Reverse Transcription-polymerase Chain Reaction Results Research Articles

Astragaloside IV Alleviates Podocyte Injury in Diabetic Nephropathy through Regulating IRE-1α/NF-κ B/NLRP3 Pathway.

To investigate the effects of astragaloside IV (AS-IV) on podocyte injury of diabetic nephropathy (DN) and reveal its potential mechanism. In in vitro experiment, podocytes were divided into 4 groups, normal, high glucose (HG), inositol-requiring enzyme 1 (IRE-1) α activator (HG+thapsigargin 1 µmol/L), and IRE-1α inhibitor (HG+STF-083010, 20 µmol/L) groups. Additionally, podocytes were divided into 4 groups, including normal, HG, AS-IV (HG+AS-IV 20 µmol/L), and IRE-1α inhibitor (HG+STF-083010, 20 µmol/L) groups, respectively. After 24 h treatment, the morphology of podocytes and endoplasmic reticulum (ER) was observed by electron microscopy. The expressions of glucose-regulated protein 78 (GRP78) and IRE-1α were detected by cellular immunofluorescence. In in vivo experiment, DN rat model was established via a consecutive 3-day intraperitoneal streptozotocin (STZ) injections. A total of 40 rats were assigned into the normal, DN, AS-IV [AS-IV 40 mg/(kg·d)], and IRE-1α inhibitor [STF-083010, 10 mg/(kg·d)] groups (n=10), respectively. The general condition, 24-h urine volume, random blood glucose, urinary protein excretion rate (UAER), urea nitrogen (BUN), and serum creatinine (SCr) levels of rats were measured after 8 weeks of intervention. Pathological changes in the renal tissue were observed by hematoxylin and eosin (HE) staining. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect the expressions of GRP78, IRE-1α, nuclear factor kappa Bp65 (NF-κBp65), interleukin (IL)-1β, NLR family pyrin domain containing 3 (NLRP3), caspase-1, gasdermin D-N (GSDMD-N), and nephrin at the mRNA and protein levels in vivo and in vitro, respectively. Cytoplasmic vacuolation and ER swelling were observed in the HG and IRE-1α activator groups. Podocyte morphology and ER expansion were improved in AS-IV and IRE-1α inhibitor groups compared with HG group. Cellular immunofluorescence showed that compared with the normal group, the fluorescence intensity of GRP78 and IRE-1α in the HG and IRE-1α activator groups were significantly increased whereas decreased in AS-IV and IRE-1α inhibitor groups (P<0.05). Compared with the normal group, the mRNA and protein expressions of GRP78, IRE-1α, NF-κ Bp65, IL-1β, NLRP3, caspase-1 and GSDMD-N in the HG group was increased (P<0.05). Compared with HG group, the expression of above indices was decreased in the AS-IV and IRE-1α inhibitor groups, and the expression in the IRE-1α activator group was increased (P<0.05). The expression of nephrin was decreased in the HG group, and increased in AS-IV and IRE-1α inhibitor groups (P<0.05). The in vivo experiment results revealed that compared to the normal group, the levels of blood glucose, triglyceride, total cholesterol, BUN, blood creatinine and urinary protein in the DN group were higher (P<0.05). Compared with DN group, the above indices in AS-IV and IRE-1α inhibitor groups were decreased (P<0.05). HE staining revealed glomerular hypertrophy, mesangial widening and mesangial cell proliferation in the renal tissue of the DN group. Compared with the DN group, the above pathological changes in renal tissue of AS-IV and IRE-1α inhibitor groups were alleviated. Quantitative RT-PCR and Western blot results of GRP78, IRE-1α, NF-κ Bp65, IL-1β, NLRP3, caspase-1 and GSDMD-N were consistent with immunofluorescence analysis. AS-IV could reduce ERS and inflammation, improve podocyte pyroptosis, thus exerting a podocyte-protective effect in DN, through regulating IRE-1α/NF-κ B/NLRP3 signaling pathway.

Molecular mechanism of the influence of related genes expression in synovium tissue around shoulder joint of secondary frozen shoulder model rats on angiogenesis.

The study aimed to explore the pathogenesis of secondary frozen shoulder and its influence on synovium tissue and angiogenesis by constructing a rat secondary frozen shoulder model along with transforming growth factor. 40 healthy male rats aged 8 weeks were divided into Sham group (n=10, no modeling treatment), Control group (n=10, modeling treatment), Low group (n=10, modeling treatment, and 10 mL/d transforming growth factor), and High group (n=10, modeling treatment, and 20 mL/d transforming growth factor). Hematoxylin and Eosin (HE) method was used for histological detection, and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and immunohistochemical staining method were adopted to detect the expression of Matrix metalloproteinase-14 (MMP-14), mitogen-activated protein kinase (p38MAPK), and Vascular endothelial growth factor (VEGF). Compared with Sham group, the range of abduction and external rotation of rat glenohumeral joint in Control group, Low group, and High group was significantly reduced, and High group had the smallest range. Compared with the Sham group, the synovium in the Control group, the Low group, and the High group had obvious hyperplasia, and the blood vessels were significantly increased. Immunohistochemical staining and RT-PCR results showed that compared with Sham group, MMP-14, p38 MAPK, and VEGF in Control group, Low group, and High group all increased significantly, among which High group increased most. The secondary frozen shoulder is mainly manifested as synovial hyperplasia and increased blood vessels, which are related to the induction of MMP-14, p38 MAPK, and VEGF by transforming growth factor, which reveals the pathogenesis of secondary frozen shoulder to a certain extent, and lays a foundation for subsequent clinical treatment of secondary frozen shoulder.

Detection method for reverse transcription recombinase-aided amplification of avian influenza virus subtypes H5, H7, and H9

BackgroundAvian influenza virus (AIV) not only causes huge economic losses to the poultry industry, but also threatens human health. Reverse transcription recombinase-aided amplification (RT-RAA) is a novel isothermal nucleic acid amplification technology. This study aimed to improve the detection efficiency of H5, H7, and H9 subtypes of AIV and detect the disease in time. This study established RT-RAA-LFD and real-time fluorescence RT-RAA (RF-RT-RAA) detection methods, which combined RT-RAA with lateral flow dipstick (LFD) and exo probe respectively, while primers and probes were designed based on the reaction principle of RT-RAA.ResultsThe results showed that RT-RAA-LFD could specifically amplify H5, H7, and H9 subtypes of AIV at 37 °C, 18 min, 39 °C, 20 min, and 38 °C, 18 min, respectively. The sensitivity of all three subtypes for RT-RAA-LFD was 102 copies/µL, which was 10 ∼100 times higher than that of reverse transcription polymerase chain reaction (RT-PCR) agarose electrophoresis method. RF-RT-RAA could specifically amplify H5, H7, and H9 subtypes of AIV at 40 °C, 20 min, 38 °C, 16 min, and 39 °C, 17 min, respectively. The sensitivity of all three subtypes for RF-RT-RAA was 101 copies/µL, which was consistent with the results of real-time fluorescence quantification RT-PCR, and 100 ∼1000 times higher than that of RT-PCR-agarose electrophoresis method. The total coincidence rate of the two methods and RT-PCR-agarose electrophoresis in the detection of clinical samples was higher than 95%.ConclusionsRT-RAA-LFD and RF-RT-RAA were successfully established in this experiment, with quick response, simple operation, strong specificity, high sensitivity, good repeatability, and stability. They are suitable for the early and rapid diagnosis of Avian influenza and they have positive significance for the prevention, control of the disease, and public health safety.

The Diagnostic Performance of Lung Sonography to Assess Pregnant Patients Infected With SARS-CoV-2 Based on Varied Clinical Severity

Objective: The aim of this study was to assess the diagnostic accuracy of lung ultrasonography (LUS) and a local clinical triage protocol complicated by varied clinical severities of COVID-19, in pregnant patients who had computed tomography (CT) and reverse transcription polymerase chain reaction (RT-PCR) results. Materials and Methods: This retrospective cohort study was conducted in a tertiary maternity unit, during the pandemic, between March 2020 and November 2021. The pregnant patients included in the study were diagnostically categorized as asymptomatic, mild, moderate, severe, and critical, upon hospital admission and their presenting disease severity. All of the patients, as well as those with mild and asymptomatic severity, LUS were evaluated accuracy based on their RT-PCR results. Results: In total, 1531 pregnant patients underwent a primary emergency department clinical evaluation, in the pandemic clinic. The sensitivity, specificity, positive predictive value, and negative predictive values for LUS were 70.5%, 72.45%, 88.1%, and 45.8%, respectively. Conclusion: In this large cohort of patients, LUS was shown to be quick, safe, effective, and reproducible diagnostic tool for the screening of COVID-19 pneumonia, in this obstetric practice. This clinical evidence also provided a new set of triage strategies, during the pandemic, for pregnant patients.

SARS-CoV-2 Viral Shedding and Rapid Antigen Test Performance - Respiratory Virus Transmission Network, November 2022-May 2023.

As population immunity to SARS-CoV-2 evolves and new variants emerge, the role and accuracy of antigen tests remain active questions. To describe recent test performance, the detection of SARS-CoV-2 by antigen testing was compared with that by reverse transcription-polymerase chain reaction (RT-PCR) and viral culture testing during November 2022-May 2023. Participants who were enrolled in a household transmission study completed daily symptom diaries and collected two nasal swabs (tested for SARS-CoV-2 via RT-PCR, culture, and antigen tests) each day for 10 days after enrollment. Among participants with SARS-CoV-2 infection, the percentages of positive antigen, RT-PCR, and culture results were calculated each day from the onset of symptoms or, in asymptomatic persons, from the date of the first positive test result. Antigen test sensitivity was calculated using RT-PCR and viral culture as references. The peak percentage of positive antigen (59.0%) and RT-PCR (83.0%) results occurred 3 days after onset, and the peak percentage of positive culture results (52%) occurred 2 days after onset. The sensitivity of antigen tests was 47% (95% CI=44%-50%) and 80% (95% CI=76%-85%) using RT-PCR and culture, respectively, as references. Clinicians should be aware of the lower sensitivity of antigen testing compared with RT-PCR, which might lead to false-negative results. This finding has implications for timely initiation of SARS-CoV-2 antiviral treatment, when early diagnosis is essential; clinicians should consider RT-PCR for persons for whom antiviral treatment is recommended. Persons in the community who are at high risk for severe COVID-19 illness and eligible for antiviral treatment should seek testing from health care providers with the goal of obtaining a more sensitive diagnostic test than antigen tests (i.e., an RT-PCR test).

The application of targeted RNA sequencing for the analysis of fusion genes, gene mutations, IKZF1 intragenic deletion, and CRLF2 overexpression in acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is characterized by highly genetic heterogeneity, owing to recurrent fusion genes, gene mutations, intragenic deletion, and gene overexpression, which poses significant challenges in clinical detection. RNA sequencing (RNA-seq) is a powerful tool for detecting multiple genetic abnormalities, especially cryptic gene rearrangements, in a single test. Sixty samples (B-ALL, n = 49; T-ALL, n = 9; mixed phenotype acute leukemia (MPAL), n = 2) and 20 controls were analyzed by targeted RNA-seq panel of 507 genes developed by our lab. Of these, 16 patients were simultaneously analyzed for gene mutations at the DNA level using a next-generation sequencing panel of 51 genes. Fusion genes, CRLF2 expression, and IKZF1 intragenic deletion were also detected by reverse transcription-polymerase chain reaction (RT-PCR). Karyotype analysis was performed using the R-banding and G-banding technique on bone marrow cells after 24 hours of culture. Partial fusion genes were analyzed using fluorescence in situ hybridization (FISH). Compared with the results of Karyotype analysis, FISH, and RT-PCR, the detection rate of fusion genes by targeted RNA-seq increased from 48.3% to 58.3%, and six unexpected fusion genes were discovered, along with one rare isoform of IKZF1 intragenic deletion (IK10). The DNA sequencing analysis of 16 ALL patients revealed that 96.2% (25/26) of gene mutations identified at the DNA level were also detectable at the RNA level, except for one mutation with a low variant allele fraction. The detection of CRLF2 overexpression exhibited complete concordance between RT-PCR and RNA-seq. The utilization of RNA-seq enables the identification of clinically significant genetic abnormalities that may go undetected through conventional detection methods. Its robust analytical performance might bring great application value for clinical diagnosis, prognosis, and therapy in ALL.

The regulatory effects of electroacupuncture on type 2 innate lymphoid cell (ILC2) function and browning of white adipose tissue in obese rats: 电针通过调控ILC2功能和白色脂肪棕色化治疗肥胖大鼠的作用机制

ObjectiveTo observe the effects of electroacupuncture (EA) on type 2 innate lymphoid cell (ILC2) function in white adipose tissue (WAT) of high-fat diet (HFD)-induced obese rats to reveal the mechanism underlying weight loss. MethodsMale Winstar rats (n=15) were randomly divided into control, HFD, and HFD+EA groups, with five rats in each group. An obesity model was established by feeding rats with a HFD. EA (2 Hz, 1 mA) was applied to “Zhongwan (CV12)” bilateral “Tianshu (ST25)” “Zusanli (ST36)” and “Sanyinjiao (SP6)” for 15 min, 7 days a week, for a total of 5 weeks. During EA treatment, the HFD group was fed a HFD, whereas rats in the control group were maintained on a normal diet. There was no intervention in either the control or the HFD groups. Body weight was measured weekly. Full-field adipocyte morphology, adipocyte volume, and the number of abdominal WAT were observed under a microscope after sectioning and hematoxylin and eosin staining. The levels of IL-5 (interleukin-5), IL-13 (interleukin-13), IL-33 (interleukin-33), and ST2 (growth stimulating gene 2 protein) in the WAT were assayed using enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction (RT-PCR). The expression levels of browning markers including PGC1α（peroxisome proliferator-activated receptor gamma coactivator 1-alpha）, PPARγ (peroxisome proliferator-activated receptor gamma), PRDM16 (histone-lysine N-methyltransferase), and UCP1（uncoupling protein 1 in WAT were detected by western blot analysis and RT-PCR. ResultsAfter a 7-week induction period, rats in both the HFD and HFD+EA groups were 20% heavier than their initial weights before modeling (P<0.01, P<0.001). Following a 5-week EA intervention, the body mass and Lee index of obese rats were significantly reduced (P<0.05). The HE results showed that adipocytes in the HFD group displayed a significant increase in diameter and a decrease in number compared to the control group. However, in the HFD+EA group, white adipocytes exhibited reduced diameter and appeared more regular in shape. In the control group, the proportion of ILC2 was (86.53±2.55)%, whereas in the HFD group, it decreased to (73.62±2.52)%, indicating a significant reduction in ILC2 in the WAT of obese rats. Following EA treatment, the proportion of ILC2 was elevated to (80.54±4.25)%. This analysis revealed a significant decrease in the expression levels of IL-5, IL-13, IL-33, and ST2 in the WAT of obese rats in the HFD group; however, these levels increased after EA intervention. The mRNA levels of PPARγ, PGC1α, PRDM16, and protein levels of UCP1 were decreased in the HFD group (P<0.01, P<0.05, and P<0.001), and the trends were significantly reversed with EA manipulation (P<0.05). RT-PCR and western blotting results revealed that EA remarkably upregulated the mRNA expression of PGC1α, PPARγ, and PRDM16, as well as the protein expression of UCP1 induced by HFD. In the HFD group, PRDM16 expression showed a strong correlation with UCP1 expression, and ILCs were strongly correlated with PGC1α expression. However, these strong correlations did not occur in HFD+EA group. Following EA treatment, the expression of PPARγ displayed a strong correlation with ILC2s, indicating that the altered ILC2 proportion was closely related to browning in WAT. ConclusionEA treatment effectively alleviated obesity by restoring ILC2 function and promoting browning of white adipocytes. ILC2s may play an important role in the effects of EA treatment, but this requires further study.

Cytokine profiles in dengue fever and dengue hemorrhagic fever: A study from Indonesia.

Recent studies have demonstrated that cytokine dysregulation has a critical role in the pathogenesis of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The aim of this study was to investigate the association between tumor necrosis factor (TNF- α), interleukin 6 (IL-6), interleukin 10 (IL-10), and interleukin 17 (IL-17) with infection status, and severity of dengue. A prospective cross-sectional study was conducted at three hospitals in Gianyar regency and Denpasar municipality, Bali, Indonesia, from June to December 2022. Sixty-four dengue infected patients were involved. Patients' serum was tested for dengue infection using NS1 antigen rapid test, dengue virus immunoglobulin M (IgM) and immunoglobulin G (IgG) test, and reverse transcription polymerase chain reaction (RT-PCR). Cytokine levels (TNF-α, IL-6, IL-10, and IL-17) were measured using enzyme-linked immunosorbent assay (ELISA). Infection status was determined by combining serological and RT-PCR results, categorizing patients into primary and secondary infections. The present study found that DF patients had lower TNF-α, IL-6, and IL-17 but higher IL-10 levels compared to DHF patients (p<0.001). Elevated TNF-α, IL-6, and IL-17 levels were higher in secondary infection, while IL-10 level was higher in primary infection (p<0.001). In conclusion, cytokines play a crucial role in the interplay between cytokine dysregulation and dengue infection dynamics.

Estimating infection prevalence using the positive predictive value of self-administered rapid antigen diagnostic tests: An exploration of SARS-CoV-2 surveillance data in the Netherlands from May 2021 to April 2022.

Measuring the severity of the disease of SARS-CoV-2 is complicated by the lack of valid estimations for the prevalence of infection. Self-administered rapid antigen diagnostic tests (Ag-RDTs) were available in the Netherlands since March 2021, requiring confirmation by reverse-transcription polymerase chain reaction (RT-PCR) for positive results. We explored the possibility of utilizing the positive predictive value (PPV) of Ag-RDTs to estimate SARS-CoV-2 prevalence. We used data from all Public Health service testing facilities between 3 May 2021 and 10 April 2022. The PPV was calculated by dividing the number of positive RT-PCR results by the total number of confirmation tests performed, and used to estimate the prevalence and compared with the number of COVID-19 hospital admissions. In total 3,599,894 cases were included. The overall PPV was 91.8% and 88.8% were symptomatic. During our study period, the estimated prevalence ranged between 2-22% in symptomatic individuals and 2-14% in asymptomatic individuals, with a correlation between the estimated prevalence and hospital admissions two weeks later (r = 0.68 (p<0.01) and r = 0.60 (p<0.01) for symptomatic/asymptomatic individuals). The PPV of Ag-RDTs can help estimate changes in SARS-CoV-2 prevalence, especially when used in conjunction with other surveillance systems. However, the used method probably overestimated the true prevalence because of unmonitored differences in test propensity between individuals.

Abnormal S100A8 expression associates with postoperative recurrence in chronic rhinosinusitis with nasal polyps

ObjectiveTo investigate the role of S100A8 in chronic rhinosinusitis with nasal polyps (CRSwNP) and assess its value in predicting disease recurrence after surgery. MethodsThirty healthy controls (HC), 30 patients with chronic rhinosinusitis without nasal polyp (CRSsNP), and 60 patients with CRSwNP were enrolled. Serum S100A8 concentration was measured by ELISA. Immunohistochemistry (IHC), western blotting (WB), and reverse transcription-polymerase chain reaction (RT-PCR)were performed to examine tissue expression levels of S100A8. The potential values of S100A8 in predicting postoperative recurrence of CRSwNP were assessed by the receiver operating characteristic (ROC)curve. ResultsSerum S100A8 concentrations in the CRSwNP group were higher than the HC group and the CRSsNP group， especially in the recurrent CRSwNP group (P < 0.05). Serum S100A8 levels were positively correlated with peripheral blood eosinophil numbers (r = 0.263, P = 0.043) and percentages (r = 0.336, P = 0.009), tissue eosinophil percentages (r = 0.273, P = 0.035), VAS score (r = 0.385, P = 0.002) and Lund-Kennedy score (r = 0.283, P = 0.029). IHC, WB, and RT-PCR results showed tissue S100A8 expression was significantly enhanced in the CRSwNP group, especially in the recurrence group (P < 0.05). Binary regression analysis showed that serum S100A8 concentration and tissue eosinophil percentage were correlated with postoperative recurrence of CRSwNP. ROC curve analysis showed that compared with tissue eosinophil percentage, the S100A8 level had a higher value for postoperative recurrence of CRSwNP. ConclusionSerum and tissue S100A8 levels were elevated in patients with CRSwNP， especially in the recurrent CRSwNP patients, and were correlated with the degree of peripheral blood and tissue eosinophilic inflammation. S100A8 seemed to be a potential objective biomarker to predict the postoperative recurrence of CRSwNP.

Effect of a 630 nm light on vasculogenic mimicry in A549 lung adenocarcinoma cells in vitro

ObjectiveThe objective of this study was to investigate the effect of photodynamic therapy (PDT) on the formation of vasculogenic mimicry (VM) in the human lung adenocarcinoma A549 cell line in vitro. MethodsThe participants were divided into a blank control group, a photosensitizer group, a light group, and a PDT group. Cells from each group were cultured in three dimensions using Matrigel, and vasculogenic mimicry generation was observed microscopically. Periodic Acid-Schiff (PAS) staining was used to verify the vasculogenic mimicry structure. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was used to detect the expression levels of cellular osteopontin (OPN) and vascular endothelial growth factor (VEGF) mRNA. Western blotting was used to detect the expression levels of cellular OPN and VEGF protein. ResultsA549 cells cultured on Matrigel for about six hours revealed VM on PAS staining, and the number of formations was significantly reduced in the PDT group compared with other groups (P < 0.05). The RT-PCR results showed that the PDT group downregulated OPN and VEGF mRNA expression compared with each control group (P < 0.05). Western blot results showed that OPN and VEGF protein expression was downregulated in the PDT group compared with each control group (P < 0.05). The results of RT-PCR showed that the expression of OPN and VEGF mRNA was downregulated in the PDT group compared with each control group (P < 0.05). The results of Western blotting showed that the expression of OPN and VEGF was downregulated in the protein PDT group compared with each control group (P < 0.001). ConclusionPhotodynamic therapy significantly inhibited the formation of vasculogenic mimicry in human lung adenocarcinoma A549 cells in vitro and downregulated the expression of OPN, VEGF mRNA, and protein levels.

Diagnostic performance of rapid antigen tests for SARS-CoV-2 transmission risk based on cycle threshold values in the emergency department

BackgroundThis study aimed to investigate the diagnostic performance of the rapid antigen test (RAT) for screening patients with cycle threshold (Ct) values of SARS-CoV-2 reverse transcription-polymerase chain reaction (RT-PCR) in the emergency department. Previous studies have shown that Ct values could be used as indicators of infectiousness. Therefore, we considered the Ct value an indicator of potential infectiousness. MethodsThis single-center retrospective observational study was conducted between January 1, 2020, and March 31, 2022. Patients who underwent both RT-PCR and RAT for the diagnosis of COVID-19 were included. Patients with negative RT-PCR results were excluded. Patients with Ct values lower than 26 and 30 were considered potentially infectious for COVID-19. ResultA total of 386 patients were analyzed. At Ct value cutoffs of 26 and 30, the result of the RAT showed a sensitivity of 82% and 74%, specificity of 84% and 89%, and area under the curve (AUC) of 0.829 and 0.813, respectively, in the receiver operating characteristic curve. However, the NPV was relatively low at 55% and 25%. ConclusionThe RAT might be a rapid screening tool for detecting patients with the infectiousness of SARS-CoV-2. However, considering the low NPV, it is challenging to depend only on a negative test result from an antigen test to terminate quarantine. Clinicians should consider additional factors, such as the duration of symptoms and the immunocompromised state, for SARS-CoV-2 transmission.

Imipenem resistance associated with amino acid alterations of the OprD porin in Pseudomonas aeruginosa clinical isolates.

Globally, the spread of carbapenem-resistant strains has limited treatment options for multidrug-resistant (MDR) Pseudomonas aeruginosa infections. This study aimed to determine the role of point mutations as well as the expression level of the oprD gene in the emergence of imipenem-resistant P.aeruginosa strains isolated from patients referred to Ardabil hospitals. A total of 48 imipenem-resistant clinical isolates of P. aeruginosa collected between June 2019 and January 2022 were used in this study. Detection of the oprD gene and its amino acid alterations was performed using the polymerase chain reaction (PCR) and DNA sequencing techniques. The expression level of the oprD gene in imipenem-resistant strains was determined using the real-time quantitative reverse transcription PCR (RT-PCR) method. All imipenem-resistant P. aeruginosa strains were positive for the oprD gene based on the PCR results, and also five selected isolates indicated one or more amino acid alterations. Detected amino acid alterations in the OprD porin were Ala210Ile, Gln202Glu, Ala189Val, Ala186Pro, Leu170Phe, Leu127Val, Thr115Lys, and Ser103Thr. Based on the RT-PCR results, the oprD gene was downregulated in 79.1% of imipenem-resistant P. aeruginosa strains. However, 20.9% of strains showed overexpression of the oprD gene. Probably, resistance to imipenem in these strains is associated with the presence of carbapenemases, AmpC cephalosporinase, or efflux pumps. Owing to the high prevalence of imipenem-resistant P. aeruginosa strains due to various resistance mechanisms in Ardabil hospitals, the implementation of surveillance programs to reduce the spread of these resistant microorganisms along with rational selection and prescription of antibiotics is recommended.

Pyrolae herba alleviates cognitive impairment via hippocampal TREM2 signaling modulating neuroinflammation and neurogenesis in lipopolysaccharide-treated mice

Ethnopharmacological relevancPyrolae herba (PH), a kind of Chinese herb, has been identified to have an anti-inflammatory effect, while the potential for treating cognitive impairment (CI), as well as the underlying mechanisms, is unclear. Currently, the interaction between neuroinflammation and neural function play a critical role in pathophysiology of CI. Aim of the studyTo elucidate therapeutic effect of PH for CI as well as its underlying mechanisms with LPS-treated mice model. Methods and materialsIn this study, male C57BL6/J mice received lipopolysaccharide (LPS) injection for 10 days to establish CI model and were administrated with PH for 14 days. We used piracetam as a positive control. Memory and spatial function was tested by Morris water maze (MWM). The level of inflammation-related cytokines (TNF-α, IL-1β, IL-10, IL-6) were determined by enzyme-linked immunosorbent assay (ELISA) in serum and western blot in hippocampus. Immunofluorescence (IF) was used to measure the levels of ionized calcium binding linker molecule 1 (IBA-1), glial fibrillary acidic protein (GFAP), BrdU, Ki67 and doublecortin (DCX) in hippocampus. The mRNA sequencing was used to screen the potential target of PH with therapeutic CI. Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the gene alteration of triggering receptor expressed on myeloid cells 2 (TREM2) in hippocampus. We used western blot to determine protein expressions of TREM2 and its related signaling, as well as synaptic proteins in hippocampus. ResultsThe results revealed that LPS contributed to CI, and PH or piracetam treatment significantly ameliorated CI in MWM test. LPS contributed to increasing expressions of TNF-α and IL-1β in serum and hippocampus, which both reversed by PH or piracetam. PH or piracetam could inhibit the activation of glial cells including microglia and astrocyte in the hippocampus in LPS-induced CI model. The mRNA sequencing and RT-PCR results showed that LPS significantly increased the gene expression of TREM2, which was reversed by PH. The alteration of TREM2 expression was the most significant among the 10 genes (TREM2, Slc24a2, Ptch2, Gck, Il1rapl1, Cadps2, Btbd11, Secisbp2l, Tenm3 and Prepl) in hippocampus. Protein results showed that LPS upregulated the expressions of TREM2 and its related proteins including DAP12, spleen tyrosine kinase (SYK) phosphorylation and ADAM 10, which were all reversed by PH or piracetam in hippocampus. Furthermore, LPS was capable of reducing the expression of BrdU and DCX co-labeled positive cells in hippocampal dentate gyrus (DG), which was reversed only by PH. Moreover, PH or piracetam treatment significantly increased the expression of Ki67 and DCX co-labeled positive cells in hippocampal DG. The expression of synapsin1 was obviously decreased by LPS and was significantly reversed by PH or piracetam. ConclusionsPH could alleviate CI by suppressing the secretion of pro-inflammatory cytokines and mitigating astrocyte activity by restraining microglia's activation in hippocampus, further facilitating neurogenesis and proliferation, thereby enhancing pre-synaptic protein. This study highlighted on the clinical application of PH, which might promote the use of phytomedicine in CI patients.

First Report of Passiflora latent virus in Passion fruit (Passiflora edulis) in China.

Passion fruit (Passilora edulis, family Passifloraceae.) is an economically important fruit crop in many tropical and subtropical regions worldwide. It is widely planting in southern China, and in greenhouses throughout the country. In Mar 2022, symptoms of a viral-like infection were observed on the leaves of passion fruit plants in a 3-hectare greenhouse complex in Hohhot, China. Chlorotic lesions were observed on leaves of two vines of passion fruit and symptomatic leaves developed chlorotic spots, followed by systemic leaf chlorosis and necrosis. Dark ringed spots emerged on the surface of matured fruits (Figure 1). To confirm infectivity, mechanical transmission of the virus was performed by grinding leaves from two symptomatic passion fruit vines in 0.1M phosphate buffer pH 7, and the resulting two samples were each used to rub-inoculate carborundum-dusted leaves of three healthy passion fruit seedlings. Newly emerging leaves of inoculated plants developed mild mosaic symptoms 30-days after inoculation. Three samples from each of the two original symptomatic plants and two samples from each inoculated seedling tested positive using a Passiflora latent virus (PLV) ELISA Kit (Creative Diagnostics, USA). To further confirm the virus identity, total RNA from leaf samples from one of the original greenhouse symptomatic plants and one of the inoculated seedlings were extracted using an TaKaRa MiniBEST Viral RNA Extraction Kit (Takara, Japan). The two RNA samples were subjected to reverse transcription-polymerase chain reaction (RT-PCR) tests with virus specific primers PLV-F (5'-ACACAAAACTGCGTGTTGGA-3') and PLV-R (5'-CAAGACCCACCTACCTCAGTGTG-3') (Cho et al., 2020). RT-PCR products of the expected 571 bp were obtained from both the original greenhouse sample and inoculated seedling. Amplicons were cloned into the pGEM-T Easy Vector, and two clones per sample were Sanger sequenced bidirectionally (Sangon Biotech, China), and the sequence of one clone from one of the original symptomatic sample was uploaded to NCBI (GenBank OP320922.1). This accession had 98% nucleotide sequence identity with a PLV isolate from Korea (GenBank: LC556232.1). RNA extracts from two asymptomatic samples tested negative for PLV with both ELISA and RT-PCR tests. We also tested the original symptomatic sample for common occurring passion fruit viruses, including passion fruit woodiness virus (PWV), cucumber mosaic virus (CMV), east asian passiflora virus (EAPV), telosma mosaic virus (TeMV), papaya leaf curl Guangdong virus (PaLCuGdV), and the RT-PCR results were negative for those viruses. However, based on the systemic leaf chlorosis and necrosis symptoms, we cannot preclude a mixed infestation of other viruses. PLV affects fruit quality and has high potential to reduce market value. To our knowledge, this is the first report of PLV in China, which could provide a reference basis to PLV identification, prevention and control. Acknowledgments This research was carried out with the support of Inner Mongolia Normal University High-level Talents Scientific Research Startup Project (Grant no. 2020YJRC010). Supplementary material Figure 1. Mottle, leaf distortion, puckering symptoms on old leaf (A), mild puckering symptom on young leaf (B), and ring-striped spots symptoms on fruit (C) of the PLV infected passion fruit plant in China.

Diagnostic accuracy of Panbio™ rapid antigen test for SARS-CoV-2 in paediatric population

BackgroundRapid antigen-detection tests (Ag-RDTs) are used to diagnose SARS-CoV-2 infection. Real-world studies of Ag-RDTs are necessary to evaluate their diagnostic yield in paediatric patients. Our aim was to evaluate the accuracy of the Panbio™ Rapid Antigen Test for SARS-CoV-2 in the setting of a primary health care centre (PHC), with use of the Reverse Transcription-Polymerase Chain Reaction (RT-PCR) as gold standard.MethodsThis prospective diagnostic study was conducted at PHCs in Mallorca, Spain. Patients were ≤ 18 years-old that attended sites for RT-PCR testing due to symptoms suggestive of infection (fever, headache, nasal congestion and dry cough, among others) or epidemiological exposure (close contacts). Two samples were collected: a nasal mid-turbinate sample for Ag-RDTs and a nasopharyngeal swab for RT-PCR testing. The sensitivity, specificity, and predictive values of the AgRDT were calculated using the RT-PCR results as the reference.ResultsWe examined 1142 participants from 0 to 18 years (47.5% female, mean age 8.9 ± 4.8 years, median 9.0 [5.0–13.0]). There were 84 positive RT-PCR results (pre-test probability of 7.3%) and 52 positive Ag-RDT results. The sensitivity of the Ag-RDT was 59.5% (95% Confidence Interval (CI): 48.2–69.9%), the specificity was 99.8% (95%CI: 99.2–99.9%), the positive predictive value was 96.1% (95%CI: 85.6–99.4%), and the negative predictive value was 96.8% (95%CI: 95.6–97.7%). The sensitivity for individuals referred by a general practitioner (GP) or paediatrician due to symptoms was 71.4% (95%CI: 51.5–86.0%) and for asymptomatic individuals was 50.0% (95%CI: 9.1–90.8%). The specificity was greater than 98.9% overall and in all subgroups. The sensitivity was 73.0% (95%CI: 52.0–87.5%) for referred patients due to symptoms and who were tested within 5 days since symptom onset. No significant statistical differences between any groups were found. There were 34 false-negative Ag-RDT results (40.5%) and 2 false-positive Ag-RDT results (0.2%).ConclusionThe sensitivity of the Panbio™ Test in paediatric individuals is below the minimum of 80% recommended by the World Health Organization for Ag-RDTs. This test had better accuracy in individuals referred by a GP or paediatrician due to symptoms, rather than those who were asymptomatic or referred due to epidemiological exposure. The RT-PCR test using a nasopharyngeal swab is accurate, but a less invasive alternative that has better sensitivity than the Panbio™ Test is needed for paediatric populations.

Diagnostic Performance of the Radiological Society of North America Consensus Statement for Reporting COVID-19 Chest CT Findings: A Revisit.

Coronavirus disease 2019 (COVID-19) is a highly contagious respiratory disease that leads to variable degrees of illness, and which may be fatal. We evaluated the diagnostic performance of each chest computed tomography (CT) reporting category recommended by the Expert Consensus of the Radiological Society of North America (RSNA) in comparison with that of reverse transcription polymerase chain reaction (RT-PCR). We aimed to add an analysis of this form of reporting in the Middle East, as few studies have been performed there. Between July 2021 and February 2022, 184 patients with a mean age of 55.56 ± 16.71 years and probable COVID-19 infections were included in this retrospective study. Approximately 64.67% (119 patients) were male, while 35.33% (65 patients) were female. Within 7 days, all patients underwent CT and RT-PCR examinations. According to a statement by the RSNA, the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of each CT reporting category were calculated, and the RT-PCR results were used as a standard reference. The RT-PCR results confirmed a final diagnosis of COVID-19 infection in 60.33% of the patients. For COVID-19 diagnoses, the typical category (n = 88) had a sensitivity, specificity, PPV, and accuracy of 74.8%, 93.2%, 94.3%, and 92.5%, respectively. For non-COVID-19 diagnoses, the PPVs for the atypical (n = 22) and negative (n = 46) categories were 81.8% and 89.1%, respectively. The PPV for the indeterminate (n = 28) category was 67.9%, with a low sensitivity of 17.1%. However, the RSNA's four chest CT reporting categories provide a strong diagnostic foundation and are highly correlated with the RT-PCR results for the typical, atypical, and negative categories, but they are weaker for the indeterminate category.

Rapid screening of SARS-CoV-2 variants, a key tool for pandemic surveillance

The utility of reverse transcription-polymerase chain reaction (RT-PCR) in analysis SARS-COV-2 variants was evaluated. RT-PCR tests were used to analyse the majority of new SARS-CoV-2 cases (n = 9315) in a tertiary hospital (Madrid, Spain) throughout 2021. Subsequently, whole genome sequencing (WGS) was conducted on 10.8% of these samples (n = 1002). Notably, the Delta and Omicron variants emerged rapidly. There were no discrepancies between RT-PCR and WGS results. Continuous surveillance of SARS-CoV-2 variants is essential, and RT-PCR is a highly useful method, specially during periods of high COVID-19 incidence. This feasible technique can be implemented in all SARS-CoV-2 laboratories. However, WGS remains the gold standard method for comprehensive detection of all existing SARS-CoV-2 variants.

SARS-CoV-2 Genome Structure, Pathogenesis, Issues, and Challenges in Laboratory Diagnosis

AbstractSevere acute respiratory syndrome coronavirus 2 causing coronavirus disease 2019 pandemic disease is an enveloped virus, showing genome similarity with bat coronavirus. This virus initially infects the upper respiratory tract, with subsequent spread to the lower respiratory tract. Despite the availability of antigen and antibody detection methods, reverse transcription-polymerase chain reaction (RT-PCR) is the diagnostic test of choice for this novel coronaviral infection. Care must be taken while interpreting the RT-PCR results, as single RT-PCR, especially in early days of infection, maybe false negative. The availability of cartridge-based nucleic acid amplification test has improved the diagnostic facilities in a peripheral setting of developing countries.

A Review of Crucial Radiological Investigations in the Management of COVID-19 Cases.

Chest X-ray, chest CT, and lung ultrasound are the most common radiological interventions used in the diagnosis and management of coronavirus disease 2019 (COVID-19) patients. The purpose of this literature review, which was performed according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, is to determine which radiological investigation is crucial for that purpose.PubMed, Medline, American Journal of Radiology (AJR), Public Library of Science (PLOS), Elsevier, National Center for Biotechnology Information (NCBI), and ScienceDirect were explored. Seventy-two articles were reviewed for potential inclusion, including 50 discussing chest CT, 15 discussing chest X-ray, five discussing lung ultrasound, and two discussing COVID-19 epidemiology. The reported sensitivities and specificities for chest CT ranged from 64 to 98% and 25 to 88%, respectively. The reported sensitivities and specificities for chest X-rays ranged from 33 to 89% and 11.1 to 88.9%, respectively. The reported sensitivities and specificities for lung ultrasound ranged from 93 to 96.8% and 21.3 to 95%, respectively. The most common findings on chest CT include ground glass opacities and consolidation. The most common findings on chest X-rays include opacities, consolidation, and pleural effusion. The data indicate that chest CT is the most effective radiological tool for the diagnosis and management of COVID-19 patients. The authors support the continued use of reverse transcription polymerase chain reaction (RT-PCR), along with physical examination and contact history, for such diagnosis. Chest CT could be more appropriate in emergency situations when quick triage of patients is necessary before RT-PCR results are available. CT can also be used to visualize the progression of COVID-19 pneumonia and to identify potential false positive RT-PCR results. Chest X-ray and lung ultrasound are acceptable in situations where chest CT is unavailable or contraindicated.