of viruses been of interest. Noroviruses e.g., can be transmitted via smear infection, are relatively stable in the environment and very resistant to chemical disinfection. Some studies determined the virucidal efficacy of laundering processes, but few studies focused on the virucidal efficacy of dishwashing processes. Here, especially consumer related conditions are of interest. Households for example are a hotspot of norovirus infection and thus a sufficient reduction of these and other viruses from dishes must be insured to avoid an infection via this route. The likelihood of such an event should not be underestimated, since it was shown that the washing machine can be a reservoir for the transmission of extended spectrum beta-lactamase producing bacteria in newborns. Although viruses do not replicate in these devices a transmission via contaminated cutlery e.g., cannot be excluded. Using a consumer related approach to determine the virucidal efficacy of dishwashers, we found a combination of a bleach containing dishwasher detergent, a cleaning temperature of 45 C for 45 min and a rinsing temperature of 50 C, to be sufficient to reduces viral titer of bovine corona virus, murine norovirus and modified vaccinia virus by 4.8, 4.2 and 3.8 logarithmic stages respectively.Copyright © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

Multicenter post-marketing clinical evaluation on the clinical performance of the five domestic fast nucleic acid detection reagents for 2019-nCoV.

Objective This multicenter clinical evaluation analyzed the clinical performance of five fast nucleic acid detection systems for 2019-nCoV. Methods Clinical performance of the five fast nucleic acid detection reagents approved in China was evaluated in the present study. Fifty-seven throat swabs samples from COVID-19 patients and fifteen throat swabs samples from healthy people were collected from the First Affiliated Hospital of Zhejiang University school of Medicine, Tongji Hospital of Tongji Medical College of HUST, and National Institute of Viral Disease Control and Prevention of CDC to evaluate the positive coincidence rate, negative coincidence rate, total coincidence rate, the detection time and retest rate as well as the relation between positive intensity and positive coincidence rate of the five fast nucleic acid detection systems in November 2020. Results The positive coincidence rates of the five kits were 92.59% (50/54), 83.64% (46/55), 98.25% (56/57), 94.44% (51/54) and 98.18% (54/55);and the negative coincidence rates were 93.33% (14/15), 93.33% (14/15), 86.67% (13/15), 100% (14/14) and 93.33% (14/15);and the total coincidence rates were 92.75% (64/69), 85.71% (60/70), 95.83% (69/72), 94.20% (65/69) and 97.14% (68/70), respectively. The positive coincidence rate of the five kits reached 100% for the strong-positive (90/90) and medium-positive samples (84/84), but only 82.18% (83/101) for weak-positive samples (cycle threshold value>33), and the retest rate of two kits were 15.28% (11/72) and 12.50% (9/72), which were both higher than 10%. Total time from sample extraction to amplification was between 32.33-65.33 minutes for these five kits. Conclusion The five fast nucleic acid detection reagents have good performance and can be used as a supplement to routine nucleic acid detection reagents.Copyright © 2022 Chinese Journal of Laboratory Medicine. All rights reserved.

Genetic Association of Angiotensin-converting enzyme 2 ACE-2 (rs2285666) Polymorphism with the Susceptibility of COVID-19 Disease in Iraqi Patients

Significant risks to human health are posed by the 2019 coronavirus illness (COVID-19). SARS coronavirus type 2 receptor, also known as the major enzyme in the renin-angiotensin system (RAS), angiotensin-converting enzyme 2 (ACE-2), connects COVID-19 and RAS. This study was conducted with the intention of determining whether or not RAS gene polymorphisms and ACE-2 (G8790A) play a part in the process of predicting susceptibility to infection with COVID-19. In this study 127 participants, 67 of whom were deemed by a physician to be in a severe state of illness, and 60 of whom were categorized as "healthy controls".The genetic study included an extraction of genomic DNA from blood samples of each covid 19 patients and healthy controls, then amplification the site of SNP (rs2285666) Within the ACE2 gene by using specific primers, sequencing PCR products, and genotyping to detect the role of the ACE-2 gene (rs2285666) in the incidence of COVID-19. ACE-2 (rs2285666) is statistically associated to COVID-19. The COVID-19 group had 65.67 %of individuals with the wild-type homozygous genotype (GG) and 20% in the control group, while the control group had 63.33% of individuals with the mutant genotype (AA). Consequently, the wild-type homozygous (GG) and allele (G) may be considered a risk factor (etiological fraction E. F) for COVID-19 in Iraqi patients, whereas the mutant homozygous (AA) and allele (A) may be considered a protective factor (preventive fraction). The findings of the present study reveal that carriers of the GG genotype of ACE2 (rs2285666) are substantially more susceptible to COVID-19.Copyright © 2023 Allami et al.

Multicenter post-marketing clinical evaluation on the clinical performance of the five domestic fast nucleic acid detection reagents for 2019-nCoV

Objective This multicenter clinical evaluation analyzed the clinical performance of five fast nucleic acid detection systems for 2019-nCoV. Methods Clinical performance of the five fast nucleic acid detection reagents approved in China was evaluated in the present study. Fifty-seven throat swabs samples from COVID-19 patients and fifteen throat swabs samples from healthy people were collected from the First Affiliated Hospital of Zhejiang University school of Medicine, Tongji Hospital of Tongji Medical College of HUST, and National Institute of Viral Disease Control and Prevention of CDC to evaluate the positive coincidence rate, negative coincidence rate, total coincidence rate, the detection time and retest rate as well as the relation between positive intensity and positive coincidence rate of the five fast nucleic acid detection systems in November 2020. Results The positive coincidence rates of the five kits were 92.59% (50/54), 83.64% (46/55), 98.25% (56/57), 94.44% (51/54) and 98.18% (54/55);and the negative coincidence rates were 93.33% (14/15), 93.33% (14/15), 86.67% (13/15), 100% (14/14) and 93.33% (14/15);and the total coincidence rates were 92.75% (64/69), 85.71% (60/70), 95.83% (69/72), 94.20% (65/69) and 97.14% (68/70), respectively. The positive coincidence rate of the five kits reached 100% for the strong-positive (90/90) and medium-positive samples (84/84), but only 82.18% (83/101) for weak-positive samples (cycle threshold value>33), and the retest rate of two kits were 15.28% (11/72) and 12.50% (9/72), which were both higher than 10%. Total time from sample extraction to amplification was between 32.33-65.33 minutes for these five kits. Conclusion The five fast nucleic acid detection reagents have good performance and can be used as a supplement to routine nucleic acid detection reagents.

Present status of microfluidic PCR chip in nucleic acid detection and future perspective

Polymerase chain reaction (PCR) amplifies specific fragment of DNA molecules and has been extensively applied in fields of pathogens and gene mutation detection, food safety and clinical diagnosis which on the other hand, holds the drawbacks of large size instrument, high heat dissipation etc. It has been demonstrated that microfluidics technique coupling with PCR reaction exhibits characteristics of integration, automatization, miniaturization, and portability. Meanwhile, various designed fabrication of microchip could contribute to diverse applications. In this review, we summarized major works about a variety of microfluidic chips equipped with several kinds of PCR techniques (PCR, RT-PCR, mPCR, dPCR) and detection methods like fluorescence, electrochemistry, and electrophoresis detection. The development and application of PCR-based microfluidic chip in pathogen and gene mutation detection, diseases prevention and diagnosis, DNA hybridization and low-volume sample treatment were also discussed. Copyright © 2022 Elsevier B.V.

Standing up FungiNet: Genomic epidemiology and surveillance of fungal diseases

Objectives: With the advent of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing, the public health landscape for genomic epidemiology and surveillance has transformed for a variety of pathogens. For fungal diseases, the U. S. Centers for Disease Control and Prevention (CDC) is working with global partners to stand up FungiNet, a network that aims to equip scientists with laboratory, bioinformatics, and informatics resources to harness genomic data. FungiNet partners will use genomic and epidemiologic data to detect outbreaks, identify introductions, and characterize transmission of fungal infections. In 2022, FungiNet aims to onboard nine state and local health departments in the United States and two global partners, the Instituto Nacional de Salud in Colombia and the National Institute for Communicable Diseases in South Africa, with a focus on Candida auris. Method(s): To streamline the onboarding process, CDC generated standardized operating procedures (SOPs) specific to C. auris. For DNA extraction, SOPs were created for workflows using the Zymo Research Quick-DNA TM (ZR) Fungal/Bacterial Miniprep, Qiagen Dneasy Blood and Tissue, and Epicentre (Illumina) MasterPure Yeast DNA Purification kits. For library preparation and Illumina sequencing, PulseNet methods used for foodborne pathogens were validated for C. auris. For NCBI data submissions, required data elements were defined. For SNP and phylogenetic analyses, the bioinformatics workflow My-coSNP was adapted to use Nextflow software and the Terra platform. For visualization with epidemiologic data, guidance documents and tutorials for Microreact were created. Finally, for data reporting, processes are being designed in REDCap and in laboratory information management systems to rapidly share genomic-related data. Result(s): To date, 11 partners have committed to building capacity for C. auris genomic sequencing and analysis as a FungiNet partner. Of these, seven have validated methods for DNA extraction, and nine have generated high-quality sequencing data. Only one partner has installed and locally run MycoSNP, and none have submitted raw sequence data to NCBI. Conclusion(s): Currently, 11 FungiNet partners are working to onboard C. auris genomic sequencing and bioinformatics analysis in 2022. This process is complex, requiring several laboratories, bioinformatics, and informatics workflows. For many partners, bioinformatics analysisand NCBIsubmission are themost challenging activities with the installationof MycoSNPand the ability to batch upload data to NCBI as the main barriers. Next steps will focus on the validation of informatics methods to link genomic and epidemiologic data.

Molecular investigation of Campylobacter spp. and salmonella infection in children with community acquired diarrhea

Introduction: Acute gastroenteritis is a typical disorder that accounts for 8-12% of pediatric outpatient visits. Campylobacter and Salmonella infections account for about 8.4% and 11% of global diarrhea cases. Due to the importance of these bacteria in pediatric diseases, the aim of this study was to determine the infectious rate of Salmonella and Campylobacter species and also the frequency of the gene encoding Cytholethal distending toxin in children with community-acquired diarrhea. Material(s) and Method(s): Stool samples of children under 5 years of age with diarrhea were collected. The samples were related to children referred to hospitals in Hamadan, Ardabil, Bandar Abbas and two hospitals in Tehran. DNA was extracted from the samples using a DNA extraction kit from stool. The presence of Campylobacter in the studied samples was detected by polymerase chain reaction using specific primers. A control stool sample was spiked with 10-fold dilution of C. jejuni suspension for LOD (detection limit determination) measurement. Result(s): In this study, PCR results showed a LOD of 100 CFU per gram in the spiked feces sample. Accordingly, out of 144 fecal samples of children with acute diarrhea, one case was positive for Campylobacter jejuni;this sample was also positive for the presence of cdtB gene. Presence of Salmonella was confirmed in two samples of the patients (1.4%). Conclusion(s): Low prevalence of Campylobacter and Salmonella was detected in symptomatic children under 5 years of age during the Covid-19 pandemic. Examination of these samples for viruses and other microbial agents can clarify the etiology of diarrhea in children referred to the hospitals. Copyright © 2022, Semnan University of Medical Sciences. All rights reserved.

Assessing the potential correlation of polymorphisms in the TMPRSS2 gene with severity of COVID 19 patients

Introduction and Aim: Coronavirus disease (COVID-19) is an infectious disease caused by the SARS-CoV-2 virus. Once infected this virus induces several clinical disorders in humans. SARSCoV-2 enters cells via TMPRSS2. Genetic variation in TMPRSS2 could affect the severity of infection. The purpose of this study was to investigate how the (TMPRSS2) gene polymorphism affected COVID-19 severity in patients as well as the effect of age and comorbidities on infection. Material(s) and Method(s): This cross-sectional analytical study comprised of 400 (185 male, 215 female) Covid-19-infected patients between ages 18-65 receiving treatment in hospitals at Baghdad, Iraq. The patients were divided into three groups: mild, moderate, and severe based on the severity of Covid-19 infection. Baseline data was collected for each patient through interview and questionnaire. Blood collected from patients was subjected to DNA extraction and detecting polymorphisms within SNPs of the TMPRSS2 gene. Result(s): The present investigation indicated higher age to be significantly associated with severe COVID-19 infection when compared to moderate and mild infection (36.14 +/- 12.716 vs. 48.52 +/- 17.513 vs. 59.26 +/- 16.035) (F= 3.697, df: 64, P= 0.000). Patients with comorbidities was associated with a greater rate of severe Covid-19 infection (74.2% vs. 25.8%). However, individuals without comorbidities had a considerably lower rate of mild and moderate Covid-19 infection (13.9% vs. 86.1%) and (36% vs. 64%), respectively (x

Impact of lab experiences on student knowledge and confidence

Due to the COVID-19 pandemic, the sophomore class of 2021 spent very little time on campus as freshmen and missed the benefits and advantages of in-person learning. Three labs were added to a required sophomore biomedical engineering (BME) course, Biochemistry and Molecular Engineering. The objectives of these labs were three-fold: (i) reinforce the content presented in the online lecture, (ii) provide students with hands-on laboratory skills, and (iii) offer students engaged in an online course in-person experiences and the corresponding academic and social benefits. The objective of this course is to develop critical thinking, teamwork and collaborative skills, as well as the ability to use physical laboratory equipment to obtain and analyze data. We eliminated some of the homework problems to account for the time spent in preparation for lab, the lab sessions, and writing the post-lab reports. The labs accounted for 10% of the total grade and included a prelab quiz, a post-lab report, and one or two exam questions for each lab. The three labs were: 1. Introduction to Pipetting 2. DNA Extraction, PCR, and Gel Electrophoresis 3. Bacterial Transformation with a Plasmid After completing the labs, a student survey indicated that over 80% of students agreed or strongly agreed that they felt confident using all these techniques. Comparing confidence levels, 63% of the students were somewhat familiar with pipetting prior to Lab 1 which increased to over 90% post-lab. After Lab 2 (DNA extraction, PCR, and gel electrophoresis), confidence levels more than doubled, from 40% in pre-lab survey results to 86% afterwards. Student confidence in the final lab, which involved using a plasmid for genetic transfection of bacteria, went from 33% pre-lab to 81% post-lab. While the prelab census indicated that a significant percentage of the students had some prior lab proficiency in high school, a quarter of the students lacked experience in pipetting and a majority of the students were unfamiliar with PCR/gel electrophoresis and plasmids. As one student noted, “The labs were a good way to build basic lab skills and exposure for students who were previously unable to work in a college lab”. By the end of the course, 87% agreed or strongly agreed that “Gaining hands-on lab skills is an important part of this course. I believe it should be continued, possibly with more labs, in future years.” In addition to gaining hands-on laboratory experience, students enjoyed working with their classmates in-person and benefited from “learning by doing”. Student comments strongly indicated that the labs reinforced the lecture content. The labs “helped me retain the information better than simply reading about it.” The combination of replacing homework with labs, and reducing the total amount of time per week students spent on the course, did not result in significant differences in the quiz scores from pre-pandemic years. Given the success of this year's lab experiences, we plan to update the labs and include additional topics for future course offerings. © American Society for Engineering Education, 2022

Development and application of LAMP assays for the detection of enteric adenoviruses in feces.

Loop-mediated isothermal amplification (LAMP) is an alternative to PCR that is faster and requires fewer resources. Here, we describe two LAMP assays for the detection of human adenoviruses in the feces of children with acute intestinal infections. We designed сolorimetric LAMP (c-LAMP) and real-time LAMP (f-LAMP) with fluorescent probes to detect the DNA of the adenovirus F human adenovirus 40/41 (hAdV40/41) hexon gene. The detection limit of both developed methods was 103 copies/mL, which is comparable to the sensitivity of PCR. The specificities of both c-LAMP and f-LAMP were high, with no false-positive results for clinical samples that do not contain adenovirus F, when testing other viruses and microorganisms. Comparative tests of PCR and LAMP on clinical samples from patients with acute gastroenteritis were carried out. For all samples with a PCR threshold cycle (CT) of up to 36, the PCR and LAMP results completely coincided; however, at low viral loads, the diagnostic sensitivity of LAMP, especially c-LAMP with colorimetric detection, was inferior to that of PCR. The combination of LAMP with modern methods of nucleic acid extraction, both in manual and automatic modes, can reduce the time for a complete study, including extraction of nucleic acid material and amplification, to 60 min. IMPORTANCE In April 2022, several cases of acute hepatitis of unknown origin were reported in children from 12 countries. In many cases, enteric adenovirus or SARS-CoV-2 and adenovirus coinfection were detected. It is known that human adenoviruses can cause different infections of varying severity, from asymptomatic to severe cases with lethal outcomes. There is a need to increase the diagnostic capabilities of clinical laboratories to identify such an underestimated pathogen as adenovirus. Although PCR remains the gold standard for pathogen detection, this method requires specialized equipment and has a long turnaround time to process samples. Previously, LAMP assays for the detection of human adenovirus have been based on measuring the turbidity, the fluorescence of intercalated dyes, or electrophoretic separation. Herein, we present LAMP-based assays with colorimetric or fluorescent detection and perform a detailed assessment of their sensitivity, specificity, and diagnostic performance.

The Surgical Mask Microbiome Among Children with Asthma

Rationale: Exposure to respiratory pathogens, aeroallergens, and air pollution can lead to asthma exacerbations. The SarsCoV2 (COVID-19) pandemic led to widespread public health mandates including mask-wearing. We hypothesize that mask-wearing sequesters respiratory pathogens, leading to observed reduction in asthma exacerbations. The goal of this study was to characterize the bacterial microbiome from surgical masks in a cohort of school children with and without asthma. By identifying what is on both inside and outside the masks, we will be able to build a catalogue as a baseline for future analyses. Methods: We performed a cross-sectional study of children (4-18 years) attending an inner-city public school district. Students wore a surgical mask for a minimum of one school day. Parents completed a questionnaire about their child's demographics, respiratory history, and level of asthma impairment. To establish the protocol, we piloted the extraction and sequencing procedures among a sample of used masks from a hospital clinical personnel. DNA was extracted using a commercial DNA extraction kit on separated mask layers: inner, middle, and outer. 16S rRNA gene sequencing was then performed and then mapped against the most recent Greengene 16S rRNA gene database.Results: Recruitment and mask wearing occurred during an 8-week period (May 2021-July 2021). 34 students (18 with asthma;16 without asthma) from four schools were enrolled and completed the study. 74% of participants were in grades K-4, mean age was 8.4 years, and 53% identified as Hispanic/Puerto Rican. 59% of participants wore the mask one school day. 44% reported an asthma-related ED visit in their lifetime, while only 16% reported an ED visit in the past 12-months;53% of participants reported asthma symptoms with upper respiratory infections, however 77% reported zero respiratory infections in the past 12-months. In the masks worn by medical staff, bacterial genera including Staphyloccus, Haemophilus, Lawsonella, Streptococcus as well as Actinomyces, were identified similarly on inner and outer layers in the masks worn by clinicians. (Figure 1).Conclusions: We have demonstrated that recruiting and enrolling students from a medium-sized, inner-city public school district and obtaining facial mask samples is feasible. We demonstrate that self-reported rates of asthmarelated ED visits and respiratory infections differed pre-pandemic as compared to during. In addition, identifying the microbiome from surgical masks is possible. Bacteria genera identified were similar to known human nasal, oral and skin microbiomes. Current work is now in process characterizing and comparing the mask microbiomes among students with and without asthma.

WHOLE EXOME SEQUENCING IDENTIFIES A RARE NONSENSE MUTATION IN NACAD AS A POSSIBLE CAUSE OF COVID ORCHITIS IN BROTHERS

OBJECTIVE: This project sought to uncover genetic explanations as to why certain men face increased susceptibility to developing COVID orchitis. Our goal was to identify genetic variants associated with COVID orchitis in a group of patients, aided by whole-exome sequencing and protein phenotyping of affected patients. MATERIALS AND METHODS:We identified and examined six COVID- 19 patients who all were confirmed with polymerase chain reaction (PCR), including three COVID-19 (+) men without orchitis (controls) and three COVID (+) men with orchitis (bilateral testicular pain for at least 5 days around the time of testing PCR positive). Of note, among the three men with COVID-19 who had orchitis, two of them were siblings.DNA extraction and whole exome sequencing were performed on blood using the QIAmp blood maxi kit on five of the six patients. Variants were prioritized by being shared between the three patients affected with orchitis, absent in controls, and introducing nonsense, frameshift, splicing or non-synonymous amino acid changes and less than 10% in population prevalence. Based on WES findings, DuoSet® Human ACE2 reagent kit 2 (catalog number: DY933- 05) was purchased from R&D Systems, USA, and used to measure the level of soluble ACE2 in the plasma samples. RESULTS: The average age of the men in the study was 25 years old. The average duration of COVID symptoms (fever, sore throat, cough, body aches) were 7 days. Among the men who developed bilateral testis pain, the symptoms lasted for an average of 22 days. The median sperm concentration and sperm motility was 19 million/cc and 60% around 3 months after original infection. A list of 16 variants was generated that found to be shared between the two siblings with COVID orchitis along with the unrelated subject with COVID orchitis, and not present in the two controls. Among the 16 variants, a nonsynonymous non-frameshit deletion in NACAD variant on chromosome 7 with a frequency of 3.9% prevalence in ExAC was prioritized based on known involvement in the ACE2 pathway, read depth, and genotype quality. Phenotypically, we found that circulating levels of solubleACE2 was 3.72 ng/ml among men who had COVID orchitis and was lower than men who developed COVID without orchitis. CONCLUSIONS: We observed a stop mutation in NACAD in 2 brothers and 1 unrelated man who developed COVID orchitis. Interestingly, we found lower circulating ACE2 serum levels in both brothers with orchitis and the one nonrelated orchitis subject but normal serum levels in all controls. NACAD when involved with cellular ability to shuttle out ACE2 becomes critical for COVID symptomatology. With decreased transcellular and extracellular transport of ACE2 being possible in subjects with the gene mutation, it can be postulated more ACE2 will be found intracellularly leading to increased cellular entry of SARS CoV-2 and possibility of orchitis sequelae. IMPACT STATEMENT: These findings provide an explanation as to why genetic variations can lead to some patients developing comorbidities such as orchitis from COVID-19.

Clinical Characteristics and Laboratory Testing of Patients With COVID-19: A Retrospective Analysis

Objective: To explore the early warning signs of deterioration of patients with COVID-19. Methods: The data of thirty-six patients who were admitted to Handan Infectious Disease Hospital was collected. The clinical features and laboratory testing were analyzed retrospectively. The initial laboratory testing included blood chemistries, blood routine, D-dimer, coagulation function, etc. The patients were divided into mild/common group and severe/critical group. Results: The lymphocyte count, monocyte count, hemoglobin, and albumin levels in severe/critical group were lower compared with those in mild/common group, while the fibrinogen was higher. The lymphocyte count and monocyte count were positively correlated with hemoglobin, pre-albumin respectively. Conclusion: In conclusion, patients with lower initial prealbumin and hemoglobin level were more likely to progress into severe conditions. Decreased prealbumin and hemoglobin, combined with lymphocyte count and monocyte count, could be the early warning signs of deterioration of patients with COVID-19.

Real-time genotyping-based breast cancer risk assessment in MyPeBS, an international randomized trial in the general population comparing risk-stratified to standard breast cancer screening (BCS)

Background: Risk-stratified BCS, integrating personal, familial variables and a polygenic risk score (PRS) is a promising strategy that may improve current BCS outcomes. Real-time risk assessment and field implementation are some of the main challenges for such an approach. Methods: MyPeBS is an ongoing EU-funded international randomized trial running in 6 countries. Eligible women (wn) aged 40-70 are randomized 1:1 between continuing standard organized BCS as recommended in their participating country/region and switching to risk-stratified BCS, in which BCS schedule and modalities are adapted to the individual predicted 5-year risk of invasive BC (IBC). Primary endpoint is 4-year incidence of stage 2 and higher BC. Secondary endpoints include PROs. 5-year IBC risk is estimated using the Mammorisk® BCSC-derived or the Tyrer Cuzick risk score and the centrally-determined PRS313 obtained from a saliva sample and calibrated for national BC incidence and age. We aim to describe 1) the feasibility of real-time assessment of BC risk and 2) the characteristics and risk profiles of the participants. Results: As of Sept. 7, 2021, 16,550 wn had been randomized. 29% were aged <50 (median age 54 (range 40-70), 13% had a previous benign breast biopsy, 40% a mammographic breast density C or D, 19% a 1st degree family history of breast or ovarian cancer;72% had tertiary education. 36% were estimated at low risk (<1% risk of IBC at 5 years), 29% at average risk, and 35% at high (34%) or very high risk (1%) (>1.67% and >6% risk, respectively). Only 2.5% of DNA extractions were not usable for genotyping, due to DNA concentration or quality;and 98.8% of the eligible DNA samples were successfully genotyped. Median turnover time from saliva sampling to risk result available was 11 weeks despite the COVID pandemic (currently 7 weeks). Conclusions: Real-time BC risk assessment based on a large set of polymorphisms, family, screening and hormonal history, and breast density is feasible within organized screening programmes. Participants are so far representative of different categories with some over-representation of highly educated participants. Clinical trial identification: NCT03672331. Legal entity responsible for the study: Unicancer. Funding: European Commission and French National Cancer Institute. Disclosure: S. Delaloge: Financial Interests, Institutional, Advisory Board: AstraZeneca;Financial Interests, Institutional, Invited Speaker: Exact Sciences;Financial Interests, Institutional, Advisory Board: Novartis;Financial Interests, Institutional, Advisory Board: Pierre fabre;Financial Interests, Institutional, Advisory Board: Orion;Financial Interests, Institutional, Advisory Board: Sanofi;Financial Interests, Institutional, Advisory Board: Rappta;Financial Interests, Institutional, Advisory Board: Cellectis;Financial Interests, Institutional, Advisory Board: Isis/servier;Financial Interests, Institutional, Invited Speaker: Pfizer;Financial Interests, Institutional, Invited Speaker: Seagen;Financial Interests, Institutional, Invited Speaker: Lilly;Financial Interests, Institutional, Invited Speaker: AstraZeneca;Financial Interests, Institutional, Invited Speaker: MSD;Financial Interests, Institutional, Advisory Board, ad board: Besins Healthcare;Financial Interests, Institutional, Invited Speaker: Roche Genentech;Financial Interests, Institutional, Invited Speaker: BMS;Financial Interests, Institutional, Invited Speaker: Puma;Financial Interests, Institutional, Invited Speaker: AstraZeneca;Financial Interests, Institutional, Invited Speaker: Orion;Financial Interests, Institutional, Invited Speaker: Sanofi;Financial Interests, Institutional, Funding: GE;Financial Interests, Institutional, Invited Speaker: Pfizer;Financial Interests, Institutional, Invited Speaker, clinical research funding to my institution: Taiho;Non-Financial Interests, Invited Speaker, Société Française de Sénologie et Pathologie Mammaire: SFSPM. D. Keatley: Financial Interests, Personal, Advisory Board: Public Advisory Board of Heealth Data UK. E. Gauthier: Financial Interests, Personal, Stocks/Shares: Predilife;Financial Interests, Personal, Full or part-time Employment: Predilife. S. Michiels: Financial Interests, Personal, Advisory Role: IDDI;Financial Interests, Personal, Advisory Role: Amaris;Financial Interests, Personal, Advisory Role: Roche;Financial Interests, Personal, Advisory Role: Sensorion;Financial Interests, Personal, Advisory Role: Biophytis;Financial Interests, Personal, Advisory Role: Servier;Financial Interests, Personal, Advisory Role: Yuhan. All other authors have declared no conflicts of interest.