Toscana virus (TOSV), a sandfly-borne virus, is an important etiological agent in human acute meningitis and meningoencephalitis in the Mediterranean area during the summer. However, the actual number of TOSV infections is underestimated. Laboratory confirmation is necessary because TOSV infection has overlapping clinical features with other neuro-invasive viral infections. Nowadays, the reference test for direct diagnosis in the acute phase of TOSV infection is the PCR based method for detecting TOSV in cerebrospinal fluid and/or plasma, serum, or blood. Although poorly employed, urine is another helpful biological matrix for TOSV detection. Urine is a matrix rich in PCR inhibitors that affect PCR efficiency; consequently, false negatives could be generated. To investigate the potential effect of urine PCR inhibitors on TOSV detection, we compared undiluted and diluted urine using 10-fold series of spiked TOSV. The results showed a significant improvement in TOSV detection performance in diluted urine (1 TCID50 vs. 1 × 104 TCID50 limit of detection and 101.35% vs. 129.62% efficiency, respectively, in diluted and undiluted urine). In conclusion, our data provide preliminary important insights into the use of diluted urine to limit the impact of the inhibitory effects of urine on the detection of TOSV in RT-PCR-based approaches.

The coronavirus infectious disease (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses. The pandemic has emerged as a global public health crisis, and the threat of fast-spreading of the latest variants of the coronavirus (such as omicron, delta) is rampant. Therefore, a fast and reliable diagnostic assay is needed to make the clinical decision for further treatment. The study aims to develop a Centers for Disease and Prevention (CDC)-modified qualitative real-time reverse transcriptase PCR (RT-qPCR) assay and parallel assessment of commercially available RT-qPCR assay (Altona, Seegene, BD, and GBC) to detect SARS-CoV-2. Two hundred nine samples were chosen randomly out of around two hundred thousand samples. The panel consisted of SARS-CoV-2-positive (n = 156) and SARS-CoV-2-negative (n = 52) nasopharyngeal swab specimens for a primary clinical evaluation. Furthermore, 29 positive samples were sequenced using Oxford Nanopore Minion technology. Two hundred nine patient sample data of the cycle threshold (Ct) readings for target genes of five assays are 100% sensitive for Ct values. Mean Ct values for N1, N2, RdRp, S, and E of the positive controls in CDC assay, RealStar®, Allplex, GBC, and SD Biosensor were 17.5 ± 0.49, 16.9 ± 0.51, 20 ± 0.49, 21.7 ± 0.38, and 23.1 ± 0.43, respectively. F test value shows ≥ 1, which was statistically significant. All assays showed an efficiency of < 120% and R squares were < 0.99, which is well above the required threshold value. Thus, when taking the CDC-modified assay as a gold standard, the other four assays demonstrated a p value of 0.0000, concordance at 100%, and a Kappa at 1.000. A maximum-likelihood (ML) tree was constructed and compared based on full-length SARS-CoV-2 with Wuhan isolate. These isolates are closely related to the B.1.617 lineage and reference sequences. Therefore, we conclude that all RT-PCR kits assessed in this study shall be used for routine diagnostics of COVID-19 in patients.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s00580-022-03356-y.

Accurate diagnosis to limit the spread of SARS-CoV-2 is crucial for the clinical management of this lethal infection. Recently, many low-cost and easy-to-use rapid test kits (RTK) have been developed in many countries for the massive screening of SARS-CoV-2. Thus, evaluating the accuracy and reliability of an RTK is critical. The current study was conducted on 157 individuals to evaluate the performance accuracy of rapid SARS-CoV-2 antigen detection kits using different clinical samples compared with qRT-PCR results. Nasopharyngeal swabs were collected from patients for qRT-PCR and RTK tests, and then buccal and nasal, and nasal swabs were collected for RTK tests separately. The nasal and buccal swabs showed high sensitivity (98%) and specificity (100%) compared with the qRT-PCR results. Meanwhile, for nasal, the sensitivity was 96% with 98% specificity, and nasopharyngeal swabs showed 98% sensitivity and 94% specificity. Fisher’s exact test revealed statistical significance (p < 0.05) between nasopharyngeal, nasal and buccal, and nasal swabs compared with qRT-PCR results. The study concludes that different clinical samples used for the rapid diagnosis of SARS-CoV-2 showed high sensitivities and specificities compared with qRT-PCR. The RTKs using nasal and buccal, nasopharyngeal, and nasal swabs are valuable tools for the early detection of SARS-CoV-2, especially when molecular detections are available with limited access and a high infectivity rate, when the timely detection of virus cases is urgently needed. These types of clinical samples are effective to be used by RTKs for surveillance among community and healthcare workers.

BACKGROUND: This study aimed to evaluate rapid antigen detection (RAD) and rapid nucleic acid amplification tests (NAATs) to detect influenza virus (IV).
METHODS: The conventional RAD test (Quick Chaser Flu A, B: QC), using silver amplified immunochromatography (Quick Chaser Auto Flu A, B: QCA), as well as two NAATs (Xpert Xpress Flu/RSV: Xpert, cobas Influenza A/B & RSV: cobas) were evaluated using nasopharyngeal swabs from suspected cases of influenza. A reference method was performed using real-time reverse transcription polymerase chain reaction according to the manual of the Japanese National Institute of Infectious Disease (NIID).
RESULTS: From a total of 177 samples, 51 were positive according to the NIID assay. The kappa (κ) coefficient in Xpert and cobas for influenza A virus (IAV)/influenza B virus (IBV) was 1.00, which was the highest among the four detection assays. However, the κ coefficients in QC and QCA for IAV/IBV were 0.71-0.77 and 0.87-0.89, respectively. The sensitivities of the RAD tests were 41.7% in QC and 50.0% in QCA at < 6 h after onset, and 100.0% in both QC and QCA at 24-48 h after onset. The cycle threshold (Ct) values were significantly lower in the group in which all detection assays were positive for IAV.
CONCLUSIONS: Xpert and cobas have comparable analytical performances and are highly useful as influenza virus detection assays. QC and QCA could show false negatives frequently in the early stage of infection and when viral load is low.

UNASSIGNED: Repurposed povidone iodine (PVP-I) has been suggested as an effective adjuvant against coronavirus disease-2019 (COVID-19).
UNASSIGNED: The aim of this study was to assess the changes in RT-PCR cycle threshold (Ct) values of severe acute respiratory Syndrome Coronavirus-2 (SARS-CoV-2) genes with PVP-I intranasal and oral application.
UNASSIGNED: A longitudinal (repeated measures) single-arm open-label interventional study was conducted for 200 samples of ten COVID-19 patients in South India.
UNASSIGNED: Demographic and clinical information were collected. Intranasal application and oral gargle with 1% PVP-I solution was done four times a day for seven days. Nasopharyngeal and oropharyngeal samples were taken for RT-PCR test at hour-0, hour-2, hour-4 on Day-0, Day-3, Day-6, and hour-0 on Day-9.
UNASSIGNED: STATA analysis software version 14.2 was used. McNemar Test was applied for paired samples. Skilling Mack Test was used to assess the association between PVP-I use (intra-day and inter-day) and E gene/N gene Ct values. Pearson correlation coefficients and Bland-Altman plots were used for further analyses.
UNASSIGNED: Mean (SD) age of the patients was 41.5 (±8.82) years. A total of 100 pairs of nasopharyngeal and oropharyngeal samples were analysed. No significant difference was observed in the Ct values of asymptomatic and symptomatic patients. E gene Ct values (nasopharyngeal) at Hour-0 increased from Day-0 to Day-9 (P = 0.005). Ct value was higher at Hour-2 for most of the samples.
UNASSIGNED: RT-PCR results (qualitative) differed at various testing points in the same patients. Lower Ct values were found in the nasopharyngeal samples. Successive increase in E gene Ct values indicates reduced viral load with natural course of COVID-19. PVP-I may have an optimal impact within 2 h of usage.
UNASSIGNED: CTRI/2020/05/024962.

The aim of the present study was to investigate the pharmacological mechanism of matrine in treatment of COVID-19 combined with liver injury. Potential targets related to matrine, COVID-19 and liver injury were identified from several databases. We constructed PPI network and screened the core targets according to the degree value. Then, GO and KEGG enrichment were carried out. Molecular docking technology was used to verify the affinity between matrine and the crystal structure of core target protein. Finally, real-time RT-PCR was used to detect the effects of matrine on hub gene expression in liver tissue of liver injury mice and lung tissue of lung injury mice to further confirm the results of network pharmacological analysis. The results show that six core targets including AKT1, TP53, TNF, IL6, BCL2L1 and ATM were identified. The potential therapeutic mechanism of matrine on COVID-19 combined with liver injury is closely related to regulate antiviral process, improve immune system and regulate the level of inflammatory factors. Molecular docking showed that matrine could spontaneously bind to the receptor protein and had strong binding force. Real-time RT-PCR demonstrated that matrine could significantly reduce the expression of AKT1, TP53, TNF, IL6 and ATM in mice with liver injury or lung injury (P < 0.05), and increase the expression of BCL2L1 to a certain extent (P > 0.05). Our results indicate that matrine can achieve simultaneous intervention of COVID-19 combined with liver injury by multi-dimensional pharmacological mechanism.

BACKGROUND: The release of miRNAs in tissue fluids significantly recommends its use as non-invasive diagnostic biomarkers for the progression and pathogenesis of mild cognitive impairment (MCI) in aged patients.
OBJECTIVE: The potential role of circulated miRNAs in the pathogenesis of MCI and its association with cellular oxidative stress, apoptosis, and circulated BDNF, Sirtuin 1 (SIRT1), and dipeptidyl peptidase-4 (DPP4) were evaluated in older adults with MCI.
METHODS: A total of 150 subjects aged 65.4±3.7 years were recruited in this study. The participants were classified into two groups: healthy normal (n=80) and MCI (n=70). Real-time PCR analysis was performed to estimate the relative expression of miRNAs; miR-124a, miR-483-5p, miR-142-3p, and miR-125b, and apoptotic-related genes Bax, Bcl-2, and caspase-3 in the sera of MCI and control subjects. In addition, oxidative stress parameters; MDA, NO, SOD, and CAT; as well as plasma DPP4 activity, BDNF, SIRT1 levels were colorimetrically estimated.
RESULTS: The levels of miR-124a and miR-483-5p significantly increased and miR-142-3p and miR-125b significantly reduced in the serum of MCI patients compared to controls. The expressed miRNAs significantly correlated with severe cognitive decline, measured by MMSE, MoCA, ADL, and memory scores. The expression of Bax, and caspase-3 apoptotic inducing genes significantly increased and Bcl-2 antiapoptotic gene significantly reduced in MCI subjects compared to controls. In addition, the plasma levels of MDA, NO, and DPP4 activity significantly increased, and the levels of SOD, CAT, BDNF, and SIRT1 significantly reduced in MCI subjects compared to controls. The expressed miRNAs correlated positively with NO, MDA, DPP4 activity, BDNF, and SIRT-1, and negatively with the levels of CAT, SOD, Bcl-2, Bax, and caspase-3 genes.
CONCLUSIONS: Circulating miR-124a, miR-483-5p, miR-142-3p, and miR-125b significantly associated with severe cognitive decline, cellular oxidative stress, and apoptosis in patients with MCI. Thus, it could be potential non-invasive biomarkers for the diagnosis of MCI with high diagnostic performance.

Infectious bronchitis is one of the most common diseases in the poultry industry in many countries, especially in the regions with a dense poultry farming industry. Gammacoronavirus is the etiologic agent of this disease, with the chickens and poultries as the natural reservoirs of the virus. Various strains of infectious bronchitis virus have been reported in poultry around the world. In terms of pathogenicity, this virus can induce a spectrum of diseases ranging from the moderate respiratory tract to kidney and reproductive diseases. The serotypes of this virus do not cause cross-immunity against each other. This issue makes it difficult to control the disease. Based on the analysis of the highly variable region of the glycoprotein S1 gene, the isolated strains in Iran were classified into seven different phylogenetic groups, including Massachusetts, QX, IS-720, IS-1494, 793/B, IR-1, and IR-2. The D1466 genotype has not been reported in the country; however, the killed vaccine is used in broiler breeder farms. In this study, tissue specimens were collected from 700 farms (i.e., broiler, egg-laying, and broiler breeder farms) suspected of infectious bronchitis within 2013-2017. The samples were examined using real-time reverse transcription-polymerase chain reaction. The D1466 genotype was not detected in any of the studied specimens. Due to the lack of immunity of the D1466 serotype against the dominant types in the country, one has to be careful in choosing the right vaccine. It is necessary to perform continuous monitoring of the circulation status of the various serotypes of viruses in the country to identify the dominant and possible new serotypes for the utilization of the appropriate vaccine.

Lumbosacral lipomas (LSLs), one form of closed spinal dysraphism, are congenital disorders of the terminal spinal cord (SC). Delayed neurologic deterioration often occurs in the subsequent developmental course of the patient. Identifying the cellular and molecular factors underlying the progressive damage to neural structures is a prerequisite for developing treatment strategies for LSLs.
Nine LSL specimens obtained from the SC/lipoma interface during surgical resection were examined. Normal SC tissue served as a control. Clinical characteristics were obtained, and spinal magnetic resonance imaging was re-evaluated. Cellular marker profiles were established. Immunoreactivity (IR) of hypoxia-inducible factor 1α (HIF-1α/-2α), erythropoietin (Epo)/erythropoietin receptor (EpoR), interleukin-1β (IL-1β)/IL-1R1, and tumor necrosis factor α/tumor necrosis factor receptor type 1 were analyzed qualitatively and semiquantitatively by densitometry. Colabeling with cellular markers was determined by multifluorescence labeling. Cytokines were further analyzed by real-time reverse transcription polymerase chain reaction.
LSL specimens showed significant gliosis. HIF-1α/HIF-2α-IR and Epo/Epo-IR were found at significantly higher levels in the LSL specimens, as were IL-1β-/IL-1β receptor type 1 (IL1-R1) and tumor necrosis factor α/tumor necrosis factor receptor type 1 (P < 0.001), than were the controls. At the messenger RNA level, cytokines appeared partially induced. Double immunofluorescence labeling confirmed the costaining of these factors with inflammatory and glial markers.
The expression of hypoxia-related and inflammatory mediators was shown for the first time in LSL specimens. These factors might play a role in multifactorial secondary lesion cascades underlying further damage to the neural placode in closed dysraphism.

Pathologic mechanisms in cochleae immediately following the onset of noise-induced hearing loss (NIHL) remain unclear. In this study, mice were exposed to 120 dB of octave band noise for 2 h to induce NIHL. Three hours after noise exposure, expression levels of the whole mouse genome in cochleae were analyzed by RNA-seq and DNA microarray. Differentially expressed genes (DEGs) exhibiting >2-fold upregulation or downregulation in noise-exposed cochleae compared to controls without noise exposure were identified. RNA-seq and microarray analyses identified 273 DEGs regulated at 3 h post-noise (51 upregulated and 222 downregulated). Bioinformatic analysis revealed that these DEGs were associated with the functional gene pathway \"neuroactive ligand-receptor interaction\" and included 28 genes encoding receptors for neurotransmitters such as gamma-aminobutyric acid and glutamate. Other DEGs included 25 genes encoding transcription factors. Downregulation of 4 neurotransmitter receptors (Gabra3, Gabra5, Gabrb1, Grm1) and upregulations of 5 transcription factors (Atf3, Dbp, Helt, Maff, Nr1d1) were validated by RT-PCR. The differentially regulated transcription factor Atf3 immunolocalized to supporting cells and hair cells in the organ of Corti at 12-h post-noise. The present data serve as a basis for further studies aimed at developing medical treatments for acute sensorineural hearing loss.