Chronic hepatitis C virus infection impairs natural killer cells-dendritic cells cross-talk: An in vitro culture study.

To examine the cross-talk between NK cells and DCs in hepatitis C virus (HCV) infection, we isolated monocytes and NK cells from 20 chronic HCV patients and 20 healthy controls. Monocytes were used to generate immature DCs which were pulsed with HCV peptides (core, NS3-NS4, and NS5). Four different cocultures were carried out: E1, both DCs and NK cells were from a chronic HCV patient; E2, NK cells from a healthy control cocultured with DCs from a chronic HCV patient; E3, NK cells from a chronic HCV patient cocultured with DCs from a healthy control; and E4, both DCs and NK cells were from a healthy control. Using flow cytometry, we assessed the effect of these different cocultures on levels of maturation markers on DCs and levels of activation/inhibition markers on NK cells. Results showed that peptide-pulsed HCV DCs showed a maturation defect in the form of decreased HLA-DR, decreased CD86, and increased CD83 expression especially when cocultured with HCV NK. This was mainly due to core peptide pulsing and to a lesser extent due to NS5 pulsing, whereas there was no effect with NS3-NS4 pulsing. Alternatively, HCV NK cells upregulated both activation and inhibition markers especially when cocultured with healthy DCs. Compared with E2, E1 resulted in higher apoptosis of both NK cells and DCs with the percentage of NK apoptosis higher than that of DCs. Taken together, the data indicate that HCV infection impairs NK-DC cross-talk which may be a leading cause in viral persistence and chronicity.

Characterization of differential antibody production against hepatitis C virus in different HCV infection status.

BACKGROUND: The Centers for Disease Control and Prevention (CDC) issued an update on hepatitis C virus (HCV) testing approach, in which it omitted the use of recombinant immunoblot assay (RIBA) in the diagnostic algorithm and recommended that future studies are needed to evaluate the performance of HCV testing without RIBA. As Egypt has the highest prevalence of HCV worldwide, we aimed to evaluate the value of RIBA in HCV testing in a high prevalence population. Our objective was to clarify whether enzyme linked immunosorbent assay (ELISA) anti-HCV signal-to-cutoff (S/CO) ratios were able to discriminate true positive from false positive anti-HCV antibody status and to evaluate the role of RIBA in solving this problem which may lead to a redefined strategy for diagnosis of HCV infection. Our second objective was to elucidate the effects of different HCV peptides of both structural and non-structural proteins on the humoral immune response to HCV infection. METHODS: The current study drew results from 167 individuals divided into three groups: Group I: included 77 HCV antibody positive (ELISA) high risk health care workers (HCW), Group II: included 56 presumably uninfected individuals who showed normal liver enzymes, negative HCV RNA and were asymptomatic. Their ELISA HCV antibody S/C ratio ranged from 0.9 to <5. Group III: included 34 patients enrolled from outpatient clinics of Ain Shams Hospital with persistent viral replication, elevated liver enzymes, and chronic HCV related liver disease. All study participants were assessed for the presence of anti-HCV antibodies by 3(rd) generation ELISA which was confirmed by RIBA. RESULTS: Interpreting the results of both ELISA and RIBA together, false positive results were highly significantly increased in HCW when compared with the other two groups. Indeterminate and false negative results were only found in the presumably uninfected group. For differentiated antibody responses by RIBA, chronic HCV cases had the highest frequency of positive antibody response to core peptides while the presumably uninfected group had the lowest. Antibody response to E2 was found less frequently in chronic cases than Core 1, Core 2 and NS3. The specific antibody response to the different HCV peptides showed the same distribution of frequencies in both chronic HCV cases and the presumably uninfected individuals with the chronic cases having the highest frequencies. This distribution was different from the HCW. The most evident difference was the reaction towards NS3 which was the highest antibody producing peptide in chronic HCV and presumably uninfected individuals whereas in HCW Core1 was the highest. CONCLUSION: The HCV antibody immunoblot assay (RIBA) is still necessary for the detection of false positive cases which can occur quite frequently in countries of high prevalence as Egypt. Indeterminate RIBA results indicate a waning antibody response in elderly individuals who recovered from previous or distant HCV infection.

Detection of occult hepatitis C virus among healthy spouses of patients with HCV infection.

The criterion standard for the diagnosis of occult hepatitis C virus (HCV) infection is detection of HCV-RNA in liver cells. However, because of the invasive nature of liver biopsy, other methods have been studied. The present study aimed to identify subjects with occult HCV-4 infection among healthy sexual partners of patients with chronic HCV-4 infection by detecting HCV-RNA in peripheral blood mononuclear cells (PBMCs) using real-time polymerase chain reaction (PCR). Fifty healthy Egyptian spouses of patients with chronic HCV-4 infection were included in this study. Real-time PCR was used to detect HCV-RNA in PBMCs in all the study subjects. The prevalence of occult HCV-4 infection was 4%, and a statistically significant higher prevalence was found among patients with a history of sexually transmitted infection. The results of the present study indicate the importance of intra-spousal transmission of HCV-4 infection, especially in subjects with a history of sexually transmitted infection.

The ABCs of viral hepatitis that define biomarker signatures of acute viral hepatitis.

UNLABELLED: Viral hepatitis is the leading cause of liver disease worldwide and can be caused by several agents, including hepatitis A (HAV), B (HBV), and C (HCV) virus. We employed multiplexed protein immune assays to identify biomarker signatures of viral hepatitis in order to define unique and common responses for three different acute viral infections of the liver. We performed multianalyte profiling, measuring the concentrations of 182 serum proteins obtained from acute HAV- (18), HBV- (18), and HCV-infected (28) individuals, recruited as part of a hospital-based surveillance program in Cairo, Egypt. Virus-specific biomarker signatures were identified and validation was performed using a unique patient population. A core signature of 46 plasma proteins was commonly modulated in all three infections, as compared to healthy controls. Principle component analysis (PCA) revealed a host response based upon 34 proteins, which could distinguish HCV patients from HAV- and HBV-infected individuals or healthy controls. When HAV and HBV groups were compared directly, 34 differentially expressed serum proteins allowed the separation of these two patient groups. A validation study was performed on an additional 111 patients, confirming the relevance of our initial findings, and defining the 17 analytes that reproducibly segregated the patient populations. CONCLUSIONS: This combined discovery and biomarker validation approach revealed a previously unrecognized virus-specific induction of host proteins. The identification of hepatitis virus specific signatures provides a foundation for functional studies and the identification of potential correlates of viral clearance.

Apolipoprotein H expression is associated with IL28B genotype and viral clearance in hepatitis C virus infection.

BACKGROUND & AIMS: HCV requires host lipid metabolism for replication, and apolipoproteins have been implicated in the response to treatment. METHODS: We examined plasma apolipoprotein concentrations in three cohorts of patients: mono-infected patients with symptomatic acute hepatitis C (aHCV); those undergoing treatment for chronic hepatitis C (cHCV); and HIV/HCV co-infected patients being treated for their chronic hepatitis C. We also evaluated associations between apolipoproteins and IL28B polymorphisms, a defined genetic determinant of viral clearance. RESULTS: Plasma apolipoprotein H (ApoH) levels were significantly higher in patients who achieved spontaneous clearance or responded to pegylated-interferon/ribavirin therapy. Strikingly, patients carrying the IL28B rs12979860 CC SNP correlated with the plasma concentration of ApoH in all three cohorts. Both ApoH and IL28B CC SNP were associated with HCV clearance in univariate analysis. Additional multivariate analysis revealed that the association between IL28B and HCV clearance was closely linked to that of Apo H and HCV clearance, suggesting that both belong to the same biological pathway to clearance. The association between IL28B CC SNP and ApoH was not observed in healthy individuals, suggesting that early post-infection events trigger differential ApoH expression in an IL28B allele dependent manner. CONCLUSIONS: This relationship identifies ApoH as the first induced protein quantitative trait associated with IL28B, and characterises a novel host factor implicated in HCV clearance.

Preventing iatrogenic HCV infection: A quantitative risk assessment based on observational data in an Egyptian hospital.

When compliance with infection control recommendations is non-optimal, hospitals may play an important role in hepatitis C (HCV) transmission. However, few studies have analyzed the nosocomial HCV acquisition risk based on detailed empirical data. Here, we used data from a prospective cohort study conducted on 500 patients in the Ain Shams hospital (Cairo, Egypt) in 2017 with the objective of identifying (i) high-risk patient profiles and (ii) transmission hotspots within the hospital. Data included information on patient HCV status upon admission, their trajectories between wards and the invasive procedures they underwent. We first performed a sequence analysis to identify different hospitalization profiles. Second, we estimated each patient's individual risk of HCV acquisition based on ward-specific prevalence and procedures undergone, and risk hotspots by computing ward-level risks. Then, using a beta regression model, we evaluated upon-admission factors linked to HCV acquisition risk and built a score estimating the risk of HCV infection during hospitalization based on these factors. Finally, we assessed and compared ward-focused and patient-focused HCV control strategies. The sequence analysis based on patient trajectories allowed us to identify four distinct patient trajectory profiles. The risk of HCV infection was greater in the internal medicine department, compared to the surgery department (0·188% [0·142%-0·235%] vs. 0·043%, CI 95%: [0·036%-0·050%]), with risk hotspots in the geriatric, tropical medicine and intensive-care wards. Upon-admission risk predictors included source of admission, age, reason for hospitalization, and medical history. Interventions focused on the most at-risk patients were most effective to reduce HCV infection risk. Our results might help reduce the risk of HCV acquisition during hospitalization in Egypt by targeting enhanced control measures to ward-level transmission hotspots and to at-risk patients identified upon admission.

CXCL10 antagonism and plasma sDPPIV correlate with increasing liver disease in chronic HCV genotype 4 infected patients.

Egypt has the highest prevalence of hepatitis C virus infection worldwide. CXCL10 is a potent chemoattractant that directs effector lymphocytes to sites of inflammation. It has been reported that plasma CXCL10 is processed by dipeptidylpeptidase IV (DPPIV) thus leading to the generation of an antagonist form. Using Luminex-based immunoassays we determined the concentration of different forms of CXCL10 (total, agonist, and antagonist). We also evaluated plasma soluble DPPIV (sDPPIV) concentration and plasma dipeptidylpeptidase (DPP) activity. Using flow cytometry and immunohistochemistry, we analyzed the distribution of lymphocyte subsets. Plasma CXCL10 was elevated in chronic HCV patients, however the agonist form was undetectable. Increased sDPPIV concentration and DPP activity supported the NH2-truncation of CXCL10. Finally, we demonstrated an increased frequency of CXCR3(+) cells in the peripheral blood, and low numbers of CXCR3(+) cells within the lobular regions of the liver. These findings generalize the observation of chemokine antagonism as a mechanism of immune modulation in chronic HCV patients and may help guide the use of new therapeutic immune modulators.

TH1 cytokine response to HCV peptides in Egyptian health care workers: a pilot study.

Our objective was to elucidate the effects of different HCV peptides on TH1 cytokine synthesis (interleukin 2(IL2), gamma interferon (INFγ) and tumor necrosis factor α (TNF α)), in a proliferative response in a high risk population of HCV seronegative aviremic Egyptian healthcare workers (HCW). We studied the TH1 cytokine response to different HCV peptides among 47 HCW with and without evidence of HCV infection. Participants were classified according to the proliferation index (PI) in a CFSE proliferation assay as an indicator of previous exposure to HCV. Cytokines were analyzed using Luminex xMAP technology. Results showed that positive PI HCW produced a higher IL2 in response to all HCV peptides except NS4, a higher IFNγ response to NS3 and NS4 and no difference in TNFα response when compared to the negative PI HCWs. When compared to chronic HCV HCW, positive PI HCW showed no difference in the IL2 response, a higher IFNγ response to NS4 and NS5 HCV peptides and a higher TNFα response to all peptides. In conclusion the magnitude and type of cytokines produced in HCV infection is critical in determining the outcome of infection. NS4 & NS5 HCV peptides induce a protective TH1 response in positive PI HCW.

Circulating plasmacytoid dendritic cells in acutely infected patients with hepatitis C virus genotype 4 are normal in number and phenotype.

The incidence of hepatitis C virus (HCV) genotype 4 infection in Egypt provides a unique opportunity to study the innate immune response to symptomatic acute HCV infection. We investigated whether plasmacytoid dendritic cells (pDCs) are activated as a result of HCV infection. We demonstrate that, even during symptomatic acute infection, circulating pDCs maintained a similar precursor frequency and resting phenotype, compared with pDCs in healthy individuals. Moreover, stimulation with a Toll-like receptor 9 agonist resulted in an intact inflammatory response. These data support the growing consensus that pDCs are not directly activated by HCV and therefore are viable targets for immunotherapy throughout HCV infection.

Hepatitis C virus infection and risk factors among patients and health-care workers of Ain Shams University hospitals, Cairo, Egypt.

BACKGROUND: Hospitals are suspected of playing a key role in HCV epidemic dynamics in Egypt. This work aimed at assessing HCV prevalence and associated risk factors in patients and health-care workers (HCWs) of Ain Shams University (ASU) hospitals in Cairo. METHODS: We included 500 patients admitted to the internal medicine or surgery hospital from February to July, 2017, as well as 50 HCWs working in these same hospitals. Participants were screened for anti-HCV antibodies and HCV RNA. A questionnaire was administered to collect data on demographic characteristics and medical/surgical history. For HCWs, questions on occupational exposures and infection control practices were also included. RESULTS: The overall prevalence of anti-HCV antibodies was 19.80% (95% CI: 16.54-23.52) among participating patients, and 8.00% (95% CI: 0.48-15.52) among participating HCWs. In HCWs, the only risk factors significantly associated with anti-HCV antibodies were age and profession, with higher prevalence in older HCWs and those working as cleaners or porters. In patients, in a multivariate logistic regression, age over 50 (aOR: 3.4 [1.9-5.8]), living outside Cairo (aOR: 2.1 [1.2-3.4]), admission for liver or gastro-intestinal complaints (aOR: 4.2 [1.8-9.9]), and history of receiving parenteral anti-schistosomiasis treatment (aOR: 2.7 [1.2-5.9]) were found associated with anti-HCV antibodies. CONCLUSIONS: While HCV prevalence among patients has decreased since the last survey performed within ASU hospitals in 2008, it is still significantly higher than in the general population. These results may help better control further HCV spread within healthcare settings in Egypt by identifying at-risk patient profiles upon admission.

Sexual Transmission of HCV in Heterologous Monogamous Spouses.

We screened for evidence of HCV infection in healthy heterologous monogamous spouses of chronic HCV patients and studied the relation with various risk factors. A cross-sectional study of fifty healthy monogamous heterosexual spouses of HCV-positive index cases was carried out. All participants were HBV and HIV negative. The association with various risk factors was studied. Five spouses (10%) showed evidence of HCV infection. Two partners were positive for HCV antibody alone (4%) and 3 for antibody and HCV PCR (6%). No association was found between HCV infection and various sociodemographic parameters with the exception of older age categories. Intraspousal transmission of HCV may be an important source of spread of HCV infection. The reservoir of HCV-infected individuals in Egypt is sizable, and sexual transmission of HCV may contribute to the total burden of infection in Egypt.

Frequent transient hepatitis C viremia without seroconversion among healthcare workers in Cairo, Egypt.

BACKGROUNDS: With 10% of the general population aged 15-59 years chronically infected with hepatitis C virus (HCV), Egypt is the country with the highest HCV prevalence worldwide. Healthcare workers (HCWs) are therefore at particularly high risk of HCV infection. Our aim was to study HCV infection risk after occupational blood exposure among HCWs in Cairo. METHODOLOGY/PRINCIPAL FINDINGS: The study was conducted in 2008-2010 at Ain Shams University Hospital, Cairo. HCWs reporting an occupational blood exposure at screening, having neither anti-HCV antibodies (anti-HCV) nor HCV RNA, and exposed to a HCV RNA positive patient, were enrolled in a 6-month prospective cohort with follow-up visits at weeks 2, 4, 8, 12 and 24. During follow-up, anti-HCV, HCV RNA and ALT were tested. Among 597 HCWs who reported a blood exposure, anti-HCV prevalence at screening was 7.2%, not different from that of the general population of Cairo after age-standardization (11.6% and 10.4% respectively, p = 0.62). The proportion of HCV viremia among index patients was 37%. Of 73 HCWs exposed to HCV RNA from index patients, nine (12.3%; 95%CI, 5.8-22.1%) presented transient viremia, the majority of which occurred within the first two weeks after exposure. None of the workers presented seroconversion or elevation of ALT. CONCLUSIONS/SIGNIFICANCE: HCWs of a general University hospital in Cairo were exposed to a highly viremic patient population. They experienced frequent occupational blood exposures, particularly in early stages of training. These exposures resulted in transient viremic episodes without established infection. These findings call for further investigation of potential immune protection against HCV persistence in this high risk group.