SARS-COV-2 Infection in Children and Young People in Bulgaria - A Prospective, Single-Center, Cohort Study

Compared to other respiratory viruses, the proportion of hospitalizations due to SARS-CoV-2 among children is relatively low. While severe illness is not common among children and young individuals, a particular type of severe condition called multisystem inflammatory syndrome in children (MIS-C) has been reported. The aim of this prospective cohort study, which followed a group of individuals under the age of 19, was to examine the characteristics of patients who had contracted SARS-CoV-2, including their coexisting medical conditions, clinical symptoms, laboratory findings, and outcomes. The study also aimed to investigate the features of children who met the WHO case definition of MIS-C, as well as those who required intensive care. A total of 270 patients were included between March 2020 and December 2021. The eligible criteria were individuals between 0-18 with a confirmed SARS-CoV-2 infection at the Infectious Disease Hospital "Prof. Ivan Kirov"in Sofia, Bulgaria. Nearly 76% of the patients were <= 12 years old. In our study, at least one comorbidity was reported in 28.1% of the cases, with obesity being the most common one (8.9%). Less than 5% of children were transferred to an intensive care unit. We observed a statistically significant difference in the age groups, with children between 5 and 12 years old having a higher likelihood of requiring intensive care compared to other age groups. The median values of PaO2 and SatO2 were higher among patients admitted to the standard ward, while the values of granulocytes and C-reactive protein were higher among those transferred to the intensive care unit. Additionally, we identified 26 children who met the WHO case definition for MIS-C. Our study data supports the evidence of milder COVID-19 in children and young individuals as compared to adults. Older age groups were associated with higher incidence of both MIS-C and ICU admissions.Copyright © 2023 P. Velikov et al., published by Sciendo.

A novel isothermal digital amplification system and its application for absolute quantification of respiratory infectious virus

Herein, we establish a novel isothermal digital amplification system termed digital nicking and extension chain reaction system-based amplification (dNESBA) by utilizing the isothermal NESBA technique and the newly developed miniaturized fluorescence monitoring system (mFMS). dNESBA enables parallel isothermal NESBA reactions in more than 10,000 localized droplet microreactors and read the fluorescence signals rapidly in 150 s by mFMS. This system could identify the genomic RNA (gRNA) extracted from target respiratory syncytial virus A (RSV A) as low as 10 copies with remarkable specificity. The practical applicability of dNESBA was also successfully verified by reliably detecting the gRNA in the artificial sputum samples with excellent reproducibility and accuracy. Due to the intrinsic advantages of isothermal amplifying technique including the elimination of the requirement of thermocycling device and the enhanced portability of the miniaturized read-out equipment, the dNESBA technique equipped with mFMS could serve as a promising platform system to achieve point-of-care (POC) digital molecular diagnostics, enabling absolute and ultra-sensitive quantification of various infectious pathogens even in an early stage.Copyright © 2023

CLINICAL AND GENETIC DETERMINANTS OF SEVERE COURSE OF COVID-19 IN PREGNANT WOMEN.

Objectives: to determine the clinical and genetic determinants of the severe course of COVID-19 in pregnant women in order to identify a risk group and search for therapeutic targets. Materials and methods. 21 patients (group 1) with a severe course of COVID-19 who required intensive care in the Anesthesiology and Intensive Care Unit (AICU) and 126 pregnant women with moderate severity treated in the Infectious-Obstetrics Unit (IOCU) were examined (group 2). Genomic DNA for molecular genetic analysis of gene variants ACE (I/D, rs 4340), PGR (Alu insertion), ESR1 (A351G, rs 9340799), PON1 (C108T, rs 705379) was isolated from the peripheral blood of patients using a commercial Quick-DNA Miniprep Plus Kit (Zymo Research, USA). Variants of ACE and PGR genes were determined using allele-specific polymerase chain reaction;polymerase chain reaction followed by restriction analysis was used to determine ESR1 and PON1 gene variants. Results. Severe course of COVID-19 is observed in 18.2% of pregnant women, critical condition in 7.5%. A third of AICU patients are over 35 years old. Somatic anamnesis was complicated in 23.8% of patients;thyroid gland pathology (14.3%) and varicose disease (19.0%) prevailed. A significant factor in the severe course of COVID-19 is obesity of the III-IV degree in 28.5% cases. The severe course of the disease was associated with complications of pregnancy (oligohydramnios - 52.4%, ahydramnios - 14.3%, fetal growth retardation syndrome - 33.3%, circulatory disorders - 57.1%, fetal distress - 47.6%, preeclampsia - 14.3%), labor (caesarean section - 57.1%, premature birth - 28.6%), disorders of newborns state (asphyxia - 35.6%). These patients are characterized by anemia (58.7%), thrombocytopenia (23.8%), leukocytosis (33.3%), lymphopenia (90.5%), a shift of the leukocyte formula to the left (an increase of rod-nuclear leukocytes by 85.7%). There were significantly increased levels of transaminases: alanine aminotransferase in 47.6%, aspartate aminotransferase in 76.2%. Prothrombotic changes are indicated by a decrease in prothrombin time and activated partial thromboplastin time in 66.7%, which is confirmed by an increase in D-dimer in 85.7% of patients up to the maximum 15,000 ng/ml in 9.5% of women. An increase in inflammation markers (C-reactive protein and interleukin-6 in all AICU patients, procalcitonin in 66.7%) is a reflection of the destructive effect of inflammatory processes. The genetic determinants of the severe course of COVID-19 in pregnant women can be the ID genotype of the ACE I/D rs4340 polymorphism (81.0%), the T2/T2 PROGINS genotype (19.0%), the ESR1 A351G rs9340799 GG genotype (28.5%). Conclusions. The use of separate clinical, laboratory and genetic indicators in pregnant women with COVID-19 will contribute to the selection of the risk group of a coronavirus severe course and the determination of targets of therapeutic impact.Copyright © 2022 Trylyst. All rights reserved.

Comparison of Clinical and Laboratory Findings at Different Clinical Stages in Cats Naturally Infected with Feline Coronavirus

Feline coronavirus (FCoV) infections occur commonly in cats, with entrocyte and monocyte-macrophage tropism. Most FCoV-infected cats remain asymp tomatic, but up to 10% develop fatal feline infectious peritonitis (FIP). This study aims to investigate the diagnostic utility of clinical and laboratory examinations including serum and effusion AGP levels in cats either with symptomatic effusive FIP or asymptomatic feline enteric coronavirus (FECV). The study included 40 cats with effusive FIP and 10 cats with FECV infection. The FIP group was divided into two subgroups: Abdominal (AE;n=30) and thoracic effusion (TE;n=10). Clinical and laboratory examinations, including serum or effusion AGP measurement, were performed. Among all the groups, TE group had higher body temperature, heart and respiratory rates (P<0.000). Compared with the FECV group, the FIP group had lower pH and HCO3 levels and higher base excess and lactate levels (P<0.05). The leukocyte and lymphocyte counts were higher and the hematocrit was lower in the AE group among all the groups (P<0.023). MCV was lower in the FIP group compared to the FECV group (P<0.002). In the AE group, total protein level was the lowest and the AST, GGT, total bilirubin and cholesterol levels were the highest (P<0.032) among all the groups. Magnesium level was lower in the FIP group compared to the FECV group (P<0.044). Although the serum AGP level was highest in the TE group among all groups (P<0.004), the AGP levels of cats with FECV were similar to the AE group (P>0.05). Since FECV-positive cats will likely develop FIP, differences in clinical and laboratory findings in FECV-positive cats were identified. Among them, pH, HCO3, base excess, lactate, MCV and magnesium were found to be important in the course of the disease, and AGP in the evaluation of the presence of an inflammatory state. It was concluded that clinical, laboratory and serum AGP evaluation could be used in the index of suspicion of development of FIP and FECV.Copyright © 2023 Erdem Gulersoy et al., published by Sciendo.

Genomic characterization and phylogenetic analysis of sars-cov-2 in libya

The COVID-19 epidemic started in Libya in March 2020 and rapidly spread. To shed some light on the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) strains circulating in Libya, viruses isolated from 10 patients in this country were sequenced, characterized at the genomic level, and compared to genomes isolated in other parts of the world. As nine genomes out of 10 belonged to the SS1 cluster and one to SS4, three datasets were built. One included only African strains and the other two contained internationally representative SS1 and SS4 genomes. Genomic analysis showed that the Libyan strains have some peculiar features in addition to those reported in other world regions. Considering the countries in which the strains are genetically more similar to the Libyan strains, SARS-CoV-2 could have entered Libya from a North African country (possibly Egypt), sub-Saharan Africa (e.g., Ghana, Mali, Nigeria), the Middle East (e.g., Saudi Arabia), or Asia (India, Bangladesh).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.

The Quinolone Resistance Genes in the Bacteriophage DNA Fractions in the Healthy Calf Stool Samples Via qPCR

The One Health approach shows that people, animals, plants, and environmental factors can affect each other. Phages are one of the mobile genetic elements. Quinolones are a critical group of antibiotics for both human and animal health and monitoring their antimicrobial resistance is very important. The aim of the study is to determine the frequency of the quinolone resistance gene in bacteriophage DNA fractions obtained from healthy calf stool samples. In our study, 50 samples from 6-9 months old calves, which were found to be healthy and not treated with any group of antibiotics in Sanliurfa province, were included. DNA isolation was made from phage lysates of stool samples and specific primers were used qnrA, qnrB and qnrS genes. qPCR was performed on LightCycler480. Despite not receiving any antibiotic treatment, qnrB was the most detected gene among the phage DNA fractions detected in 11 calves. While qnrA, qnrB and qnrS quinolone resistance genes were detected together in one sample, qnrB and qnrS resistance genes were found together in two samples. Our data, obtained from the study in Turkiye to search for antimicrobial resistance genes in phage fractions, showed the importance of the One Health approach and determined that it was highly effective in quinolone resistance gene shedding in healthy calves that had never been treated with antibiotics. It has been concluded that in empirical treatment with quinolone, attention should be paid to all living things and unconscious antibiotic use may cause the spread of resistance genes more than expected.Copyright © 2023, Veteriner Fakultesi Dergisi. All rights reserved.

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.

Blood Transcriptomes of Kidney Recipients with Covid-19 Demonstrate Deficient Adaptive Immune Activation

Purpose: Kidney transplant recipients (KTRs) have poor outcomes compared to non-KTRs with acute COVID-19. To provide insight into management of immunosuppression (IS) during COVID-19, we studied immune signatures from the peripheral blood during and after COVID-19 infection from a multicenter KTR cohort. Method(s): Clinical data were collected by chart review. Paxgene blood RNA was polyA-selected and sequenced at enrollment Results: A total of 64 KTRs affected with COVID-19 were enrolled (31 Early cases (<4weeks from a positive SARS-CoV-2 PCR test) and 33 late cases). Out of the 64 patients, eight died and three encountered graft losses during follow-up. Among 31 early cases, we detected differentially expressed genes (nominal p-value < 0.01) in the blood transcriptome that were positively or negatively associated with the COVID-19 severity score (scale of 1 to 7 with increasing severity;Fig 1A). Enrichment analyses showed upregulation of neutrophil and innate immune pathways and downregulation of adaptive immune activation pathways with increasing severity score (Fig 1B). This observation was independent of lymphocyte count, despite reduction in immunosuppression (IS) in 75% of KTRs. Interestingly, compared with early cases, the blood transcriptome in late cases showed "normalization" of these enriched pathways after 4 weeks, suggesting return of adaptive immune system activation despite re-initiation of immunosuppression (Fig 1C). The latter analyses were adjusted for the severity score. Interestingly, similar pathway enrichment with worsening severity of COVID-19 was identifiable from a public dataset of non-KTRs (GSE152418), showing overlapped signatures for acute COVID-19 between KTRs and non-KTRs (overlap P<0.05) (Fig 1D). Conclusion(s): Blood transcriptome of COVID-KTRs shows marked decrease in adaptive immune system activation during acute COVID-19, even during IS reduction, which show recovery after acute illness. (Figure Presented).

Blood Transcriptomes of Covid-19 Infected Kidney Recipients Demonstrate Frequent and Persistent SARS-CoV-2 Virome Gene Expression

Purpose: Kidney transplant recipients (KTRs) have poor outcomes vs non-KTRs with acute COVID-19. To provide insight into management of immunosuppression during acute COVID-19, we studied peripheral blood transcriptomes during and after COVID-19 from a multicenter KTR cohort. Method(s): Clinical data were collected by chart review. Paxgene blood RNA was polyA-selected and sequenced at enrollment. Result(s): A total of 64 KTRs with COVID-19 were enrolled (31 Early cases (<4weeks from a positive SARS-CoV-2 PCR test) and 33 late cases). Out of the 64 patients, eight died and three encountered graft losses during follow-up. Due to presence of mRNA reads in the blood transcriptome unmapped to the human genome, we aligned the mRNA short reads to the SARS-CoV-2 genome. Surprisingly, our strategy detected the SARS-Cov2 mRNA, especially Spike mRNA in 27 (87%) early cases, and 18 (54%) of late cases (Fig 1A and B). We then analyzed the raw reads from a public dataset of non-KTRs with Paxgene RNA (GSE172114). The SARS-CoV-2 Spike mRNA was detected in 2/47 (4.2%) critically ill COVID-19 cases and 0/25 noncritically ill cases in this non-KTR dataset (compared to KTRs, Chi-square P<0.001;Fig 1B). Among our KTRs, the amount of Spike mRNA was associated positively with the COVID-19 severity score (scale of 1 to 7 of increasing severity;Fig 1C) and inversely with time from initial positive PCR (Fig 1D). More interestingly, 7/64 patients had detectable Spike RNA-emia beyond 60 days after COVID-19 diagnosis. Of the 3 graft losses in our cohort, 2 occurred among these 7 patients. Conclusion(s): Blood transcriptome of KTRs with COVID-19 demonstrated a risk for persistent viremia with implications for pathogenesis of COVID-19 disease. This finding also supports using passive immune strategies in COVID-KTRs. (Figure Presented).

Evaluation of CCR5-Î"32 mutation and HIV-1 surveillance drug-resistance mutations in peripheral blood mononuclear cells of long-term non progressors of HIV-1-infected individuals

Aim: This study aimed to evaluate chemokine receptor 5 delta 32 (CCR5-δ32) mutation and HIV-1 surveillance drug-resistance mutations (SDRMs) in peripheral blood mononuclear cells of long-term non progressors (LTNPs) of HIV-1-infected individuals. Materials & methods: This research was performed on 197 treatment-naive HIV-1-infected patients. After follow-up, it was determined that 15 (7.6%) of these people were LTNPs. The PCR assay was performed to identify the CCR5 genotype and HIV-1 SDRMs. Results: One (6.7%) of the LTNPs was heterozygous (wt/δ32) for the CCR5 delta 32 (CCR5δ32). However, none of the individuals was homozygous for this mutation (δ32/δ32). Moreover, none of the LTNPs showed HIV-1 SDRMs. The CRF35-AD subtype was the most dominant subtype, with a percentage of 93.3%. Conclusion: Iranian elite controllers are negative for CCR5-delta 32 homozygous genotype and drug resistance against antiretroviral drugs.

Access Profiling systemic physiology through blood-based multidimensional

The physiology of critical care patients is more complex than normally appreciated. Patients arrive at the intensive care unit (ICU) or the pediatric ICU (PICU) with a variety of infections, trauma, organ damage, and dysfunctional immune systems. This population is the prime target for testing and applying new precision medicine tools to decipher the unique biology occurring within each patient. This is particularly important as COVID-19 has made such an impact on the United States healthcare system. Thus, there is a need to develop strategies to find multiple levels of information while minimizing the number of tests performed, shifting the balance of testing to more proactive than reactive. With the collection of ∼2mL of blood (about half a teaspoon), our collaboration between Spectrum Health and Michigan State University has shown the ability to use PAXgene tubes and RNAseq to simultaneously map human gene/transcript signatures, score panels of corresponding risk genes, deconvolute immune cells, detect markers of organ/cell damage, detect RNA from bacteria/viruses/plants/fungi, profile the immune repertoire, address how patients are unique from other samples, and address common/rare genetic mutations. These tools have been applied to three cohorts of patients (and age matched controls) for critical care medicine physiology understanding for nearly all ages: 1) Infants with Respiratory syncytial virus (RSV);2) Kids with multiple organ dysfunction syndrome;and 3) Adults with hospitalized or lethal COVID-19. Our findings from these tools shows the complexity of immune system activation, secondary infections, and under appreciated interactions of the immune cell disorder overlapping COVID-19 pathology. The promise of blood-based transcriptomics to reveal cellular and cell free signatures opens a door for building more detailed physiological mechanisms from precision medicine.

LUNG ACE2 AND TMPRSS2 GENE EXPRESSION VARIES WITH ALLERGEN EXPOSURES IN MURINE MODELS OF ASTHMA

Introduction: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires angiotensin-converting enzyme 2 (ACE2) and the transmembrane protease, serine 2 (TMPRSS2) for cell entry. Prior studies have reported that allergen exposure can downregulate ACE2 expression. Here, we sought to determine if exposure to distinct combinations of allergens results in the differential expression of ACE2 and TMPRSS2 in the mouse lung. Methods: We utilized three established murine models of asthma: 1. Alum/Ovalbumin (OVA) model;2. House dust mite (HDM)/OVA model;3. Mixed-allergen (MA) model using OVA, HDM, Aspergillus fumigatus and Alternaria alternata. Phosphate-buffered saline (PBS) treated mice were used as controls. Lung RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol, and complementary DNA was synthesized. Quantitative PCR (qPCR) was performed 24 hours after the last challenge utilizing validated primers for ACE2 and TMPRSS2. Analysis was performed using one-way ANOVA. Results: Here we report that ACE2 mRNA expression was lower in the MA and Alum/OVA-treated mice compared to the controls (p-value < 0.0001). No difference was seen in the ACE2 mRNA expression between HDM/OVA and PBS-treated mice. Furthermore, HDM/OVA-treated mice expressed higher levels of TMPRSS2 mRNA compared to controls (p-value < 0.01). No difference was seen in the TMPRSS2 mRNA expression between the MA or Alum/OVA, and the PBS-treated mice. Conclusion: The exposure to distinct combinations of allergens results in unique patterns of ACE2 and TMPRSS2 gene expression in the mouse lung. Further studies are required to evaluate the effects of allergen exposure with the susceptibility to SARS-CoV-2 infection.

Qatar Biobank: COVID-19 biorepository project

Background: The rapid spread of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its resulting disease (COVID-19) is one of the greatest global public health crisis of the recent decades1. The COVID-19 Biorepository is a national project aimed to support the high demand of biomedical research by multiple groups and the need to have access to high quality, curated clinical data, and specimens contributing to the understanding of, and response to, the COVID-19 pandemic and its impacts in Qatar2, 3. Methods/Case presentation: Patients with a laboratory diagnosis of COVID-19, who were Qatar residents that could communicate in Arabic, English, Hindi, and Urdu were eligible to participate in the study. COVID-19 diagnosed patients were recruited at the time of their disease period from the main three public hospitals (Communicable Disease Center, Cuban, and Hazm Mebaireek Hospitals) serving as isolation facilities of symptomatic patients in Qatar, during a 7-month period from March 2020 until September 2020. Consented participants were followed up on a weekly basis until recovery, and then monthly for a year. Sociodemographic and clinical data were collected in electronic questionnaires via a face-to-face interview by trained Qatar Biobank (QBB) staff. Results: A total of 2097 consented participants were recruited up to September 2020, males (N=1050) and females (N=1047), with a mean age of 41 years (SD: 15.5). 61.0% of the participants had at least one follow up while 27% adhered to monthly follow up visits. Data was collected for 99.7% of the participants, while the follow up process is still ongoing. In total there are 107,171 high quality specimens in the biorepository including plasma, erythrocytes, buffy coat, serum, PAXgene whole blood, nasopharyngeal secretions, and DNA. Conclusion: The COVID-19 Biorepository is a national asset to illuminate the pathophysiological and identify markers of disease prognosis as well as to describe the clinical features and epidemiology of COVID-19 in Qatar and worldwide.