Analysis of CDR1 and MDR1 Gene Expression and ERG11 Substitutions in Clinical Candida tropicalis Isolates from Alexandria, Egypt.

INTRODUCTION: Candida tropicalis is a common non-albicans Candida (NAC) species that causes numerous fungal infections. Increasing antifungal resistance to azoles in NAC is becoming a major health problem worldwide; however, in Egypt, almost no data is available regarding fluconazole resistance mechanisms in C. tropicalis. The current study aims to investigate two possible important molecular mechanisms involved in fluconazole resistance in C. tropicalis isolates. MATERIALS: Fifty-four clinical C. tropicalis isolates were included. Identification and antifungal susceptibility profiles of the isolates were carried out using the VITEK 2 compact system. The molecular investigation of fluconazole resistance included the expression of the CDR1 and MDR1 genes by quantitative real-time RT-PCR as well as the sequence analysis of the ERG11 gene. RESULTS: Antifungal susceptibility testing identified 30 fluconazole-non-susceptible isolates. Statistically, CDR1 gene expression in fluconazole-non-susceptible isolates was significantly higher than that in fluconazole-susceptible isolates, with MDR1 gene expression levels that were similar in both non-susceptible and susceptible isolates. Sequence analysis of the ERG11 gene of 26 fluconazole-resistant isolates identified two missense mutations: A395T (Y132F) and G1390A (G464S). CONCLUSIONS: This study has highlighted the role of overexpression of the CDR1 gene and ERG11 gene mutations in fluconazole non-susceptibility. Further studies in Egypt are required to investigate other possible molecular mechanisms involved in azole resistance.

Loss of p53 Expression in Gastric Epithelial Cells of Helicobacter pylori-Infected Jordanian Patients.

Background: Around half of the global population is chronically infected with the stomach bacterium Helicobacter pylori, making it one of the most common chronic infections worldwide. H. pylori induces the production of reactive oxygen species, DNA damage, and accelerates the degradation of the tumor suppressor protein p53, which may lead to cancer development. In this study, we investigated the relationship between H. pylori infection and the expression of p53 in gastric mucosa in a group of patients from Jordan. Methods: In this retrospective case-control study, the epithelium of gastric glands in subjects chronically infected with H. pylori was examined for the expression of p53. Paraffin-embedded gastric biopsy samples from the archives for 50 Jordanian patients diagnosed with chronic H. pylori infection and 25 samples free of H. pylori infection and any other gastric abnormalities were selected. Samples were analyzed for the presence of H. pylori as well as p53 expression levels in the mucosa and submucosa by immunohistochemical analyses and Western blotting. Results: H. pylori was detected in the gastric tissues of infected individuals (n = 50); whereas, no H. pylori infection was detected in uninfected healthy individuals (n = 25) using immunohistochemistry. In contrast to the noninfected samples of gastric mucosa, no nuclear p53 expression was detected in the infected samples using immunohistochemistry. In addition, the levels of p53 in H. pylori-positive samples detected by Western blotting were significantly lower than those in the negative individuals. Conclusion: Our data reveal that p53 protein expression decreased in gastric mucosa of patients infected with H. pylori. The loss of this tumor suppressor may play a role in the increased risk for tumor initiation associated with H. pylori carriage.

Virulence Factors and Antifungal Susceptibility Profile of C. tropicalis Isolated from Various Clinical Specimens in Alexandria, Egypt.

BACKGROUND: The incidence of candidiasis caused by non-albicans Candida (NAC) species is increasing. Candida tropicalis has emerged as one of the most important NAC species. This study aims to examine the antifungal susceptibility profile and some virulence factors of C. tropicalis isolated from various clinical specimens. METHODS: A total of 71 C. tropicalis isolates from various clinical specimens (69.01%, 18.31%, 9.86%, and 2.82% of isolates were collected from urine, respiratory samples, blood, and skin and soft tissue infections, respectively) from ICU patients in Alexandria, Egypt. The isolates were identified at species level by CHROMagar Candida and VITEK 2 compact system. Furthermore, the antifungal susceptibility was determined using the VITEK 2 system AST-YS07 card containing different antifungals. Hemolysin, phospholipase, and proteinase activity and biofilm formation were also tested as virulence factors. RESULTS: Only 30 isolates (42.25%) were non-susceptible (MIC ≥ 4 µg/mL) to fluconazole, of which 28 isolates showed non-susceptibility (MIC ≥ 0.25 µg/mL) to voriconazole. All isolates showed both hemolysin and proteinase activities, while only 9 isolates (12.68%) showed phospholipase production and 70 isolates (98.59%) demonstrated biofilm formation. Strong biofilm production was observed among the blood culture isolates (85.71%), followed by the respiratory and urinary isolates (61.54% and 46.94%, respectively). CONCLUSIONS: This study sought to provide useful data on the antifungal susceptibility of C. tropicalis isolates from ICU patients suffering from invasive infections with an increased trend towards elevated MICs levels of both fluconazole and voriconazole. Due to the high incidence of systemic candidiasis and antifungal resistance, C. tropicalis is emerging as a serious root of infections. Therefore, early and accurate identification of Candida species along with susceptibility testing is of utmost importance.

Impact of Interleukin 28B and ICAM-1 Genetic Polymorphisms on Response to Direct Antiviral Treatment Among HCV Infected Patients.

BACKGROUND: Single nucleotide polymorphisms (SNPs) of IL-28B and/or ICAM-1 could have a role in expecting a response from HCV infected patients to direct antiviral agents (DAAs). OBJECTIVE: The aim of the current study was to investigate the impact of IL-28B rs12979860 and rs8099917, and, ICAM-1 rs281437 SNPs on response to treatment with sofosbuvir + Daclatsvir ± Ribavirin, among HCV-infected Egyptian patients. METHODS: Whole blood genomic DNA was extracted from 120 participants (80 HCV-infected patients and 40 healthy volunteers). HCV-infected patients were subdivided into responders and nonresponders to DAAs. Liver function testing, anti-HCV antibodies, HCV-RNA viral load and HCV genotyping were performed. IL-28B and ICAM-1 SNPs were evaluated by real-time PCR. RESULTS: ALT and AST levels were significantly higher among non-responder HCV infected patients (P = 0.001*). 90% of the patients had HCV genotype 4a and the remaining 10% had 4l genotype. Allelic discrimination revealed that IL-28B rs12979860 T, IL-28B rs809917 T and ICAM-1 rs281437 C alleles were more frequent among HCV-infected patients (responders or non-responders) than controls. However, IL-28B rs8099917 G allele was more frequent among healthy controls. Regarding the response to DAAs treatment, HCV-infected patients with IL-28B rs8099917 GG genotype showed a significantly earlier viral response compared to those carrying TT alleles. ICAM-1 rs281437 CT alleles were non significantly more frequent among responders. However, IL-28B rs12979860 alleles did not show any difference. CONCLUSION: Genotyping of IL-28B rs8099917 is a useful independent tool for expecting a response of Egyptian HCV-infected patients to DAAs.

Human papillomavirus as an independent risk factor of invasive cervical and endometrial carcinomas in Jordan.

BACKGROUND: Endometrial and cervical carcinomas are the most common gynecologic malignancies in Western world and many countries. The human papillomavirus (HPV) high-risk genotypes are associated with cervical carcinoma (CC). Chlamydia trachomatis (C. trachomatis), the most common sexually transmitted bacterial infection worldwide, considered a cofactor for HPV infection and CC. Information on HPV infection rate and type distribution among Jordanian women having CC is currently limited and unavailable among those with endometrial carcinoma. Therefore, the present study aimed to provide an updated estimate on HPV infection rate and its high-risk genotypes' distribution among Jordanian women by comparing data from invasive cervical carcinoma (ICC) to normal cervical tissues. Similarly, assessment of HPV infection rate was extended to the endometrial tissues. C. trachomatis infection was investigated as well to explore its possibility as HPV cofactor for induction of such carcinomas. METHODS: Total DNA was extracted from 144 formaldehyde-fixed paraffin-embedded cervical and endometrial tissue, equally divided between age-matched control and carcinoma cases. Polymerase chain reaction (PCR) was used for general detection of HPV-DNA, high risk HPV-16 and 18 genotypes and C. trachomatis DNA using specific primers. RESULTS: HPV infection was detected in 91.7% and 61.1% of cervical cancer patients and controls, respectively. Likewise, it was higher among cases (47.2%) than controls (13.8%) in endometrial biopsies. Significantly higher HPV infection rates were found among ICC and endometrial control biopsies of women >50 years. Out of 33 HPV positive ICC cases, single HPV-16 infections were detected in 69.7% compared to HPV-18 (15.2%), while HPV-16/18 co-infections were only found in three (9%) samples. C. trachomatis was not detected in all studied groups. CONCLUSION: The present study has successfully provided an updated estimate on HPV infection rate among Jordanian women with and without ICC and endometrial carcinoma. In addition, a lack of co-infection was observed between HPV and C. trachomatis in both cancer types.

Occult hepatitis B virus infection among chronic hemodialysis patients in Alexandria, Egypt.

The prevalence of end-stage renal disease has increased dramatically in developing countries. Hepatitis B virus (HBV) infection is a global health problem that represents a significant co-morbidity event that has led to outbreaks of hepatitis B. There are inadequate data concerning occult HBV infection among Egyptian chronic hemodialysis patients. This study aimed to detect occult HBV infection among chronic hemodialysis patients in Alexandria, Egypt. A cross-sectional study was performed on 100 patients with end-stage renal disease that received maintenance hemodialysis and had tested negative for HBV surface antigen. Blood samples were collected before the initiation of hemodialysis. Sera were tested for hepatitis C virus (HCV) and hepatitis B core (HBc) antibodies using ELISA, and HBV DNA was detected by SYBR Green real-time PCR using specific primers for the s and c genes and by nested PCR using pol gene-specific primers. The serum activity of alanine and aspartate aminotransferase (ALT and AST) were also measured. Anti-HCV and anti-HBc antibodies were detected in 34% and 48% of patients, respectively, and 70.6% of anti-HCV positive patients were also positive for anti-HBc antibodies. This association was statistically significant (p=0.001). HBV DNA was detected in 32% of the hemodialysis patients. A significant association was determined between the presence of HBV DNA and anti-HCV positivity (p=0.021). Aminotransferases were elevated in 21% of the studied patients, more often in patients with positive anti-HCV profiles than in patients negative for anti-HCV (p<0.05). In conclusion, the serological markers of HBV infection should be verified with molecular tests to investigate possible occult infections, especially among anti-HBc-positive hemodialysis patients, to improve our understanding of their clinical, laboratory, and epidemiological characteristics.

Relation of serum insulin-like growth factor-1 (IGF-1) levels with hepatitis C virus infection and insulin resistance.

The prospect of the growing worldwide epidemic of hepatitis C virus (HCV) infection and type 2 diabetes mellitus certainly merits attention toward their controversial relationship. Insulin-like growth factor-1 (IGF-1) plays an important role in glucose homeostasis. This study is a cross-sectional study considered as an initial investigation aimed to evaluate the effect of HCV infection on serum IGF-1, as well as to find out whether IGF-1 has a role in development of insulin resistance (IR) in HCV infection. A total of 45 subjects divided into 3 groups were included in the study: chronic HCV-infected patients (15 patients), chronic HCV-infected diabetic patients (15 patients), and diabetic patients without HCV infection (15 patients), along with 15 healthy controls. HCV RNA was quantified using real-time polymerase chain reaction (PCR). Serum IGF-1 levels were measured by enzyme-linked immunosorbent assay (ELISA). Homeostasis model assessment of insulin resistance [HOMA-IR], insulin sensitivity [HOMA-S], and ß-cell function [HOMA-ß] were determined by previously validated mathematic indexes. Fasting blood glucose, insulin levels, and liver parameters including alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined. IGF-1 levels were significantly lower in the 3 patient groups compared with controls (P = 0.001). The HCV group demonstrated high HOMA-IR and HOMA-ß with a positive correlation between HOMA-IR and either HOMA-ß or fasting insulin (P < 0.001). In addition, a negative correlation was found between IGF-1 levels and both AST and ALT, and HOMA-IR was correlated positively with AST activity (P < 0.05). In HCV patients with detectable viremia, IGF-1 levels were correlated negatively with HOMA-ß (P < 0.01) and with HOMA-IR. However, this correlation did not reach statistical significance (P = 0.074). No significant correlation was found between HCV viral load and the studied parameters. In conclusion, low IGF-I levels might have a role in IR among HCV viremic patients.

Potential Co-prevalence of Plasmid-Mediated Quinolone Resistance Determinant qepA and 16S rRNA Methylase rmtB among E.coli Clinical Isolates from Alexandria- Egypt.

BACKGROUND: Fluoroquinolone-resistant Gram-negative pathogens have been increasingly reported from most regions of the world over the last decade. A new plasmid-mediated fluoroquinolone efflux pump gene (qepA) is known to be associated with the 16S rRNA methylase gene (rmtB) that confers resistance to aminoglycosides. AIM: To investigate the potential co-prevalence of qepA and rmtB genes in Escherichia coli (E. coli) clinical isolates collected from Egyptian medical facilities. MATERIAL AND METHODS: A total of 225 non-duplicate E. coli isolates were collected between 2008 and 2009 from two hospitals in Alexandria. Isolates were initially tested for their antibiotic susceptibility by disc diffusion method. Isolates exhibited quinolone and aminoglycosides co-resistance profile were screened for the presence of qepA and rmtB genes. The effect of efflux pump inhibitor, phenylarginine-beta-naphthylamide (PAßN) on the minimum inhibitory concentration (MIC) of ciprofloxacin, levofloxacin and gentamicin against these strains was tested and log activity index was calculated. Using checkerboard titration method, the combinations of gentamicin with ciprofloxacin against the strains harboring qepA and rmtB genes were tested and the fractional inhibitory concentrations (FIC) were calculated. RESULTS: Forty-five E. coli isolates exhibited quinolone and aminoglycosides co-resistance profile. Of them, two E. coli isolates were positive for qepA, and three harbored rmtB genes. No association between both genes was detected. The calculated log activity index revealed a reduction in MIC of the fluoroquinolones with PAßN but not of gentamicin. FIC calculated here for gentamicin/ciprofloxacin combinations reflected either antagonism or indifference against the strains harboring qepA and rmtB genes. CONCLUSION AND RECOMMENDATIONS: qepA as well as rmtB genes-carrying E. coli strains could become a greater nosocomial infection problem with appropriate foci of selective pressure. Therefore, public health support for active surveillance for plasmid mediated fluoroquinolones, aminoglycosides resistance determinants among clinical E. coli isolates should be encouraged. In addition, the effect of efflux pumps needs to be considered in the design of future antibiotics as their synergistic role may pave the way to novel combination therapies that could be used against these strains.

Influence of HCV infection on insulin-like growth factor 1 and proinflammatory cytokines: association with risk for growth hormone resistance development.

Communications between the endocrine, immune systems and the liver have been postulated. The liver is the central organ in growth hormone/insulin-like growth factor (GH-IGF) axis. Infection with hepatitis C virus (HCV) can lead to liver problems. Although proinflammatory cytokines are an integral part of inflammation in chronic liver diseases, their involvement in mediating hepatic GH resistance during HCV infection remains to be elucidated. To address this issue, our study aimed at evaluating the influence of HCV infection on serum profile of IGF-1, TNF-alpha and IL-6 to assess their possible relation to hepatic dysfunction and GH resistance development. Twenty-five chronic HCV patients were studied together with 15 healthy control subjects. Serum concentration of IGF-1, TNF-alpha and IL-6 was determined by ELISA. HCV viral load was assessed by Real-time polymerase chain reaction using TaqMan probe technology. Basal serum GH levels were determined by a chemiluminescence assay and serum aminotransferases' activities were also measured. TNF-alpha and IL-6 demonstrated higher serum levels, while IGF-1 levels were significantly lower in HCV patients compared to healthy controls. A statistically significant positive correlation was observed between GH and IL-6 levels (P<0.05), a similar trend was found between GH levels, GH/IGF-1 ratio and AST/ALT ratio (P<0.01, P<0.01, respectively). A significant negative correlation was observed between HCV viral load and GH levels (P<0.05). The progressive increase in HCV viral load matches the decrease in circulating IGF-1 levels but without reaching statistical significance. We conclude that the GH insensitivity could be induced by HCV infection and mediated by proinflammatory cytokines through their possible role in blunting the hepatic response to GH. This crosstalk between proinflammatory cytokines and GH-IGF-1 axis could be responsible for triggering impaired glucose metabolism and diabetes later on in chronic HCV infection.

Influence of hepatitis C virus infection on circulating levels of sICAM-1 and VEGF in patients with hepatitis C and hepatocellular carcinoma (HCC) and their role in enhancing detection of HCC.

Infection with hepatitis C virus (HCV) is characterized by inflammatory liver damage and a long viral persistence associated with an increased risk of developing hepatocellular carcinoma (HCC). Intercellular adhesion molecule-1 (ICAM-1) plays a key role during liver inflammation and also expressed in HCC. Its cellular expression is associated with the release of soluble form (sICAM-1) in the peripheral blood. The process of angiogenesis plays a critical role in liver damage-associated HCV infection and in tumor growth and metastasis. Vascular Endothelial Growth Factor (VEGF) is an important angiogenic factor regulating tumor angiogenesis. This study aimed at investigating the influence of HCV infection on serum profile of sICAM-1 and VEGF in patients with hepatitis C and HCC and their diagnostic value as useful markers reflecting progressive liver damage and development of HCC. Serum levels of sICAM-1 and VEGF were determined in the serum of fifteen HCV infected patients, fifteen HCV-positive patients with superimposed HCC as well as ten healthy control subjects by enzyme linked immunosorbent assay. HCV RNA copy numbers were analyzed by Real-time polymerase chain reaction using TaqMan probe technology. Alpha-fetoprotein levels and serum aminotransferases activities were also measured. The group of patients with hepatitis C and superimposed HCC had significantly higher sICAM-1 and VEGF values than HCV infected patients (1178.113 +/- 631.87 vs. 313.67 +/- 82.72 & 320.88 +/- 117.99 vs. 132.45 +/- 91.56, p < 0.001 respectively). In comparison to healthy subjects, HCV infected patients showed dramatically elevated serum levels of VEGF (132.45 +/- 91.56 vs. 7.76 +/- 7.41, p < 0.001). On the other hand, sICAM-1 levels were elevated in patients with HCV as compared with healthy controls, but this did not reach statistical significance (313.67 +/- 82.72 vs. 230.3 +/- 47.4, p > 0.05). A highly significant correlation was found between VEGF and sICAM-1 levels in all patients (r = 0.731, p < 0.001) also between VEGF, sICAM-1 and AFP (r = 0.473, p < 0.001, r = 0.690, p < 0.001, respectively) as well as between sICAM-1 and AST activities (r = 0.367, p < 0.05). A weak correlation was found between the level of viremia and VEGF, sICAM-1 levels, yet this did not reach statistical significance (r = 0.312, p = 0.09 & r = 0.228, p > 0.05 respectively). The sensitivity of HCC detection using AFP alone was 93.3%. It yielded 100% detection sensitivity when combined with sICAM-1 and/or VEGF with diagnostic accuracy reaching 96.67%. In conclusion, HCV infection and the development of HCC on top greatly affect the serum profile of VEGF and sICAM-1. VEGF as it stimulates endothelial cell growth, it could modulate the expression of sICAM-1 and both could be considered as convenient markers of progressive liver damage, endothelial activation and therefore could improve detection and management of HCC.