Study of some potential biomarkers in Egyptian hepatitis C virus patients in relation to liver disease progression and HCC.

BACKGROUND: Egypt has the greatest prevalence of hepatitis C worldwide according to the WHO reports, accounting for 13% of the global HCV infections. HCV is a substantial precursor for fibrosis, cirrhosis, and hepatocellular carcinoma. This study aimed to investigate the potential relevance of some cytokines, miR-122 and miR-221 for the diagnosis of liver disease progression associated to HCV infection. METHODS: One hundred and twenty blood samples were collected from patients with chronic liver disease, HCC, and healthy individuals. Total bilirubin, alanine aminotransferase, aspartate aminotransferase, platelet count, albumin, and creatinine were measured. Serum level of selected cytokines was conducted by ELISA. Serum miRNA expression was detected by RT-PCR. RESULTS: IL-2R was higher among HCC patients and the mean concentration of both TNF-αRII and IL-6R was higher among cirrhotic patients. The expression of miRNA-122 showed a little fold decrease in all studied groups; the highest level was observed in HCC patients. The expression of miRNA-221 showed a significant fold increase in HCC and cirrhotic groups. CONCLUSIONS: This study revealed that there is no difference in liver disease progression in patients regarding sex and age. Routine liver function tests performed poorly in terms of early diagnosis of liver disease progression; however, serum total bilirubin gave somewhat useful guide for discrimination between fibrotic, cirrhotic and HCC cases. IL-2R showed a significant consistent increase in its level with disease progression. The miR-221 serum level showed significant fold increase with liver disease progression. Therefore, making miR-221 a potential non-invasive biomarker for liver disease progression in the diagnostic setting is recommended.

Genotyping and molecular investigation of plasmid-mediated carbapenem resistant clinical Klebsiella pneumoniae isolates in Egypt.

Klebsiella pneumoniae is a multidrug-resistant nosocomial pathogen. Carbapenem resistance is mediated mainly by enzymes carried on transmissible plasmids causing their dissemination among other members of Enterobacteriaceae. This study aimed to molecularly detect carbapenem resistance genes in K. pneumoniae clinical isolates, genotype them using ERIC-PCR, and investigate plasmid transformation of resistant genes by using ERIC-PCR and sequencing. Methods: Antimicrobial resistance of sixty carbapenem-resistant K. pneumoniae strains was evaluated by using the disc diffusion method. Five carbapenemases' genes were amplified by conventional PCR. Genotyping was performed using ERIC-PCR. Gene transformation was performed for the five genes to sensitive isolates. Wild and transformed isolates were genetically investigated using ERIC-PCR and sequencing. Results: Carbapenem resistance in our isolates was associated with high resistance to all tested antibiotics. The 60 K. pneumoniae isolates were divided into 6 resistor types. The prevalence of KPC, IMP, VIM, NDM, and OXA-48 genes were 17%, 63%, 93%, 85% and 100%, respectively. Dendrogram analysis showed 57 distinct patterns, arranged in three clusters. The five genes were transformed successfully into sensitive isolates. ERIC profiles of wild and transformed isolates showed cluster A contained all the wild isolates, and cluster B contained all transformed isolates. Genetic sequences of the 5 genes reflected high genetic similarity with the GenBank reference genes before plasmid transformation; however, a distinguishable decrease of genetic similarity was observed after transformation. Conclusion: Plasmid-mediated carbapenem resistance in K. pneumoniae and its dissemination among different strains is a real threat to public health.

Investigation of the protective and therapeutic effects of Lactobacillus casei and Saccharomyces cerevisiae in a breast cancer mouse model.

Introduction: The development of novel strategies for cancer therapy is crucial to improve standard treatment protocols. Aim: This study aimed to determine the protective and therapeutic effects of heat-killed preparations of Lactobacillus casei and Saccharomyces cerevisiae in a breast cancer mouse model. Methods: Forty-two female BALB/c mice (7-8 weeks old) were divided into six groups (seven mice per group). Four groups were injected with 107 Ehrlich ascites tumor (EAT) cells suspended in phosphate-buffered saline (PBS) subcutaneously into the left side of the mammary fat pad. Tumor growth was monitored weekly until all animals developed a palpable tumor. The tumor-bearing mice in the experimental groups received heat-killed L. casei or S. cerevisiae three times per week for 35 days. The mice in the control group received PBS. The remaining two groups received heated L. casei or S. cerevisiae and then were injected with EAT cells. After 35 days, all mice were sacrificed to determine the immune response. Results: Animals that received heated S. cerevisiae exhibited the lowest rate of tumor growth compared with the other groups. TGF-ß and IL-4 secretion was increased in all mice, whereas the secretion of INF-γ and IL-10 was decreased in breast tissues. Moreover, at the histopathological level, the volume of viable tumor in the control group was higher than in the treated groups. Conclusion: Supplementary treatment with S. cerevisiae resulted in the best outcome in the breast cancer model compared with other treated and vaccinated groups.

Identifying Different Mutation Sites Leading to Resistance to the Direct-Acting Antiviral (DAA) Sofosbuvir in Hepatitis C Virus Patients from Egypt.

The hepatitis C virus (HCV) is a major global health challenge and a leading cause of morbidity and mortality. Many direct-acting antivirals (DAAs) target essential macromolecules involved in the virus' life cycle. Although such DAAs achieve great success in reducing the viral load in genotype 1 infections, other genotypes demonstrate different levels of response. This study focused on mutation sites associated with patients with genotype 4a infections that failed to respond to treatment with sofosbuvir. The genotyping of HCV samples from patients with virological failure, and responder patients, was conducted using Geno2Pheno webserver-based full NS5B sequences. We constructed 3D structural models for all the samples and used structural analysis to investigate the effect of amino acid substitution on the observed resistance to SOF-based treatment, and the docking of sofosbuvir into the active sites of the 10 models was performed. Finally, 10 molecular dynamic (MD) simulation experiments were conducted to compare the stability of the 3D models of the resistant samples against the stability of the 3D models of the responder samples. The results highlighted the presence of HCV subtype 4a in all ten samples; in addition, an amino acid (aa) substitution in the palm region may hinder HCV polymerase activity. In this study, we provide evidence that a mutation in the NS5B gene that induces resistance to sofosbuvir in patients with the S282T/C/R mutant virus is present in the Egyptian population. Overall, the docking and MD results support our findings and highlight the significant impact of the identified mutations on the resistance of HCV NS5B RNA-dependent RNA polymerase to direct-acting antivirals (DAAs).

Ultra-deformable liposomes containing terpenes (terpesomes) loaded fenticonazole nitrate for treatment of vaginal candidiasis: Box-Behnken design optimization, comparative ex vivo and in vivo studies.

Fenticonazole nitrate (FTN) is a potent antifungal drug adopted in the treatment of vaginal candidiasis. It has inadequate aqueous solubility hence, novel ultra-deformable liposomes 'Terpesomes' (TPs) were developed that might prevail over FTN poor solubility besides TPs might abstain the obstacles of mucus invasion. TPs were assembled by thin-film hydration then optimized by Box Behnken design utilizing terpenes ratio (X1), sodium deoxycholate amount (X2), and ethanol concentration (X3) as independent variable, whereas their impact was inspected for entrapment efficiency (Y1), particle size (Y2), and polydispersity index (Y3). Design Expert® was bestowed to select the optimal TP for more studies. The optimal TP had entrapment efficiency of 62.18 ± 1.39%, particle size of 310.00 ± 8.16 nm, polydispersity index of 0.20 ± 0.10, and zeta potential of -10.19 ± 0.2.00 mV. Elasticity results were greater in the optimal TP related to classical bilosomes. Further, ex vivo permeation illustrated tremendous permeability from the optimal TP correlated to classical bilosomes, and FTN suspension. Besides, in vivo assessment displayed significant inhibition effect in rats from FTN-TPs gel compared to FTN gel. The antifungal potency with undermost histopathological variation was detected in rats treated with FTN-TPs gel. Overall, the acquired findings verified the potency of utilizing FTN-TPs gel for treatment of vaginal candidiasis.

Detection and phylogenetic analysis of Human bocavirus in children diagnosed with acute respiratory tract infection.

Introduction. Human bocavirus (HBoV) is a recently discovered parvovirus; it has been shown to be a common cause of respiratory infections and gastroenteritis in children. Since its identification, HBoV has been detected worldwide in nasopharyngeal swabs, serum and stool samples particularly those obtained from young children suffering from respiratory or gastrointestinal tract infections.Aim. The aim of this work was to determine HBoV prevalence among children with acute respiratory tract infection in Egypt, to detect the most prevalent HBoV genotype and to compare PCR and ELISA as diagnostic techniques for HBoV infection.Methods. Nasopharyngeal swabs and blood samples were obtained within the first day of admission from 75 children diagnosed with acute respiratory tract infection in El-Shatby University Hospital for Children in Alexandria, Egypt from October 2018 to March 2019. Conventional PCR was used to detect HBoV DNA, ELISA was used to detect HBoV IgM antibodies and sequencing of the VP1/2 genes was used for genotyping.Results. Seven (9.3%) of the 75 nasopharyngeal swabs obtained from patients with acute respiratory tract infection were positive for HBoV by PCR, while 5 (6.7 %) of the 75 serum samples were positive for HBoV IgM antibodies using ELISA. The correlation between PCR and ELISA results showed a highly significant association between PCR and ELISA techniques (X 2=52.041, P<0.01) and a highly significant agreement between the two methods (Kappa=81.9 %, P<0.01). Phylogenetic analysis showed that all positive samples were related to the HBoV-1 genotype.Conclusion. Human bocavirus was detected at 9.3 % prevalence in nasopharyngeal swabs obtained from children with acute respiratory tract infection. The HBoV-1 genotype was the only genotype detected, suggesting that a single genetic lineage of HBoV is circulating in Egypt. PCR and ELISA are two reliable methods for detection and diagnosis of HBoV.

Prevalence and Molecular Differentiation of Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, and Entamoeba hartmanni in Egypt.

INTRODUCTION: Entamoeba histolytica-caused amoebiasis is a major cause of mortality worldwide. E. histolytica is morphologically indistinguishable from nonpathogenic species like E. dispar, E. moshkovskii, and E. hartmanni. Polymerase chain reaction (PCR) is the approved method by World Health Organization for diagnosis and differentiation of amoebiasis. This study aims to molecularly differentiate the four Entamoeba spp. using conventional PCR and correlate their prevalence with the patients' sociodemographic data. METHODS: We collected fecal samples of 175 patients with gastrointestinal diseases at Damanhour General Hospital (El-Behira, Egypt). All microscopically positive samples were subjected to conventional PCR. RESULTS: The overall prevalence of Entamoeba infection was 65.7% (115/175). The differentiation by PCR was successfully attained in 102 samples. The species distribution was as follows: E. histolytica (14.7%), E. dispar (61.8%), E. moshkovskii (11.8%); besides, 11.8% of samples revealed mixed infection. Of note, the infection rate was higher in men, patients from rural areas and patients who did not have sanitation facilities for sewage disposal. CONCLUSION: This study demonstrates a high prevalence of infections caused by the nonpathogenic Entamoeba spp. E. dispar, E. moshkovskii, and E. hartmanni along with the pathogenic E. histolytica. Hence, we recommend PCR assay as an accurate, rapid, and effective diagnostic method for the detection and differentiation of the four morphologically indistinguishable Entamoeba spp. in both routine diagnosis of amoebiasis and epidemiological surveys.

Correlation of quorum sensing and virulence factors in Pseudomonas aeruginosa isolates in Egypt.

INTRODUCTION: Pseudomonas aeruginosa is one of the most virulent nosocomial pathogens worldwide. Quorum sensing (QS) regulates the production of pathogenic virulence factors and biofilm formation in P. aeruginosa. The four genes lasR, lasI, rhlR,and rhlI were found to regulate this QS system. In this study, we aimed to assess the correlation between these four genes and QS-dependent virulence factors and to detect the inhibitory effect of clove oil on QS. METHODOLOGY: Fifty P. aeruginosa clinical isolates were collected. Susceptibility to different antibiotics was tested. Virulence factors including biofilm formation, pyocyanin production, and twitching motility were phenotypically detected. QS genes were amplified by polymerase chain reaction (PCR), and one strain subsequently underwent sequencing. The inhibitory effect of clove oil on virulence factors was also tested. RESULTS: A positive correlation was found between biofilm formation and the presence of lasR and rhlI genes. Twitching motility was positively correlated with the presence of lasR, lasI, and rhlI genes. On the other hand, no correlation was found between pyocyanin production and any of the studied genes. Only one isolate amplified all the tested QS gene primers, but it did not express any of the tested virulence factors phenotypically. Sequence analyses of this isolate showed that the four genes had point mutations. CONCLUSIONS: Results emphasize the importance of QS in P. aeruginosa virulence; however, QS-deficient clinical isolates occur and are still capable of causing clinical infections in humans. Also, clove oil has an obvious inhibitory effect on QS, which should be clinically exploited.