Utility of comet assay of DNA damage in the detection of malignant transformation of chronic liver cirrhosis.

Early detection of hepatocellular carcinoma (HCC) remains a great challenge in laboratory medicine. This study aimed to assess the ability to use the degree of DNA damage (using the comet assay) for the early detection of malignant transformation of liver cirrhosis (LC) to HCC. We used alkaline comet assay for measuring DNA damage in peripheral blood lymphocytes in 50 patients with chronic LC and 50 patients with HCC. Fifty healthy individuals served as a control group. We compared the results of comet assay parameters with alpha fetoprotein as a relevant traditional marker. The HCC group was associated with a significantly higher tail intensity (p=.004), tail moment (p=.016), total area (p=.003), total intensity (p=.010), width (p=.005), and a significantly lower head intensity (.004) when compared to the LC group. Good areas under the curve (AUCs) were found for total area (0.890), head intensity (0.880) and tail intensity (0.880), making it useful for discrimination between HCC and LC groups. Lower head intensity, higher tail intensity, tail moment, total area and width were found to be independent risk factors for HCC on top of LC. Measuring DNA damage using the Alkaline comet assay technique can be considered a sensitive and reliable diagnostic tool for early neoplastic transformation of advanced LC.

Circulating Cell-Free DNA and DNA Integrity as Molecular Diagnostic Tools in Hepatocellular Carcinoma.

OBJECTIVES: We assessed the ability to use circulating cell-free DNA (cfDNA) and the DNA integrity index (DNAII) to detect the transition from liver cirrhosis (LC) to hepatocellular carcinoma (HCC). METHODS: Circulating cfDNA and DNAII were measured in 50 patients with advanced LC and 50 patients with HCC who were followed for 1 month after transarterial chemoembolization (TACE). Fifty healthy participants served as a control group. Real-time quantitative polymerase chain reaction (PCR) was used to measure circulating cfDNA concentration, and Alu-PCR was used to measure the concentration of Alu repeats, both short fragments (115 base pairs [bp]) and long fragments (247 bp). We compared liquid biopsy results with the relevant traditional markers. RESULTS: The HCC group showed significantly higher circulating cfDNA concentrations and DNAII values compared with the LC and control groups. No significant differences were found in circulating cfDNA concentrations and DNAII values between the LC and control groups. Circulating cfDNA concentrations decreased significantly after treatment (TACE); areas under the curve of circulating cfDNA concentration and DNAII values were significantly better than those of ɑ-fetoprotein and vascular endothelial growth factor in discriminating between LC and HCC. CONCLUSIONS: The combined use of DNAII with proteins induced by vitamin K absence or antagonist showed better diagnostic performance in HCC. Circulating cfDNA could have a potential role in monitoring HCC treatment.

Association between Toll-Like Receptor 3 (TLR3) rs3775290, TLR7 rs179008, TLR9 rs352140 and Chronic HCV.

BACKGROUND: Inconsistent results were reported on the association of TLRs polymorphisms with the risk of HCV infection and HCV-related diseases. OBJECTIVE: to assess the relation between TLR3 rs3775290, TLR7 rs17900 and TLR9 rs352140 SNPs and chronic HCV in the Egyptian cohort and to study their relation to interferon response. METHODS: TLR3 rs3775290, TLR7 rs179008 and TLR9 rs352140 gene polymorphisms were typed by RFLP for 100 patients with chronic HCV and 25 with HCC in addition to 100 healthy controls. RESULTS: A significant higher frequency has been found for the CT genotype of TLR3 rs3775290 in chronic HCV infection (p < 0.001) and CC genotype and the combined genotype CC-AT-GA ♀ in controls (p < 0.001). Non-significant associations have been found for studied SNPs and HCC and response to interferon and also the viral load or the degree of fibrosis, however, the higher HCV viral load and the higher grade of fibrosis were associated with treatment failure (p < 0.001). CONCLUSION: The heterozygous CT genotype of TLR3 rs3775290 may be a susceptibility risk factor for chronic HCV infection and the homozygous CC and the combined CC-AT-GA ♀ genotypes may be protective. The HCV viral load and the grades of liver fibrosis could be considered a risk factor for interferon treatment failure. It seems that the studied SNPs have no role in HCC development or failure of treatment. However, the small sample size is a limiting factor of the present study when interpreting the negative associations and that the current used cohort does not permit such conclusion. ABBREVIATIONS: cHCV=chronic Hepatitis C virus, HCC=hepatocellular carcinoma, TLR=Toll like Receptor, RFLP=Restriction Fragment Length Polymorphism, SNP=Single Nucleotide Polymorphism, IFN-α= interferon alpha.

Evaluation of the role of Notch1 expression in hepatic carcinogenesis with clinico-pathological correlation.

The role of Notch pathway in hepatocarcinogenesis is unclear with conflicting results reported from different researchers. This study aimed to investigate the exact role of Notch1 in hepatocarcinogenesis and its influence on survival and to determine the possibility of it being a target therapy. Differential immunohistochemical expression of Notch1 in 100 cases of hepatocellular carcinoma (HCC) and adjacent non-neoplastic liver tissue was performed. The results showed that expression of Notch1 was significantly higher in the non-neoplastic hepatic tissues than in HCC tissues (p < 0.001), but there was no significant difference in Notch1 expression between cirrhotic and non-cirrhotic liver tissue (p = 0.197). Notch1 expression was higher in low grade than in high grade HCC (p = 0.036). Notch1 expression showed reverse correlation with mitotic count (p = 0.008), and necrosis (p = 0.005). The disease free survival was shorter in patients displaying low levels of Notch1 expression (p = 0.045). The overall survival showed no significant difference between high and low levels of Notch1 expression; however, it was somewhat longer in patients with high Notch1 expression (p = 0.220). In conclusion, the tumour suppressor role of Notch1 was supported and the use of Notch1 agonists may have a role in improving the prognosis of HCC.

Assessment of auditory functions in chronic hepatitis C patients treated by sofosbuvir.

OBJECTIVE: Evaluating the auditory function in patients with chronic hepatitis C treated with sofosbuvir and ribavirin. METHODS: This study involved 80 patients with chronic hepatitis C who agreed to receive sofosbuvir and ribavirin. All participants were subjected to baseline otological and audiological assessment just before treatment. The audiological assessment included standard pure tone audiometry, extended high-frequency audiometry, immitancemetry and otoacoustic emissions (OAEs) (transient and distortion product). According to baseline hearing threshold measurements, the study population was divided into 2 groups. Group 1 included 42 patients with normal hearing sensitivity (250-8000 Hz), and Group 2 included 38 patients with sensorineural hearing loss. After 24 weeks of therapy, otological and audiological assessments were repeated and compared between the two groups and before and after therapy. RESULTS: Post-treatment hearing threshold evaluation showed no significant difference from pretreatment evaluation at all tested frequencies. There was no statistically significant difference between pre and post-treatment otoacoustic emissions results. CONCLUSION: Therapy with sofosbuvir and ribavirin in chronic hepatitis C has no noticeable effects on cochlear functions.

Do we need to screen for de-novo diabetes mellitus in chronic hepatitis C patients after a sustained virological response?

BACKGROUND AND AIM: There are millions of chronic hepatitis C (CHC) virus-infected patients who have been treated with a combination therapy (interferon and ribavirin) and have achieved a virological response (SVR) worldwide. The aim of this study is to evaluate the risk factors for de-novo diabetes mellitus in CHC patients treated with combination therapy (interferon and ribavirin) and have achieved an SVR. PATIENTS AND METHODS: A total of 214 nondiabetic CHC patients with SVR and baseline homeostasis model assessment (HOMA) less than or equal to 2 were divided into group A, which included 108 patients with a BMI less than 25, and group B, which included 106 patients with a BMI of at least 25 and less than 30. HOMA insulin resistance (IR) and BMI were measured at the baseline, at achievement of an SVR, and 1 year after achievement of an SVR. Leptin levels were assessed at baseline and 1 year after achievement of an SVR in patients with increased BMI. RESULTS: One year after SVR, 36 (33.33%) patients from group A developed increasing BMI with no significant changes in HOMA versus that at SVR (P=0.53), but showed a significant reduction versus baseline HOMA (P=0.02). In group B, 68 (64.1%) patients showed increased BMI of at least 25, with a significant increase in HOMA versus that at SVR (P=0.02), and with no significant reduction versus baseline HOMA (P=0.44). In group B, serum leptin showed a significant reduction 12 months after achievement of an SVR versus baseline in patients with increased BMI. Six patients from group B with increased BMI after 1 year developed de-novo IR and type two diabetes mellitus. CONCLUSION: In nondiabetic CHC patients with SVR and baseline BMI of at least 25, the post-SVR increase in BMI predisposed to an increase in HOMA-IR and could be considered a predisposing factor for diabetes mellitus.

Genetic Polymorphisms of Cytochrome P4501A1 (CYP1A1) and Glutathione S-Transferase P1 (GSTP1) and Risk of Hepatocellular Carcinoma Among Chronic Hepatitis C Patients in Egypt.

Cytochrome P450 1A1 (CYP1A1) and Glutathione S-transferase P1 (GSTP1) genes are involved in the metabolism of many carcinogens. Polymorphisms in these genes with altered enzyme activity have been reported. The present study evaluated the synergistic effect between CYP1A1 and GSTP1 gene polymorphisms and smoking on development of HCV-related liver disease and hepatocellular carcinoma (HCC). The patients group comprised 40 patients with HCC and 40 patients with liver cirrhosis. The control group comprised 40 healthy subjects having no history of malignancy. The genetic polymorphisms were studied using polymerase chain reaction restriction fragment length polymorphism (PCR RFLP) technique on blood samples. The number of current or former smoker among HCC and cirrhotic patients as well as the median Pack/year of cigarette smoked were significantly higher in HCC and liver cirrhotic patients than in control group. Subjects with CYP1A1 gene variants (m1 and m3) had no significant risk to develop cirrhosis or HCC compared to control group. Individuals carrying the Ile/Val genotype of GSTP1 had a significant increased risk of HCC (OR of 2.2, 95 % CI 1.143-4.261) and had larger tumor size. No significant risk was observed on combining both genes variants or on combining smoking with variants of both genes. In conclusion, the GSTP1 Ile/Val genotype and Val allele are associated with an increased risk of HCC. CYP1A1 and GSTP1 genes variants interaction did not increase the risk of HCC.

Toxoplasma gondii infection among chronic hepatitis C patients: a case-control study.

OBJECTIVE: To determine the detection rate of anti-Toxoplasma gondii (T. gondii) IgG and IgM in chronic HCV patients attending the Department of Tropical Medicine Mansoura University hospital in Egypt. METHODS: This study included 120 adult chronic HCV patients, 81 decompensate cirrhosis (late-stage) and 39 chronic HCV non cirrhotic patients (early-stage) and 40 healthy blood donors as controls. Serum samples were examined for anti-Toxoplasma IgM and anti-Toxoplasma IgG antibodies by ELISA. Real-time RT-polymerase chain reaction assay was done for quantitation of hepatitis C virus. RESULTS: Anti-T. gondii IgG antibodies were detected in 75 (92.6%) of 81 late-stage cirrhotic patients, 30 (76.9%) of the 39 chronic HCV non cirrhotic patients (early-stage) and in 6 (15%) of 40 controls with statistically significant difference (P<0.001). Anti-T. gondii IgM antibodies were found in 11 (13.6%) in late stage patients, 5 (12.8%) in early stage and in 3 (7.5%) of controls with no statistical significant difference (P=0.610). There was no correlation between stage of fibrosis and IgM or IgG antibodies positivity in our studied groups (P=0.526). High IgG levels significantly correlated with high viral load (P=0.026). CONCLUSIONS: Our findings suggest that the serious opportunistic T. gondii infection represent a potential significant risk for chronic HCV patients. So, toxoplasmosis should be considered in their investigations and follow-up.

Human leukocyte antigen class I alleles can predict response to pegylated interferon/ribavirin therapy in chronic hepatitis C Egyptian patients.

BACKGROUND: Racial differences and broad spectrum response to anti-hepatitis C (anti-HCV) therapy suggest a possible role for host genetic diversity in treatment outcomes. We aim to determine the association and predictive value of certain human leukocyte antigen (HLA) class I alleles with either susceptibility to viral clearance or persistence following pegylated interferon (Peg-IFN) plus ribavirin therapy in chronic hepatitis C (HCV) genotype 4 patients in Egypt.  METHODS: This study included 200 unrelated chronic HCV patients who received Peg-IFN plus ribavirin therapy [112 patients with sustained virological response (SVR) and 88 non-responders (NR)]. Serological testing of HLA class I antigens (HLA-A and HLA-B alleles) were performed by standard complement-dependent microlymphocytotoxicity assay.  RESULTS: The frequency of HLA-A01 was significantly higher in SVR than in NR cases [OR: 0.51; 95% CI: 0.27-0.981; P = 0.042], while the frequency of alleles B38 (P = 0.011), B40 (P < 0.001) and B41 (P < 0.001) was significantly higher in NR cases (OR/95% CI: 7.05/(1.39-18.01), 10.31/3.14-36.1 . On logistic regression analysis, presence of the HLA-A01 allele was associated with SVR  (OR: 0.50; 95% CI: 0.28-0.89; P = 0.02) and HLA-B38 can predict non response to therapy (OR: 7.92; 95% CI: 1.67-37.54; P = 0.009) with an overall accuracy of 60%.Severe fibrosis (OR: 3.035; 95% CI: 1.521-6.091; P = 0.002), high viremia (OR: 2.69; 95% CI: 1.11-6.53; P = 0.005) and steatosis (OR: 2.1; 95% CI: 1.002-3.90; P = 0.041) predicted no response with an overall accuracy of 81.8%.  CONCLUSION: HLA-A01 and HLA-B38 alleles are associated with and may have a role in the outcome of response to Peg-IFN plus ribavirin therapy in Egyptian patients diagnosed with chronic HCV infection. The use of immunologic markers to predict the outcome of treatment may help pharmacogenetic personalization of treatment for HCV infection.

Colorectal schistosomiasis: Is it still endemic in delta Egypt, early in the third millennium?

BACKGROUND: Several governmental efforts have been exerted toward controlling schistosomiasis during the last decades in Egypt. This work was designed to study the prevalence of colorectal schistosomiasis in patients with different gastrointestinal symptoms. MATERIALS AND METHODS: Patients presented to the gastroenterology unit with different gastrointestinal symptoms were endoscopically examined, where three to six tiny biopsies were taken from those with visible, suspected schistosomal lesions for histopathological examination and two additional rectal biopsies were taken from the apparently normal colonic mucosa. Form each patient, at least three stool samples were examined by the formal-ether concentration method for schistosoma ova. RESULTS: Colonic abnormalities were detected in 510 out of 984 patients presented with different gut symptoms. Schistosoma mansoni was detected in 205 patients (180 males, 25 females) with an age range (18-65years). Six patients only had schistosomal polyps and excised successfully by snare polypectomy. The squash technique established the diagnosis of schistosomiasis in all endoscopically normal 118 (50.75%) cases by demonstrating the schistosomiasis ova and their associated histopathological findings showed no or minimal reaction in 96 (46.82%) cases and variable degrees of submucosal granulomata in the remaining cases. Stool examination detected the schistosomiasis ova in 25 (9.83%) patients only of the biopsy-positive cases. CONCLUSIONS: Our data revealed that despite governmental efforts, the prevalence of colorectal schistosomiasis (20.83%) is significant among patients with gut symptoms. Gaps in health care services should be detected and filled appropriately.

Clinical significance of antibody to hepatitis B core antigen in multitransfused hemodialysis patients.

BACKGROUND: In spite of the progress made in the prevention of transfusion transmitted infections over the last few years, transmission of HBV infection through transfusion of HBsAg negative blood has been documented. OBJECTIVES: To assess the frequency and clinical significance of anti-HBc in multitransfused hemodialysis patients. MATERIALS AND METHODS: One hundred and forty-three hemodialysis patients who had been receiving blood regularly with an average of 39.4 +/- 7.579 months on hemodialysis were enrolled in this study. HBV markers (HBsAg, anti-HBc, anti-HBs) were measured in these patients and in 100 healthy controls by the ELISA technique. The following data were obtained for all patients: socio demographic data, number of blood transfusions and some laboratory investigations. RESULTS: In our patients, anti-HBc was positive in 9%, anti HBs in 7%, coexistant HbsAg/anti-HBc in 2.8% and anti HBc/anti HBs in 18.9%, meanwhile no patients were positive for HBsAg alone. In patients with only positive anti-HBc, the levels of anti-HBc were significantly related to abnormal results of liver function. In patients with positive anti-HBs/anti-HBc (n = 27), 18 patients had abnormal liver function, and 9 patients had normal liver function with no significant difference between them. CONCLUSIONS: This study suggests that hepatitis B prevalence in our multitransfused hemodialysis patients is far in excess of that anticipated on the basis of HBsAg prevalence. Absence of HBsAg in the blood of hemodialyzed patients may not be sufficient to ensure lack of circulating HBV, and isolated positivity of anti-HBc may be a possible indicator of active hepatitis B infection.

Study of some immune aspects in patients with fascioliasis before and after Chommiphora molmol (Mirazid) treatment.

This study was planned to evaluate the in vitro production of IL-1 beta and IL-4 by peripheral blood mononuclear cells (PBMCs) and total IgE in patients with fascioliasis before and 3 months after treatment with purified extract of myrrh from Commiphora molmol tree (Mirazid), to determine the role of these variables in immunopathogenesis of the disease in relation to this new drug. The study was carried out in Departments of Tropical Medicine, Al-Azhar University Hospitals in the period from March 2002 to November 2003. A total of 35 patients with chronic fascioliasis with age range from 9-45 years in addition to 10 healthy subjects with matched age and sex serving as controls were studied. Serum IgE and in vitro IL-1 and IL-4 were estimated by enzyme immuno-assay (ELISA) before and 3 months after therapy. Results revealed significant increase in IL-1 beta in patients before treatment than control (p<0.001) but it decreased significantly after therapy (p<0.001) to reach the control level (p=0.16). In contrast, IL-4 was significantly lower than control before therapy (p=0.04) and increased significantly after treatment (p<0.001) to reach normal levels as control (p=0.59). Total IgE was significantly elevated in patients before treatment (p<0.001) and it did decrease significantly with treatment (p<0.001), although it remained significantly higher than the control level. In conclusion, Mirazid is an effective fasciolicidal drug. IL-1 may be involved in disease immunopathogenesis and the depressed IL-4 may be a phenomenon of parasite immune suppression. Complete decline of total IgE is not an early criterion of cure.