MicroRNAs and cytokines as potential predictive biomarkers for COVID-19 disease progression.

Host microRNAs can influence the cytokine storm associated SARS-CoV-2 infection and proposed as biomarkers for COVID-19 disease. In the present study, serum MiRNA-106a and miRNA-20a were quantified by real time-PCR in 50 COVID-19 patients hospitalized at Minia university hospital and 30 healthy volunteers. Profiles of serum inflammatory cytokines (TNF-α, IFN-γ, and IL-10) and TLR4 were analyzed by Eliza in patients and controls. A highly significant decrease (P value = 0.0001) in the expressions of miRNA-106a and miRNA-20a was reported in COVID-19 patients compared to controls. A significant decrease in the levels of miRNA-20a was also reported in patients with lymphopenia, patients having chest CT severity score (CSS) > 19 and in patients having O2 saturation less than 90%. Significantly higher levels of TNF-α, IFN-γ, IL-10 and TLR4 were reported in patients compared to controls. IL-10 and TLR4 levels were significantly higher in patients having lymphopenia. TLR-4 level was higher in patients with CSS > 19 and in patients with hypoxia. Using univariate logistic regression analysis, miRNA-106a, miRNA-20a, TNF-α, IFN-γ, IL-10 and TLR4 were identified as good predictors of disease. Receiver operating curve showed that the downregulation of miRNA-20a in patients having lymphopenia, patients with CSS > 19 and patients with hypoxia could be a potential biomarker with AUC = 0.68 ± 0.08, AUC = 0.73 ± 0.07 and AUC = 0.68 ± 0.07 respectively. Also, ROC curve showed accurate association between the increase of serum IL-10 and TLR-4 and lymphopenia among COVID-19 patients with AUC = 0.66 ± 0.08 and AUC = 0.73 ± 0.07 respectively. ROC curve showed also that serum TLR-4 could be a potential marker for high CSS with AUC = 0.78 ± 0.06. A negative correlation was detected between miRNA-20a with TLR-4 (r = - 0.30, P value = 0.03). We concluded that, miR-20a, is a potential biomarker of COVID-19 severity and blockade of IL-10 and TLR4 may constitute a novel therapy for COVID-19 patients.

Effect of ZnO nanoparticles on methicillin, vancomycin, linezolid resistance and biofilm formation in Staphylococcus aureus isolates.

BACKGROUND: Multidrug resistant (MDR) and biofilm producing Staphylococcus aureus strains are usually associated with serious infections. This study aimed to evaluate the antibacterial and antibiofilm-formation effects of zinc oxide nanoparticles (ZnO-NPs) against staphylococcus aureus (S. aureus) isolates. METHODS: A total of 116 S. aureus isolates were recovered from 250 burn wound samples. The antimicrobial/antibiofilm effects of ZnO-NPs against methicillin, vancomycin and linezolid resistant S. aureus (MRSA, VRSA and LRSA) isolates were examined using phenotypic and genotypic methods. The minimum inhibitory concentration (MIC) of ZnO-NPs was determined by microdilution method. The effects of sub-MIC concentrations of ZnO-NPs on biofilm formation and drug resistance in S. aureus were determined by the microtiter plate method. The change in the expression levels of the biofilm encoding genes and resistance genes in S. aureus isolates after treatment with ZnO-NPs was assessed by real time reverse transcriptase PCR (rt-PCR). RESULTS: MICs of ZnO-NPs in S. aureus isolates were (128-2048 µg/ml). The sub-MIC of ZnO-NPs significantly reduced biofilm formation rate (the highest inhibition rate was 76.47% at 1024  µg/ml) and the expression levels of biofilm genes (ica A, ica D and fnb A) with P < 0.001. Moreover, Sub-MIC of ZnO-NPs significantly reduced the rates of MRSA from 81.9 (95 isolates) to 13.30% (15 isolates), VRSA from 33.60 (39 isolates) to 0% and LARSA from 29.30 (34) to 0% as well as the expression levels of resistance genes (mec A, van A and cfr) with P value < 0.001. CONCLUSION: ZnO-NPs can be used as antibiofilm and potent antimicrobial against MRSA, VRSA and LRSA isolates.

Serum MicroRNAs as predictors for fibrosis progression and response to direct-acting antivirals treatment in hepatitis C virus genotype-4 Egyptian patients.

BACKGROUND: MicroRNA (miRNAs) are small non-coding molecules that play an important role in hepatitis C virus (HCV) replication and liver diseases progression. The current study aimed to evaluate serum miRNAs as potential biomarkers for diagnosis, monitoring of fibrosis progression and prediction of responses to direct-acting antivirals (sofosbuvir + daclatasvir + ribavirin) in HCV genotype-4 patients. METHODS: The serum levels of four miRNAs (miRNA-21, 199, 448 and 181c) were assessed in 150 HCV patients and 50 healthy controls using quantitative real-time PCR. The diagnostic accuracy was determined using receiver operating characteristic (ROC) curve. RESULTS: The four studied miRNAs showed significant upregulation in HCV patients compared with controls. There were significant upregulation of MiR-199 and significant downregulation of miR-448 in late stages of fibrosis with high diagnostic accuracy (area under the curve "AUC" = 0.989%; P < .001) and (AUC = 0.0.672; P > .001), respectively. Regarding response to treatment, only miR-199 showed a significant upregulation in non-responder patients with high diagnostic accuracy (AUC = 0.968; P < .001). CONCLUSION: miR-199 and miR-448 could serve as valuable non-invasive biomarkers for assessment of liver fibrosis progression. Additionally, miR-199 could be also a potential biomarker for assessment of treatment efficacy among HCV patients. Therefore, miR-199 and miR-448 serum levels should be considered during the treatment of HCV genotype-4 patients in Egypt and the world.

Prevalence and molecular characteristics of ESBL and AmpC ß -lactamase producing Enterobacteriaceae strains isolated from UTIs in Egypt.

BACKGROUND: Infections caused by Enterobacteriaceae are mainly treated with the ß-lactam antibiotics, nevertheless, the emergence of species with plasmid-borne ß-lactamases has decreased the efficacy of these antibiotics. Therefore, continuing studies on the resistance pattern of different regions is important for assessment of proper antimicrobial therapy protocols. The study aimed to characterize extended-spectrum ß-lactamase (ESBL) and AmpC ß -lactamase (AmpC) producing Enterobacteriaceae isolated from community-acquired UTIs in Egypt. METHODS: Out of 705 urine samples, 440 Enterobacteriaceae isolates were investigated to detect ESBL and AmpC ß -lactamases producers by phenotypic and molecular methods. RESULTS: Out of 440 Enterobacteriaceae isolates, 311 were identified as ESBL producers by phenotypic testing. ESBL genes were detected in 308 isolates. BlaCTX-M-type was the most prevalent 254 (81.6%), out of them blaCTXM-15 was the commonest (152, 48.8%) followed by blaCTX-M-1 (140, 45%), blaCTX-M-8 (72, 23.1%) and lastly blaCTX-M-2 (4, 1.3%). blaTEM gene also was detected in a high rate (189, 60.7%). Two hundred and thirty-five (75.5%) of ESBL producers harbored blaCTX-M in combination with blaTEM and/or blaSHV genes. Multiple drug resistance in the ESBL-producers was significantly (P < 0.05) higher than in non-ESBL producers. Imipenem was the most effective drug against ESBL producers. Among 35 cefoxitin resistant isolates, 18 (51.4%) identified as carrying AmpC genes by multiplex PCR. Within AmpC ß -lactamase genes, DHA gene was the predominant gene (15, 42.3%). CIT and MOX genes were also present, but in a low rate (5, 14.2% and 4, 11.4%) respectively. Co-existence of multiple AmpC genes was detected exclusively in K. pneumoniae isolates. E. coli isolates harbored DHA gene only. However, FOX gene was not detected in the study isolates. Seventeen of isolates carrying AmpC genes were also positive for ESBL genes. CONCLUSION: The study shows that the prevalence of ESBL producing Enterobacteriaceae spread in south Egypt is alarming, however AmpC ß -lactamase production is not so high.

Prevalence, phylogeny, and antimicrobial resistance of Escherichia coli pathotypes isolated from children less than 5 years old with community acquired- diarrhea in Upper Egypt.

BACKGROUND: Diarrhoea, affecting children in developing countries, is mainly caused by diarrheagenic Escherichia coli (DEC). This study principally aimed to determine the prevalence of DEC pathotypes and Extended-spectrum ß-lactamase (ESBL) genes isolated from children under 5 years old with diarrhea. METHODS: A total of 320 diarrhoea stool samples were investigated. E. coli isolates were investigated for genes specific for enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC) and enterohemorrhagic E. coli (EHEC) using polymerase chain reaction (PCR). Furthermore, antimicrobial susceptibility testing, detection of antibiotic resistance-genes and phylogenetic typing were performed. RESULTS: Over all, DEC were isolated from 66/320 (20.6%) of the children with diarrhoea. EAEC was the predominant (47%), followed by typical EPEC (28.8%) and atypical EPEC (16.6%). Co-infection by EPEC and EAEC was detected in (7.6%) of isolates. However, ETEC, EIEC and EHEC were not detected. Phylogroup A (47%) and B2 (43.9%) were the predominant types. Multidrug-resistance (MDR) was found in 55% of DEC isolates. Extended-spectrum ß-lactamase (ESBL) genes were detected in 24 isolates (24 blaTEM and 15 blaCTX-M-15). Only one isolate harbored AmpC ß-lactamase gene (DHA gene). CONCLUSION: The study concluded that, EAEC and EPEC are important causative agents of diarrhoea in children under 5 years. MDR among DEC has the potential to be a big concern.

Multidrug-resistant Klebsiella pneumoniae in hospital-acquired infections: Concomitant analysis of antimicrobial resistant strains.

BACKGROUND: Hospital-acquired infections caused by K pneumoniae are difficult to eradicate since K pneumoniae carries resistance genes for many antimicrobials, including carbapenems. The study aimed to determine the prevalence of hospital-acquired infections caused by multiple drug-resistant K pneumoniae and identify carbapenem and fluoroquinolone resistance by phenotypic and genotypic methods amongst hospitalised patients. METHODS: Two hundred and fifty samples from patients with hospital-acquired infections were included. Identification and susceptibility testing for K pneumoniae isolates was performed by standard methods. The detection of carbapenemase resistance (blaKPC , blaVIM-1 and blaOXA-48 ) and plasmid-mediated quinolone resistance (PMQR; qnrA, qnrB and qnrS) genes was performed using PCR assay. RESULTS: Out of 250 samples, 42 (16.8%) were multiple drug-resistant K pneumoniae, and the frequency of K Pneumoniae isolation was higher in urine samples, in the age group (<10 years), in ICU and in patients with longer hospital stay. Twenty-four (57%) of the isolates were resistant to Meropenem, 13 (31%) were resistant to Imipenem and 35 (83.3%) were resistant to Ciprofloxacin. blaOXA-48 gene was detected in 9 (21.4%) of isolates, and blaVIM-1 gene was detected in 6 (14.3%) of isolates. However, no isolate harboured blaKPC gene. PMQR genes were detected in 100% of ciprofloxacin resistant isolates, and qnrS was the dominant. CONCLUSION: Multidrug-resistant K pneumoniae isolates harbouring blaOXA-48, blaVIM-1 and PMQR genes are emerging in hospitals particularly with long hospital stays.

Impact of co-existence of PMQR genes and QRDR mutations on fluoroquinolones resistance in Enterobacteriaceae strains isolated from community and hospital acquired UTIs.

BACKGROUND: Fluoroquinolones are commonly recommended as treatment for urinary tract infections (UTIs). The development of resistance to these agents, particularly in gram-negative microorganisms complicates treatment of infections caused by these organisms. This study aimed to investigate antimicrobial resistance of different Enterobacteriaceae species isolated from hospital- acquired and community-acquired UTIs against fluoroquinolones and correlate its levels with the existing genetic mechanisms of resistance. METHODS: A total of 440 Enterobacteriaceae isolates recovered from UTIs were tested for antimicrobial susceptibility. Plasmid-mediated quinolone resistance (PMQR) genes and mutations in the quinolone resistance-determining regions (QRDRs) of gyrA and parC genes were examined in quinolone-resistant strains. RESULTS: About (32.5%) of isolates were resistant to quinolones and (20.5%) were resistant to fluoroquinolones. All isolates with high and intermediate resistance phenotypes harbored one or more PMQR genes. QnrB was the most frequent gene (62.9%) of resistant isolates. Co-carriage of 2 PMQR genes was detected in isolates (46.9%) with high resistance to ciprofloxacin (CIP) (MICs > 128 µg/mL), while co-carriage of 3 PMQR genes was detected in (6.3%) of resistant isolates (MICs > 512 µg/mL). Carriage of one gene only was detected in intermediate resistance isolates (MICs of CIP = 1.5-2 µg/mL). Neither qnrA nor qnrC genes were detected. The mutation at code 83 of gyrA was the most frequent followed by Ser80-Ile in parC gene, while Asp-87 Asn mutation of gyrA gene was the least, where it was detected only in high resistant E. coli isolates (MIC ≥128 µg/mL). A double mutation in gyrA (Lys154Arg and Ser171Ala) was observed in high FQs resistant isolates (MIC of CIP < 128 µg/mL). CONCLUSION: FQs resistance is caused by interact between PMQR genes and mutations in both gyrA and parC genes while a mutation in one gene only can explain quinolone resistance. Accumulation of PMQR genes and QRDR mutations confers high resistance to FQs.

Phylogenic classification and virulence genes profiles of uropathogenic E. coli and diarrhegenic E. coli strains isolated from community acquired infections.

The emergence of E.coli strains displaying patterns of virulence genes from different pathotypes shows that the current classification of E.coli pathotypes may be not enough, the study aimed to compare the phylogenetic groups and urovirulence genes of uropathogenic Escherichia coli (UPEC) and diarrheagenic E.coli (DEC) strains to extend the knowledge of E.coli classification into different pathotypes. A total of 173 UPEC and DEC strains were examined for phylogenetic typing and urovirulence genes by PCR amplifications. In contrast to most reports, phylogenetic group A was the most prevalent in both UPEC and DEC strains, followed by B2 group. Amplification assays revealed that 89.32% and 94.29% of UPEC and DEC strains, respectively, carried at least one of the urovirulence genes, 49.5% and 31.4% of UPEC and DEC strains, respectively, carried ≥ 2 of the urovirulence genes, fim H gene was the most prevalent (66.9% and 91.4%) in UPEC and DEC strains respectively. Twenty different patterns of virulence genes were identified in UPEC while 5 different patterns in DEC strains. Strains with combined virulence patterns of four or five genes were belonged to phylogenetic group B2. Our finding showed a closer relationship between the DEC and UPEC, so raised the suggestion that some DEC strains might be potential uropathogens. These findings also provide different insights into the phylogenetic classification of E. coli as pathogenic or commensals where group A can be an important pathogenic type as well as into the classification as intestinal or extra- intestinal virulence factors.

Association of IL-1ß, IL-1RN, and ESR1 genes polymorphism with risk of infection and response to sofosbuvir plus daclatasvir combination therapy in hepatitis C virus genotype-4 patients.

Sofosbuvir (SOF) and daclatasvir combination with or without ribavirin proved to be effective and safe in the treatment of hepatitis C virus infection. The study aimed to assess the efficacy of (SOF plus daclatasvir) combination + ribavirin in the treatment of treatment-experienced HCV genotype 4 Egyptian patients and to investigate the impact of IL-1ß _31, IL-1ß _511, and IL-1RN and T29C of ESR1 genes polymorphisms on treatment outcome. The study also aimed to assess the effect of the treatment on liver fibrosis. The sustained virologic response was assessed at 0, 4, 12, and 24 weeks from the beginning of treatment by RT-PCR. IL-1ß _31, IL-1ß _511, IL-1RN, and T29C genes polymorphisms were examined by PCR-based techniques in two groups of patients (responders and non-responders) and a control group of healthy subjects. A significant association between IL-1ß_511 gene polymorphism and SOF/DAV-induced response was observed, where the "CC" genotype was the most frequent in responders while the "CT" genotype was the most frequent among non-responders (P = 0.0001, OR = 39; 95% CI = 4.7-316). IL-1RN gene polymorphism also showed significant associations with response to treatment, genotypes that include allele "1" were the most frequent in responders, particularly the homozygous genotype "1/1" (P = 0.0001, OR = 2.3; 95% CI = 1.57-3.2). However, the genotypes "4/4" and "2/4" were the most frequent in non-responders; (P = 0.0001). At least 71% of the responders carry allele "1" while 54% of non-responders were allele "4" carriers (P value = 0.0001. OR = 2.8; 95% CI = 6.4-134.2). Liver fibrosis is significantly improved regardless of the response. © 2018 IUBMB Life, 70(11):1156-1163, 2018.

Genotyping of occult hepatitis B virus infection in Egyptian hemodialysis patients without hepatitis C virus infection.

BACKGROUND: Occult hepatitis B viral infection is the presence of hepatitis B viral nucleic acids in the serum and/or liver in the absence of hepatitis B surface antigen. AIM: The study aimed to determine the prevalence of occult hepatitis B virus infection among hepatitis C virus-negative hemodialysis patients and to identify their genotypes. METHODS: of 144 patients on maintenance hemodialysis, 50 hepatitis B surface antigen and hepatitis C virus nucleic acid-negative patients were selected according to strict inclusion criteria to avoid the effect of confounding variables. The following investigations were done: serum AST and ALT; HBsAg; HBcAb; HCV-Ab; HCV-RNA; and HBV-DNA. RESULTS: Positive hepatitis B viral nucleic acid was confirmed in 12/144 (8.3%) hemodialysis patients and 12/50 (24%) in our study group (occult infection). Mean hemodialysis periods for negative patients and occult hepatitis B virus patients were 27.3±18.8 and 38.4±8.14 months, respectively, and this difference was significant (p-value=0.02). Mean alanine transaminase levels were 20.27±5.5IU/L and 25.3±9.6 in negative patients and occult infection patients, respectively. This difference was non-significant. Aspartate transaminase levels were 21.4±10.2IU/L and 27.3±4.6IU/L, respectively, in negative patients and infected patients; this difference was significant (p-value=0.03). Half (6/12) of the positive samples belonged to genotype 'B', 33.3% (4/12) to 'C', and 16.6% (2/12) to genotype 'D'. CONCLUSION: OBI is likely among hemodialysis patients even without HCV coinfection (24%). Genotype D cannot be the only genotype distributed in Upper Egypt, as the current study reported relatively new results that 50% of the patients with occult B carry genotype B, 33.3% carry genotype C and only 16.6% carry genotype D.

Changing patterns of drug-resistant Shigella isolates in egypt.

BACKGROUND: The emergence of multidrug resistance (MDR) is a serious problem in treating shigellosis. There are limited existing data examining the change in the antimicrobial resistance profile of Shigella in Egypt. We previously reported that 58% of the Shigella isolates in Egypt were resistant to at least one member of the three different antimicrobial groups. This study was performed to determine the antimicrobial resistance profile of Shigella, determine their possible mechanisms of resistance, and compare their resistance profile to those reported 20 years ago. PATIENTS AND METHODS: Stool samples were collected from 500 subjects and processed for the isolation and identification of Shigella. The susceptibility of the isolates to 11 different antimicrobials was determined using the disc diffusion method. RESULTS: Of 500 stool cultures, 24 (4.8%) samples were positive for Shigella. There was a high percentage of resistance to ampicillin (88%), tetracycline (83%), and sulfamethoxazole-trimethoprim (75%). Also, there was a moderate percentage of resistance to chloramphenicol (46%), streptomycin (42%), ceftazidime (33%), and cefotaxime (25%). A lower percentage of resistance was recorded for amikacin, nalidixic acid (17% each), and ofloxacin (7%), while no resistance was found to ciprofloxacin (0%). Twenty-one of the isolates (88%) were resistant to at least three different antimicrobial groups (indicating MDR). The average number of antimicrobial agents to which the Shigella isolates were resistant was 4.3±1.4, while it was 3.4±1.5 in the same locality in 1994. CONCLUSION: These data demonstrate that there is a marked increase in MDR and change in the resistance patterns of Shigella over the past 20 years.

Increased quinolone resistance among typhoid Salmonella isolated from Egyptian patients.

INTRODUCTION: Typhoid fever is endemic in Egypt; and quinolones are the empirical treatment of choice. There are very limited data reporting quinolone resistance among Egyptian typhoidal Salmonella isolates. We previously reported that all typhoidal Salmonella were sensitive to quinolones. This study aimed to isolate and identify typhoidal Salmonella from cases suffering from enteric fever at Minia Governorate, Egypt, determine their quinolone resistance patterns, compare them to those reported 20 years ago, and test gyrA mutation as a possible mechanism for quinolone resistance. METHODOLOGY: Stool samples from Widal-positive subjects were screened by culture on suitable media and were identified biochemically. The identified isolates were tested for resistance against three representatives of the first three quinolone generations, namely nalidixic acid (NAL), levofloxacin (LEV), and norfloxacin (NOR). The gyrA gene was amplified and sequenced to detect point mutation(s) conferring quinolone resistance. RESULTS: Out of 230 stool samples (from patients with Widal anti-O titers of ≥ 1/160), 40 isolates were S. enterica serovar Typhi (97.5%) and Paratyphi A (2.5%). Six (15%) isolates were resistant to at least one of the quinolones, compared to 0% in 1993. In this regard, 15%, 7.5%, and 2.5% of the isolates were resistant to NAL, both NAL and LEV, and all three quinolones tested, respectively. Sequencing of the gyrA gene revealed point mutations at position 83 and/or 87 of the gyrA gene only among the resistant isolates. CONCLUSION: There has been an increase in quinolone-resistant typhoidal Salmonella in Egypt over time.

The Frequency of Methicillin-Resistant Staphylococcus aureus and Coagulase Gene Polymorphism in Egypt.

The current study aimed to use Coagulase gene polymorphism to identify methicillin-resistant Staphylococcus aureus (MRSA) subtypes isolated from nasal carriers in Minia governorate, Egypt, evaluate the efficiency of these methods in discriminating variable strains, and compare these subtypes with antibiotypes. A total of 400 specimens were collected from nasal carriers in Minia governorate, Egypt, between March 2012 and April 2013. Fifty-eight strains (14.5%) were isolated and identified by standard microbiological methods as MRSA. The identified isolates were tested by Coagulase gene RFLP typing. Out of 58 MRSA isolates 15 coa types were classified, and the amplification products showed multiple bands (1, 2, 3, 4, 5, and 8 bands). Coagulase gene PCR-RFLPs exhibited 10 patterns that ranged from 1 to 8 fragments with AluI digestion. Antimicrobial susceptibility testing with a panel of 8 antimicrobial agents showed 6 different antibiotypes. Antibiotype 1 was the most common phenotype with 82.7%. The results have demonstrated that many new variants of the coa gene are present in Minia, Egypt, different from those reported in the previous studies. So surveillance of MRSA should be continued.