High spontaneous clearance of symptomatic iatrogenic acute hepatitis C genotype 4 infection.

Acute hepatitis C (AHC) infection resolves spontaneously in 15% to 40% of patients. Factors favoring spontaneous viral clearance remain undefined. In this study, predictors of spontaneous viral clearance in patients with symptomatic AHC were investigated. Epidemiological, clinical, and virologic parameters were also examined. Patients with symptomatic AHC were enrolled and followed up prospectively. The patients were followed up every 2 weeks in the first month and then monthly for the following 5 months, with a follow-up visit 6 months after the last hepatitis C virus (HCV)-RNA negative sample for those who had cleared the virus. Interleukin (IL)-28B.rs12979860 single-nucleotide polymorphism and HCV genotype were tested at baseline. HCV-RNA was tested during each visit. Patients who remained RNA-positive at 24 weeks were treated with pegylated interferon plus ribavirin for 24 weeks. A total of 30 patients, mostly with iatrogenically acquired AHC genotype 4 infections completed 6-months' follow-up, to either spontaneous clearance or start of treatment. The mean age of the patients was 37 ± 13 years. In total, 67% of patients were females, and the mean incubation period was 7.6 ± 3.5 weeks. Viral clearance occurred spontaneously in 19 (63.3%) patients. The average time to clearance was 24.3 ± 9.6 weeks. A total of 11 patients received therapy, and 8 (72.7%) cleared the virus and had a sustained virologic response to the treatment 24 weeks after the therapy. A total of three patients were treatment nonresponders. IL28B.rs12979860 CC genotype, female gender, and viremia level were not associated with self-limiting AHC in this cohort. In conclusion, patients with symptomatic AHC genotype 4 infection caused by an iatrogenic exposure had higher rates of spontaneous resolution than previously reported. Predicting spontaneous viral clearance after iatrogenic AHC exposure was not possible in this population.

Association of Toll-like receptor 3 and Toll-like receptor 9 single-nucleotide polymorphisms with hepatitis C virus persistence among Egyptians.

Toll-like receptors (TLRs) give the innate immune system a considerable specificity for a large range of pathogens. TLR3 detects dsRNA of viruses while TLR9 recognizes bacterial and viral unmethylated CpG motifs. This study examined whether there is a potential association between single-nucleotide polymorphisms (SNPs) in the TLR3.rs3775290 (c.1377C/T), TLR9.rs5743836 (-1237T→C) and TLR9.rs352140 (G2848A) genes and HCV infection among Egyptian patients and healthcare workers (HCWs). We enrolled 546 subjects (409 HCWs and 137 patients) divided into four groups: group 1 included 265 seronegative, aviremic subjects; group 2 included 25 seronegative, viremic subjects; group 3 included 87 subjects with spontaneously resolved HCV infection; and group 4 included 169 chronic HCV patients. All subjects were genotyped for TLR3.rs3775290, TLR9.rs5743836 and TLR9.rs352140 SNPs by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis. TLR3.rs3775290 "CC" genotype was associated with chronic HCV infection, where there was a significantly greater frequency of this genotype among chronic patients when compared to subjects with spontaneously resolved infection (63.9% vs. 51.9%; p = 0.033; OR = 1.639 and 95% CI = 0.94-2.84). However, this SNP did not correlate with the HCV RNA load among the chronic subjects (p > 0.05). There was no significant difference in TLR9.rs5743836 and TLR9.rs352140 genotype distribution between groups (p > 0.05). Lack of association between the three SNPs was found, as the three SNPs are located on two different chromosomes. In conclusion, the TLR3.rs3775290 "CC" genotype was associated with HCV chronicity, while the TLR9 gene may not play a major role in HCV infection.

Differential distribution of IL28B.rs12979860 single-nucleotide polymorphism among Egyptian healthcare workers with and without a hepatitis C virus-specific cellular immune response.

The CC genotype of the interleukin (IL)-28B.rs12979860 gene has been associated with spontaneous hepatitis C virus (HCV) clearance and treatment response. The distribution and correlation of an IL28B.rs12979860 single-nucleotide polymorphism (SNP) with HCV-specific cell-mediated immune (CMI) responses among Egyptian healthcare workers (HCWs) is not known. We determined this relationship in 402 HCWs who serve a patient cohort with ~85% HCV prevalence. We enrolled 402 HCWs in four groups: group 1 (n = 258), seronegative aviremic subjects; group 2 (n = 25), seronegative viremic subjects; group 3 (n = 41), subjects with spontaneously resolved HCV infection; and group 4 (n = 78), chronic HCV patients. All subjects were tested for an HCV-specific CMI response using an ex-vivo interferon-gamma (IFNγ) ELISpot assay with nine HCV genotype-4a overlapping 15-mer peptide pools corresponding to all of the HCV proteins. All subjects were tested for IL28B.rs12979860 SNP by real-time PCR. An HCV-specific CMI was demonstrated in ~27% of the seronegative aviremic HCWs (group 1), suggesting clearance of infection after low-level exposure to HCV. The frequency of IL28B.rs12979860 C allele homozygosity in the four groups was 49%, 48%, 49%, and 23%, while that of the T allele was 14%, 16%, 12 and 19%, respectively, suggesting differential distributions among subjects with different HCV status. As reported, IL28B.rs12979860 predicted the outcome of HCV infection (p < 0.05), but we did not find any relationship between the IL28B genotypes and the outcome of HCV-specific CMI responses in the four groups (p > 0.05). The data show differential IL28B.rs12979860 genotype distribution among Egyptian HCWs with different HCV status and could not predict the outcome of HCV-specific CMI responses.

Interleukin 28B.rs12979860 genotype does not affect hepatitis C viral load in Egyptians with genotype 4 chronic infection.

Several host and viral factors affect the natural history of Hepatitis C Virus (HCV) infection. Interleukin 28B (IL28B).rs12979860 single nucleotide polymorphism (SNP) was found to predict viral clearance with and without therapy. Subjects with the CC (favorable) genotype of IL28B.rs12979860 were more likely to spontaneously clear the infection and respond favorably to therapy. These data suggest that subjects with the "favorable" CC genotype might have a lower viral load when compared to those with the "unfavorable" TT genotype. Therefore, we examined the effect of IL28B.rs12979860 SNP on HCV viral load and clearance among HCV-infected Egyptians. This cross sectional study was conducted on 375 HCV antibody-positive subjects. Detection and quantification of HCV-RNA was determined by RT-PCR. IL28B.rs12979860 genotyping was performed using SYBR green real-time PCR and specific primers. Of 375 HCV-antibody positive subjects, 239 (63.7%) had chronic HCV infection while the remaining 136 (36.3%) subjects had spontaneously cleared the virus. The frequency of IL28-B CC, CT, and TT genotypes among spontaneous resolvers were 54.4%, 39.0%, and 6.6% while among the chronically infected subjects, they were 31.4%, 49.8%, and 18.8%, respectively. As expected, IL28 genotype predicted spontaneous HCV clearance (p < 0.001). The average HCV viral loads were 1.5 ± 0.69 x 10(6), 0.62 ± 0.11 x 10(6) and 0.51 ± 0.14 x 10(6) IU/ml among chronic subjects with the IL28B.rs12979860 CC, CT and TT genotypes, respectively (p > 0.05). In conclusion, our results show that IL28B.rs12979860 genotype does not affect viral load among chronic HCV infected Egyptians. These findings further confirm the complexity of viral host interactions in determining HCV infection outcome.

Hepatitis C virus-specific cell-mediated immune responses in children born to mothers infected with hepatitis C virus.

OBJECTIVE: To investigate the association between hepatitis C virus (HCV)-specific cell-mediated immunity (CMI) responses and viral clearance in children born to mothers infected with HCV. STUDY DESIGN: A cross-sectional study of children from a mother-infant cohort in Egypt were enrolled to detect CMI responses to recombinant core and nonstructural HCV antigens (nonstructural segments NS3, NS4a/b, and NS5 of the HCV genome) using an interferon-gamma enzyme-linked immunospot assay. Children born to mothers with chronic HCV were enrolled into 3 groups: transiently viremic (n = 5), aviremic (n = 36), and positive control (n = 6), which consisted of 1 child with chronic HCV from this cohort and another 5 children with chronic HCV from a companion study. Children without HCV born to mothers without HCV (n = 27) served as a negative control group. Wilcoxon rank sum test was used to compare the magnitude of CMI responses between groups. RESULTS: None of the 6 control children who were positive for HCV responded to any HCV antigen, and 4 (80%) of 5 children with transient viremia responded to at least one HCV antigen, compared with 5 (14%) of 36 and 3 (11%) of 27 children in the aviremic and negative control groups, respectively. Children with transient viremia elicited stronger responses than did negative controls (P = .005), positive controls (P = .011), or children without HCV viremia (P = .012), particularly to nonstructural antigens. CONCLUSIONS: HCV-specific CMI responses were significantly higher in magnitude and frequency among transiently infected children compared with those persistently infected. This suggests CMI responses may be associated with past viral clearance and can identify children at high risk of infection, who can be targeted for health education, screening, and follow-up.

Strong hepatitis C virus (HCV)-specific cell-mediated immune responses in the absence of viremia or antibodies among uninfected siblings of HCV chronically infected children.

BACKGROUND: Cell-mediated immune (CMI) responses to hepatitis C virus (HCV) antigens in adults without seroconversion or viremia are biomarkers for prior transient infection. We investigated HCV-specific CMI responses in seronegative children living with HCV-infected siblings. METHODS: Children 3-18 years of age living with HCV-infected siblings were screened for HCV antibodies and HCV RNA. Peripheral blood mononuclear cells (PBMCs) were evaluated for HCV-specific CMI responses by interferon γ (IFN-γ) enzyme-linked immunospot assay using 3 recombinant HCV protein antigens. Flow cytometry phenotypically characterized IFN-γ-secreting cells. RESULTS: Forty-five seronegative children and 5 seropositive viremic siblings had functionally viable PBMCs. Among the 45 seronegative siblings, 15 (33.3%) had positive HCV-specific IFN-γ responses, and subsequent RNA testing revealed that 3 were viremic. Compared with the 5 seropositive viremic children, the median number of HCV-specific spot-forming units was significantly higher in the 12 seronegative aviremic children (P = .002) and in the 3 seronegative viremic children (P = .025). Flow cytometric analysis revealed that IFN-γ was synthesized mainly by CD4(+) T cells. CONCLUSION: Strong HCV-specific CD4(+) T cell responses were detectable in higher frequency among seronegative, aviremic children compared with persistently infected siblings. Further studies are needed to determine whether these immune responses are protective against HCV infection.

Capturing and characterizing immune cells from breast tumor microenvironment: an innovative surgical approach.

BACKGROUND: In breast cancer patients, venous drainage of the breast may contain cells of immunological importance, tumor cells undergoing dissemination, and other biological factors derived from the tumor microenvironment. Collecting axillary venous blood during modified radical mastectomy and thus before dilution in the circulation may allow us to define biological properties of the tumor microenvironment. Aims were to (1) develop a surgical approach to collect blood from the breast tumor microenvironment through tributaries of the axillary vein and (2) characterize and compare immune cells collected from the axillary vein with those in peripheral blood of breast cancer patients. MATERIALS AND METHODS: We enrolled 17 women aged 30-50 years and diagnosed with breast cancer by mammography, ultrasound, and biopsy (stages II-III). All patients were, preoperatively, treatment-naive. During routine surgical dissection, blood was collected in heparin tubes, 10 mL from tributaries of the axillary vein and 10 mL from peripheral blood. Mononuclear cells were separated, and percentages of different leukocyte populations were determined by flow cytometry. RESULTS: We detected a significant increase in the percentage of total T lymphocytes and T helper cells collected from axillary tributaries, but not in the percentages of cytotoxic T cells, monocytes, natural killer, or B cells compared with peripheral blood. CONCLUSIONS: The present study validated using an intraoperative surgical approach to collect leukocytes drained from the tumor microenvironment through axillary tributaries. Our results showed an increase in the infiltration of total T-lymphocytes and T helper cells in the tumor microenvironment, suggesting that they may contribute to tumor pathogenesis.

Incidence of hepatitis C virus infection among Egyptian healthcare workers at high risk of infection.

BACKGROUND: Hepatitis C virus (HCV) is a global health threat with Egypt having the highest worldwide prevalence. Evaluation of the efficacy of a preventive HCV vaccine, such as those currently in Phase I/II trials, requires a cohort with a high-risk exposure to HCV. OBJECTIVE: To identify a reliable cohort for evaluating preventive HCV vaccines, we studied HCV incidence among HCW in a hospital where almost 85% of patients are HCV-infected. STUDY DESIGN: Of 717 HCW negative for HCV-antibodies (anti-HCV) at baseline, 651 were followed up and tested for seroconversion twice annually for an average of 504 ± 154 days. Those reporting a needle-stick injury were additionally tested for both HCV antibodies and RNA monthly for a total of four months. RESULTS: Two subjects (0.31%) had anti-HCV and HCV-RNA seroconversion with an overall incidence of 2.04/1000 person-years and a 4.8% incidence among the 21 subjects who reported a needle-stick injury. Two additional subjects had viremia without detectable anti-HCV. Two of the four subjects were among 21 with reported needle-stick injuries (9.5%) and another had surgery. All four were nurses providing direct patient care. CONCLUSIONS: Our results show that both transient and persistent viremia were detectable in this high-risk cohort of HCW and suggest that absence of anti-HCV in two of the subjects may be due to low-dose viral exposures. These data indicate that HCV infections acquired from documented injuries during direct patient care are frequent in Egypt and can guide selection of eligible HCW suitable for preventive HCV vaccine trials.

Risk factors for hepatitis C virus infection among Egyptian healthcare workers in a national liver diseases referral centre.

Little is known about the prevalence of hepatitis C virus (HCV) among healthcare workers (HCW) in Egypt, where the highest worldwide prevalence of HCV exists. The prevalence of HCV, hepatitis B virus and Schistosoma mansoni antibodies was examined in 842 HCWs at the National Liver Institute in the Nile Delta, where >85% of patients are HCV antibody-positive. The mean age of HCWs was 31.5 years and they reported an average of 0.6±1.2 needlesticks/HCW/year. The prevalence of anti-HCV, hepatitis B surface antigen (HBsAg) and co-infection was 16.6%, 1.5% and 0.2%, respectively. HCV-RNA was present in 72.1% of anti-HCV-positive HCWs, and all but one subject were infected with HCV genotype 4. Schistosoma mansoni antibodies were present in 35.1%. The anti-HCV rate increased sharply with age and employment duration, but not among those with needlestick history. After adjusting for other risk factors, the anti-HCV rate was higher among older HCWs [P<0.001; risk ratio (RR) = 1.086, 95% CI 1.063-1.11], males (P=0.002; RR=1.911, 95% CI 1.266-2.885) and those with rural residence (P<0.001; RR=2.876, 95% CI 1.830-4.52). Occupation (P=0.133), duration of employment (P=0.272) or schistosomal antibody positivity (P=0.152) were not significant risk factors for anti-HCV positivity. In conclusion, although one in six HCWs had been infected with HCV, the infections were more likely to be community-acquired and not occupationally related.

Hepatitis C virus-multispecific T-cell responses without viremia or seroconversion among Egyptian health care workers at high risk of infection.

Hepatitis C virus (HCV)-specific cell-mediated immunity (CMI) has been reported among exposed individuals without viremia or seroconversion. Limited data are available regarding CMI among at-risk, seronegative, aviremic Egyptian health care workers (HCW), where HCV genotype 4 predominates. We investigated CMI responses among HCW at the National Liver Institute, where over 85% of the patients are HCV infected. We quantified HCV-specific CMI in 52 seronegative aviremic Egyptian HCW using a gamma interferon (IFN-γ) enzyme-linked immunospot assay in response to 7 HCV genotype 4a overlapping 15-mer peptide pools covering most of the viral genome. A positive HCV-specific IFN-γ response was detected in 29 of 52 HCW (55.8%), where 21 (40.4%) had a positive response for two to seven HCV pools and 8 (15.4%) responded to only one pool. The average numbers of IFN-γ total spot-forming cells (SFC) per million peripheral blood mononuclear cells (PBMC) (± standard error of the mean [SEM]) in the 29 responding and 23 nonresponding HCW were 842 ± 141 and 64 ± 15, respectively (P < 0.001). Flow cytometry indicated that both CD4(+) and CD4(-) T cells produced IFN-γ. In summary, more than half of Egyptian HCW demonstrated strong HCV multispecific CMI without viremia or seroconversion, suggesting possible clearance of low HCV exposure(s). These data suggest that detecting anti-HCV and viremia to determine past exposure to HCV can lead to an underestimation of the true disease exposure and that CMI response may contribute to the low degree of chronic HCV infection in these HCW. These findings could have strong implications for planning vaccine studies among populations with a high HCV exposure rate. Further studies are needed to determine whether these responses are protective.

Protective role of humoral immune responses during an outbreak of hepatitis E in Egypt.

Although the seroprevalence of hepatitis E virus (HEV) is approximately 80% in adult Egyptians living in rural areas, symptomatic HEV-caused acute viral hepatitis (AVH) is sporadic and relatively uncommon. To investigate the dichotomy between HEV infection and clinical AVH, HEV-specific immune responses in patients with symptomatic and asymptomatic HEV infection during a waterborne outbreak in Egypt were examined. Of 235 acute hepatitis patients in Assiut hospitals screened for HEV infection, 42 (17.9%) were acute hepatitis patients confirmed as HEV-caused AVH; 37 (88%) of the 42 patients were residents of rural areas, and 14 (33%) were from one village (Kom El-Mansoura). Another 200 contacts of AVH cases in this village were screened for HEV and 14 (7.0%), all of whom were family members of AVH cases, were asymptomatic HEV IgM-positive. HEV infections in this village peaked during the summer. Asymptomatic HEV seroconverters had significantly higher levels of epitope-specific neutralising (p=0.006) and high avidity (p=0.04) anti-HEV antibodies than the corresponding AVH cases. In conclusion, naturally acquired humoral immune responses appear to protect HEV-exposed subjects from AVH during an HEV outbreak in Egypt.