Nucleic acid-based electrochemical biosensor for detection of influenza B by gold nanoparticles.

The influenza virus is a pervasive pathogen that exhibits increased prevalence during colder seasons, resulting in a significant annual occurrence of infections. Notably, pharmaceutical interventions effective against influenza A strains often exhibit limited efficacy against influenza B variants. Against this backdrop, the need for innovative approaches to accurately and swiftly differentiate and detect influenza B becomes evident. Biosensors play a pivotal role in this detection process, offering rapid, specific, and sensitive identification of the virus, facilitating timely intervention and containment efforts. Oligonucleotide sequences targeting the conserved B/Victoria/2/87 influenza virus NP region were designed. Nasopharyngeal swabs were collected from patients suspected of influenza virus infection, and viral RNA was extracted. RNA quality was assessed through one-step PCR. cDNA synthesis was performed using random hexamers, and real-time PCR quantified the influenza genome. Gold nanoparticles were immobilized on a surface to immobilize the specific DNA probe, and electrochemical hybridization was electrochemically followed. The biosensor exhibited high selectivity and effective distinction of complementary sequences from mismatches and influenza virus cDNA genome. The biosensor successfully detected the influenza B virus genome in real samples. Non-influenza samples yielded no significant hybridization signals. The comparison between the results obtained from the biosensor and real-time PCR revealed full agreement of these methods. The biosensor utilized electrochemical detection of hybridization and proved effective in detecting the influenza B virus genome with high specificity, sensitivity, and selectivity. Comparative analysis with real-time PCR underscored the accuracy and potential applicability of the biosensor in rapid and specific virus detection. This innovative approach holds promise for future diagnostic and epidemiological applications in detecting influenza B virus and other pathogens.

Human adenoviruses in children with gastroenteritis: a systematic review and meta-analysis.

PURPOSE: Human adenoviruses (HAdVs) have always been suggested as one of the main causes of gastroenteritis in children. However, no comprehensive report on the global epidemiology of these viruses in pediatric gastroenteritis is available. METHODS: A systematic search was conducted to obtain published papers from 2003 to 2023 in three main databases PubMed, Scopus, and Web of Science. RESULTS: The estimated global pooled prevalence of HAdV infection in children with gastroenteritis was 10% (95% CI: 9-11%), with a growing trend after 2010. The highest prevalence was observed in Africa (20%, 95% CI: 14-26%). The prevalence was higher in inpatients (11%; 95% CI: 8-13%) and patients aged 5 years old and younger (9%; 95% CI: 7-10%). However, no significant difference was observed between male and female patients (P = 0.63). The most prevalent species was found to be the species F (57%; 95% CI: 41-72%). The most common HAdVs observed in children with gastroenteritis were types 40/41, 38, and 2. Analysis of case-control studies showed an association between HAdV and gastroenteritis in children (OR: 2.28, 95% CI; 1.51-3.44). CONCLUSION: This study provided valuable insights into the importance of HAdVs in children with gastroenteritis, especially in hospitalized and younger children. The results can be used in future preventive measurements and the development of effective vaccines.

Sequence variation of the Epstein-Barr virus nuclear antigen 1 (EBNA1) gene in chronic lymphocytic leukemia and healthy volunteer subjects.

Epstein-Barr virus-related malignancies have been linked to variations in the sequences of EBV genes, notably EBNA1. Therefore, the purpose of this study was to examine the DBD/DD domain and USP7 binding domain sequences at the C-terminus of the EBNA1 gene in patients with chronic lymphocytic leukemia (CLL). This study included 40 CLL patients and 21 healthy volunteers. Using commercial kits, total DNA was extracted from buffy coat samples, and each sample was tested for the presence of the EBV genome. The C-terminus of EBNA1 was then amplified from positive samples, using nested PCR. Sanger sequencing was used to identify mutations in the PCR products, and the results were analyzed using MEGA11 software. The mean ages of CLL patients and healthy individuals were 61.07 ± 10.2 and 59.08 ± 10.3, respectively. In the EBNA-1 amplicons from CLL patients and healthy individuals, 38.5% and 16.7%, respectively, harbored mutations in the DBD/DD domain of the C-terminal region of the EBNA1 gene (P = 0.378). The mutation frequency at locus 97,320 was significantly higher in CLL patients than in healthy individuals (P = 0.039). Three EBV subtypes based on residue 487 were detected. The frequency of alanine, threonine, and valine in both groups was 88, 8, and 4 percent, respectively (P = 0.207). Moreover, all of the isolates from healthy donors had alanine at this position. The findings indicated that the presence of threonine or valine at residue 487 as well as a synonymous substitution at residue 553 in the C-terminal region of EBNA1 might be involved in the pathogenesis of EBV in CLL patients.

Investigation of genetic variation: Neuraminidase gene of influenza A virus H1N1/pdm09, Shiraz, Iran (2015-2016).

Oseltamivir and antiviral agents are frequently used for the prevention and treatment of influenza infection. However, resistance to oseltamivir has been reported globally due to a mutation in the Influenza virus neuraminidase gene. Such resistance will be detected by genotyping and phenotyping studies of viral isolates. The recent study aimed to determine the genetic mutation of neuraminidase gene in influenza A (H1N1) viruses isolated from children referred to Shiraz tertiary hospitals during 1 year (2015-2016) with influenza-like symptoms. A total of 300 patients were registered and throat samples were taken. The throat swabs were used for viral RNA extraction. Detection of influenza A (H1N1) was performed using the one-step real-time polymerase chain reaction (qRT-PCR) method. From positive isolates for H1N1, 51 random samples were evaluated for neuraminidase gene mutation with the nested PCR-sequencing method. Of 300 cases, 102 (34%) isolates were detected as influenza A (H1N1) pdm09. Based on sequencing results, 2 of the 44 sequenced isolates exhibited H275Y substitution, which presented oseltamivir resistance. In comparison with reference strain, the phylogenetic analysis of sequenced isolates was classified in genogroup 6B. While this result is the first report of emerging oseltamivir-resistant in the southwest of Iran, it is highly recommended to perform these evaluations on the different geographical regions in any prevalence area to plan treatment strategies for influenza.

The Inflammatory and Fibrotic Patterns of Hepatic Stellate Cells Following Coagulation Factors (VII or X)-Shielded Adenovirus Infection.

The role of coagulation factors on the inflammatory effect of adenovirus (Ad) is an unresolved question that was considered herein. Adenovirus-36(Ad36) and adenovector-5-GFP(Ad5-GFP) were prepared; then, they were loaded with VII or FX factors. The size/charge parameters and transduction efficiency were evaluated using fluorescent microscopy and Zetasizer, respectively. The Ad36-coagulation factor complexes were added on the stellate cells, LX-2. Thereafter, the expression levels of inflammatory and fibrotic genes including PKR, IL-1ß, TNF-α, TIMP-1, collagen, and TGF-ß were measured by qPCR and ELISA assays. The loading of FVII or FX factors not only increased the size/charge of Ad5-GFP but also enhanced the transduction rate up to 60% and 75%, respectively, compared to the controls (45%). The PKR expression analysis showed an upregulation following treatment with all Ad36 forms (P = 0.0152). The IL-1ß and TNF-α cytokines analyses demonstrated that the Ad36-FVII complex elicited the highest inflammatory response (P = 0.05). Similarly, the fibrosis-related expression analysis revealed a more inductive role of FVII when loaded on Ad36, compared to the FX factor. The findings suggested that adenovirus elicited the innate inflammatory and activation state in the hepatic stellate cell. In addition, adenovirus shielded by FVII exhibited more innate inflammation as well as activation of the stellate cells than the FX-loaded virus.

Characterization of the neuraminidase genes from human influenza A viruses circulating in Iran from 2010 to 2015.

BACKGROUND: Characterization of influenza viruses is critical for detection of new emerging variants. Herein, we analyzed the genetic diversity and drug susceptibility of the neuraminidase gene (NAs) expressed by influenza A/H1N1pdm09 and A/H3N2 viruses circulating in Iran from 2010 to 2015. METHODS: We genetically analyzed the NAs of 38 influenza A/H1N1pdm09 and 35 A/H3N2 isolates. RESULTS: The Iranian A/H1N1pdm09 viruses belonged to seven genogroups/subgenogroups, with the dominant groups being genogroups 6B and 6C. The A/H3N2 isolates fell into six gneogroups/subgenogroups, with the dominant genogroups being 3C and 3C.2a. The most common mutations detected among the A/H1N1pdm09 viruses included N44S, V106I, N200S, and N248D. All H1N1pdm09 viruses were genetically susceptible to the NAIs. However, one A/H1N1pdm09 virus from the 2013-2014 season possessed an NA-S247N mutation, which reduces the susceptibility to oseltamivir. In case of H3N2, none of the analyzed Iranian strains carried a substitution that might affect its susceptibility to NAIs. CONCLUSION: The ongoing evolution of influenza viruses and the detect of influenza viruses with reduced susceptibility to NAIs warrants continuous monitoring of the circulating strains.

Molecular characterization and phylogenetic analysis of human influenza A viruses isolated in Iran during the 2014-2015 season.

Influenza A viruses are an important cause of severe infectious diseases in humans and are characterized by their fast evolution rate. Global monitoring of these viruses is critical to detect newly emerging variants during annual epidemics. Here, we sought to genetically characterize influenza A/H1N1pdm09 and A/H3N2 viruses collected in Iran during the 2014-2015 influenza season. A total of 200 nasopharyngeal swabs were collected from patients with influenza-like illnesses. Swabs were screened for influenza A and B using real-time PCR. Furthermore, positive specimens with high virus load underwent virus isolation and genetic characterization of their hemagglutinin (HA) and M genes. Of the 200 specimens, 80 were influenza A-positive, including 44 A/H1N1pdm09 and 36 A/H3N2, while 18 were influenza B-positive. Phylogenetic analysis of the HA genes of the A/H1N1pdm09 viruses revealed the circulation of clade 6C, characterized by amino acid substitutions D97N, V234I and K283E. Analysis of the A/H3N2 viruses showed a genetic drift from the vaccine strain A/Texas/50/2012 with 5 mutations (T128A, R142G, N145S, P198S and S219F) belonging to the antigenic sites A, B, and D of the HA protein. The A/H3N2 viruses belonged to phylogenetic clades 3C.2 and 3C.3. The M gene trees of the Iranian A/H1N1pdm09 and A/H3N2 mirrored the clustering patterns of their corresponding HA trees. Our results reveal co-circulation of several influenza A virus strains in Iran during the 2014-2015 influenza season.

The Impact of IFN-γ Gene Polymorphisms on Spontaneous Clearance of HCV Infection in Fars Province, Southern of Iran.

BACKGROUND: Certain polymorphisms in cytokine genes such as IFN-γ may influence the outcome of hepatitis C virus (HCV) infection. Here the frequency of the genotype, allele, and haplotype of IFN-γ gene at some loci is investigated in HCV-infected patients. METHODS: Totally 255 patients with chronic HCV infection and 44 spontaneously cleared individuals were included. The chronic or clearance states were confirmed using enzyme-linked immunosorbent assay (ELISA) and two different qualitative reverse transcriptase polymerase chain reaction (RT-PCR) techniques. IFN-γ gene polymorphisms were performed by PCR using sequence-specific primers and PCR-RLFP on extracted genomic DNA. RESULTS: The frequency of GG genotype (P = 0.0001, OR: 5.69 and CI: 2.21-14.62) and allele (P = 0.0003, OR: 2.73 and CI: 1.54-4.83) of IFN-γ gene at +2109 locus was significantly higher in cases that spontaneously cleared the infection. Haplotype analysis showed the association of AG haplotype (P = 0.0046, OR = 6.14 and CI = 1.56-25) with spontaneous clearance of the infection. CONCLUSION: Our finding indicated that individuals with GG genotype at +2109 loci of IFN-γ gene and also AG haplotype (A allele at +874 loci and G allele at +2109 loci) may clear HCV infection more frequently than those with AA and AG genotype at +2109 loci and AA, TA, and TG haplotype.

In Silico Functional and Structural Characterization of H1N1 Influenza A Viruses Hemagglutinin, 2010-2013, Shiraz, Iran.

Hemagglutinin (HA) is a major virulence factor of influenza viruses and plays an important role in viral pathogenesis. Analysis of amino acid changes, epitopes' regions, glycosylation and phosphorylation sites have greatly contributed to the development of new generations of vaccine. The hemagglutinins of 10 selected isolates, 8 of 2010 and 2 of 2013 samples were sequenced and analyzed by several bioinformatic softwares and the results were compared with those of 3 vaccine isolates. The study detected several amino acid changes related to altered epitopes' sites, modification sites and physico-chemical properties. The results showed some conserved modification sites in HA structure. This study is the first analytical research on isolates obtained from Shiraz, Iran, and our results can be used to better understand the genetic diversity and antigenic variations in Iranian and Asian H1N1 pathogenic strains.

The antibacterial effect of four mouthwashes against streptococcus mutans and escherichia coli.

OBJECTIVE: To evaluate the antimicrobial properties of several mouthwash concentrations on oral Streptococcus mutans and Escherichia coli. METHODS: The study was conducted at Shiraz Medicine School in 2011. Serial dilutions of Chlorohexidin, Oral B and Persica and Irsha (2,4,8,16,64,128) were prepared in Muller-Hinton media. Minimum inhibitory concentration was visually determined and defined as the lowest concentration of each oral washing which inhibited > 95% growth reduction compared to the growth control well. RESULTS: Chlorhexidine, Oral B and Irsha mouthwash inhibited Streptococcus mutans even with diluted concentrations. Also, Chlorhexidine and Oral B prohibited Escherichia coli with different potencies. But Persica had no antimicrobial activity against either Escherichia coli or Streptococcus mutans. CONCLUSIONS: Chlorhexidine, Irsha, and Oral B mouthwashes can be used for antimicrobial effects, especially on Streptococcus mutans. This chemical activity of mouthwashes is an adjuvant for mechanical removing of plaque. However, the antimicrobial effect of Persicaremains controversial.

Effects of hypo- and hyperthyroid states on herpes simplex virus infectivity in the rat.

OBJECTIVE: Available data from in vitro studies show that thyroid hormones (THs) regulate herpes simplex virus (HSV) gene expression and may modulate latency/reactivation of the virus. Whether infectivity of the virus is also affected by THs is not known. Using animal models (in vivo study) and Vero cell culture (in vitro study), we examined the effects of alterations in THs level on HSV-1 infectivity. METHODS: Rats were rendered hypo- and hyperthyroid by daily addition of methimazole and l-thyroxine into their drinking water, respectively. Euthyroid animals served as control. All animals were given a single dose of HSV-1 (10(7)TCID50, ip) and sacrificed 3 d later. The spleen of the animals was then removed and viral particles were recovered from the tissue extract through aseptic procedures. Serial dilution of the extracts was prepared and added to Vero cell culture. For the in vitro study, the cultures were pretreated with l-thyroxine and the viral particles were then added. Virus titration was determined by Reed-Muench quantal assay. RESULTS: The viral load of spleen in hyperthyroid rats was significantly lower (1000-fold) than that of the euthyroid rats. Similarly, in vitro presence of supraphysiologic levels of l-thyroxine in the culture media of Vero cells decreased virus infectivity. Interestingly, hypothyroid animals showed a significant increase (10-fold) in spleen viral load as compared to that of their euthyroid counterparts. CONCLUSIONS: These data clearly show that the HSV-1 infectivity is affected by THs, and suggest that THs or their analogs may have a potential application in prevention and/or treatment of viral infections.

Prevalence of Influenza Viruses A and B, Adenovirus, Respiratory Syncytial Virus, and Human Metapneumonia Viruses among Children with Acute Respiratory Tract Infection.

Background: Acute respiratory tract infection (ARTI) is a significant cause of morbidity and mortality among children worldwide. The majority of acute respiratory infections in children are caused by viruses, with respiratory syncytial virus (RSV) being the most frequently encountered. Other important viral pathogens include human metapneumovirus, human coronaviruses, adenovirus, and influenza. These infections can lead to complications such as bronchitis and pneumonia. So, this study aimed to evaluate the prevalence of influenza viruses A and B, adenovirus, respiratory syncytial virus (RSV), and human metapneumovirus (HMPV) in children with ARTI. Methods: The molecular diagnostic of polymerase chain reaction approach was used to detect influenza (A and B), metapneumovirus, respiratory syncytial virus (RSV), and adenovirus in respiratory samples of children with acute respiratory infection hospitalization in a teaching hospital of the Shiraz University of Medical Sciences in January 2016-March 2017. Results: Of the 340 patients examined, 208 (61.20%) were male and the median age was 3.13 ± 2.38 years. Respiratory viruses were found in 179 (52.64%) patients. The male-to-female ratio was 1.63 : 1 in patients who were viral positive. Detection rates for influenza A, adenovirus, influenza B, RSV, and HMPV were 28.23%, 24.70%, 8.52%, 3.23%, and 2.64%, respectively, and coinfections were detected in 24.02%. The most common combination of two-virus coinfections was IFVA/AdV, followed by IFVB/AdV, AdV, IFVB/IFVA, RSV/IFVA, HMPV/AdV, RSV/AdV, and HMPV/IFVA. Conclusion: The high prevalence of respiratory viruses in children hospitalized with ARTI suggests that viral infection may play a role in disease pathogenesis. This should be confirmed through the conduct of case-control studies and may inform the role of vaccination to prevent respiratory viral infections.

HA antigenic variation and phylogenetic analysis of influenza B virus in Shiraz, Iran.

BACKGROUND: Influenza infection is highly contagious respiratory illness triggered by the influenza virus, bearing substantial implications for global health. Influenza B viruses, specifically the Victoria and Yamagata lineages, have contributed to the disease burden, and the mismatch between circulating strains and vaccine strains has led to increased mortality and economic costs. Understanding the global epidemiology, seasonal variations, and genetic characteristics of influenza B is crucial for effective prevention and control strategies. METHODS: The study investigated influenza B viruses in Shiraz, Iran during the Oct 2017 to Jan 2018. Throat swabs were collected from 235 individuals under 15 with influenza-like symptoms including fever and cough. Samples were stored at -80°C and transported to the lab for further analysis. Viral RNA was extracted and analyzed using Real-time PCR. The hemagglutinin (HA) gene of positive samples was sequenced, and phylogenetic trees were constructed. Amino acids indicative of adaptive mutations were identified using global sequence data. RESULTS: 23 of 235 samples (9.7 %) were positive for influenza B virus. The most common clinical manifestations were rhinorrhea and myalgia, with 20 individuals (87 % of the 23 infected people) each showing these symptoms. The phylogenetic analysis of the HA gene showed that the Victoria isolates were close to the B/Brisbane/60/2008 strain (12.5 % of the positive samples) and belonged to clade-1A, while the Yamagata isolates were close to the B/Phuket/3037/2013 strain (87.5 % of the positive samples) and belonged to clade-3. CONCLUSION: The study highlights the need for importance vaccine coverage in the Shiraz region to address limited genetic diversity and strain mismatch. Continuous surveillance of mutations in the HA gene resulting in amino acid substitutions and their impact on vaccine efficacy is crucial. This study showed that the circulation of influenza B in Shiraz matched with the recommended Yamagata vaccine strain. These findings contribute to the understanding of influenza B dynamics and emphasize the importance of region-specific prevention and control strategies.

Determining the cytotoxicity of the Minimum Inhibitory Concentration (MIC) of silver and zinc oxide nanoparticles in ESBL and carbapenemase producing Proteus mirabilis isolated from clinical samples in Shiraz, Southwest Iran.

OBJECTIVE: Proteus mirabilis is related to serious infections. The present study was designed to investigate the minimum inhibitory concentration (MIC) of silver nanoparticles (AgNPs) and zinc oxide nanoparticles (ZnONPs) and cytotoxicity among P. mirabilis isolates recovered from clinical samples in Shiraz. RESULTS: A total of 100 P. mirabilis isolates were screened by biochemical tests and polymerase chain reaction (PCR). Also, 25 (25%) and 7 (7%) isolates were positive for extended-spectrum beta-lactamase (ESBLs) and carbapenemase, respectively. Synthesized nanoparticles were characterized by UV-vis spectrum, X-ray diffraction (XRD), and electron microscopy. The average size of AgNPs and ZnONPs in the present study is 48 and < 70 nm, respectively. The MIC and the MBC of the ZnONPs were in the range of 31.25 µg/ml and 62.5 µg/mL, respectively. Also, for AgNPs, the MIC and the MBC were in the range of 7.8 µg/mL and 15.6 µg/mL, respectively. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in a primary culture of fibroblast L929 cells for this MIC indicated biocompatibility and low cytotoxicity of Ag NPs and for ZnONPs indicated significant cytotoxicity. Also, a MIC of AgNPs can be used as a therapeutic concentration without the effect of cytotoxicity in human cells.

Immunological mechanisms of the nucleocapsid protein in COVID-19.

The emergence of corona virus disease 2019 (COVID-19), resulting from Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has left an indelible mark on a global scale, causing countless infections and fatalities. This investigation delves into the role of the SARS-CoV-2 nucleocapsid (N) protein within the HEK293 cells, shedding light on its influence over apoptosis, interferon signaling, and cytokines production. The N gene was amplified, inserted into the pAdTrack-CMV vector, and then transfected to the HEK293 cells. Changes in the expression of IRF3, IRF7, IFN-ß, BAK, BAX, and BCL-2 genes were evaluated. The levels of proinflammatory cytokines of IL-6, IL-12, IL-1ß, and TNF-α were also determined. The N protein exhibited an anti-apoptotic effect by modulating critical genes associated with apoptosis, including BAK, BAX, and BCL-2. This effect potentially prolonged the survival of infected cells. The N protein also played a role in immune evasion by suppressing the interferon pathway, evidenced by the downregulation of essential interferon regulatory factors of IRF3 and IRF7, and IFN-ß expression. The N protein expression led to a substantial increase in the production of proinflammatory cytokines of IL-6, IL-12, IL-1ß, and TNF-α. The N protein emerged as a versatile factor and was exerted over apoptosis, interferon signaling, and cytokine production. These findings carry potential implications for the development of targeted therapies to combat COVID-19 and mitigate its global health impact.

Epidemiological and phylogenetic assessment of human respiratory syncytial virus among pediatric patients presenting acute respiratory infections in Shiraz, Iran during 2015-2016.

Background and Objectives: The pediatric population worldwide bears a significant morbidity and death burden due to acute respiratory infections (ARIs). Human Orthopneumovirus, sometimes referred to as the Human Respiratory Syncytial Virus (HRSV), is one of the main causes of ARIs in infants. The main goal of this study was to identify the genetic diversity of HRSV strains that were circulating in the Iranian population at a certain time of year. Materials and Methods: Two hundred youngsters less than 12 years old with acute respiratory infections had samples taken from their throat and pharynx secretions. Then, external and hemi-nested PCR were employed, using specific primers targeting the G gene region to detect HRSV. Subsequently, nine randomly selected positive samples were subjected to sequencing. The results were then compared with reference strains cataloged in GeneBank, and phylogenetic tree was constructed using Chromes and MEGA7. Results: Out of 200 samples, 34 were identified as containing HRSV. Subgroup A was predominant, accounting for 61.76% of cases, followed by subgroup BA (35.29%) and subgroup B (2.94%). Phylogenetic analysis revealed five samples associated with subtype B and four with genotype A. Genomic analysis showed three samples under the GA2 subgroup and one under GA1 for subtype A, and four samples in subgroup BA and one in GB2 for subtype B. Conclusion: In this study, subgroup A strains, particularly genotype GA2, exhibited a higher prevalence compared to subgroup B strains during the specific period under investigation, shedding light on the genetic landscape of HRSV in this region.

Characteristics of Hospitalized COVID-19 Patients in a Major Referral Center in Shiraz, Iran.

Background: Several countries, including Iran, have been affected by the novel Coronavirus Disease 2019 (COVID-19) pandemic since December 2019. The aim of this study was to provide a comprehensive report on COVID-19 patients in Shiraz, Southern Iran. Materials and Methods: This study was performed on 311 hospitalized patients with COVID-19. The data on demographic, clinical, and paraclinical features were analyzed. Results: The median age of the patients was 58 years, with 42.1% of the patients being above 60 years of age. Upon admission, fever was detected in 28.2% of critically ill patients. At least one underlying disease or risk factor was also present in 75.6% of the patients. Shortness of breath was the most common clinical symptom (66.2%), dry cough (53.7%), and muscle pain (40.5%) was the second and third. Sneezing (0.3%), rhinorrhea (0.7%), and sore throat (3.09%) were observed only in non-critically ill patients. In addition, 26.9% of all patients had lymphocytopenia, 25.8% had raised C-reactive protein, and 79.9% had abnormal creatinine levels. Finally, death occurred in 39 patients (12.5%). Conclusions: Noncritically ill patients were younger than critically ill patients. The most common risk factors for getting critically ill were surgery, hypertension, diabetes mellitus, chronic heart disease, asthma, and chronic renal disease.

Th-1, Th-2, Th-9, Th-17, Th-22 type cytokine concentrations of critical COVID-19 patients after treatment with Remdesivir.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly spread around the world causing a pandemic known as coronavirus disease 2019 (COVID-19). Cytokine storm was directly correlated with severity of COVID-19 syndromes. We evaluated the levels of 13 cytokines in ICU hospitalized COVID-19 patients (n = 29) before, and after treatment with Remdesivir as well as in healthy controls (n = 29). Blood samples were obtained from ICU patients during ICU admission (before treatment) and 5 days after treatment with Remdesivir. A group of 29 age- and gender-matched healthy controls was also studied. Cytokine levels were evaluated by multiplex immunoassay method using a fluorescence labeled cytokine panel. In comparison to cytokine levels measured at ICU admission, serum levels were reduced of IL-6 (134.75 pg/mL vs. 20.73 pg/mL, P < 0.0001), TNF-α (121.67 pg/mL vs. 10.15 pg/mL, P < 0.0001) and IFN-γ (29.69 pg/mL vs. 22.27 pg/mL, P = 0.005), whereas serum level was increased of IL-4 (8.47 pg/mL vs. 12.44 pg/mL, P = 0.002) within 5 days after Remdesivir treatment. Comparing with before treatment, Remdesivir significantly reduced the levels of inflammatory (258.98 pg/mL vs. 37.43 pg/mL, P < 0.0001), Th1-type (31.24 pg/mL vs. 24.46 pg/mL, P = 0.007), and Th17-type (36.79 pg/mL vs. 26.22 pg/mL, P < 0.0001) cytokines in critical COVID-19 patients. However, after Remdesivir treatment, the concentrations of Th2-type cytokines were significantly higher than before treatment (52.69 pg/mL vs. 37.09 pg/mL, P < 0.0001). In conclusion, Remdesivir led to decrease levels of Th1-type and Th17-type cytokines and increase Th2-type cytokines in critical COVID-19 patients 5 days after treatment.

Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) RNA in the Human Eye.

PURPOSE: To determine the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in postmortem ocular specimens of patients with severe COVID-19 disease. PATIENTS AND METHODS: Postmortem conjunctival (28 samples), aqueous humor (30 samples) and vitreous humor (30 samples) specimens were obtained bilaterally from the eyes of 15 deceased COVID-19 patients within one hour of death. The presence of viral RNA was evaluated in samples using Real-time reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Positive RT-PCR SARS-COV-2 results were found in one conjunctival and 2 vitreous humor samples. All aqueous humor samples tested negative for the presence of SARS-COV-2 RNA. Of note, three positive samples were obtained from three different patients. The overall prevalence of positive RT-PCR ocular samples was 3.4% among all samples and 20% at the patient level. CONCLUSION: SARS-CoV-2 RNA is detectable in postmortem conjunctival and vitreous humor samples of patients with severe COVID-19.

Implementation of an In-House Platform for Rapid Screening of SARS-CoV-2 Genome Variations.

BACKGROUND: Global real-time monitoring of SARS-CoV-2 variants is crucial to controlling the COVID-19 outbreak. The purpose of this study was to set up a Sanger-based platform for massive SARS-CoV-2 variant tracking in laboratories in low-resource settings. METHODS: We used nested RT-PCR assay, Sanger sequencing and lineage assignment for 930-bp of the SARS-CoV-2 spike gene, which harbors specific variants of concern (VOCs) mutations. We set up our platform by comparing its results with whole genome sequencing (WGS) data on 137 SARS-CoV-2 positive samples. Then, we applied it on 1028 samples from March-September 2021. RESULTS: In total, 125 out of 137 samples showed 91.24% concordance in mutation detection. In lineage assignment, 123 out of 137 samples demonstrated 89.78% concordance, 65 of which were assigned as VOCs and showed 100% concordance. Of 1028 samples screened by our in-house method, 78 distinct mutations were detected. The most common mutations were: S:D614G (21.91%), S:P681R (12.19%), S:L452R (12.15%), S:T478K (12.15%), S:N501Y (8.91%), S:A570D (8.89%), S:P681H (8.89%), S:T716I (8.74%), S:L699I (3.50%) and S:S477N (0.28%). Of 1028 samples, 980 were attributed as VOCs, which include the Delta (B.1.617.2) and Alpha (B.1.1.7) variants. CONCLUSION: Our proposed in-house Sanger-based assay for SARS-CoV-2 lineage assignment is an accessible strategy in countries with poor infrastructure facilities. It can be applied in the rapid tracking of SARS-CoV-2 VOCs in the SARS-CoV-2 pandemic.