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.

The affordable and reliable detection of Hepatitis C Virus (HCV) RNA is a cornerstone in the management and control of infection, affecting approximately 3% of the global population. However, the existing technologies are expensive, labor intensive and time consuming, posing significant limitations to their wide-scale exploitation, particularly in economically deprived populations. Here, we utilized the unique optical and physicochemical properties of gold nanoparticles (AuNPs) to develop a novel assay platform shown to be rapid and robust in sensing and quantifying unamplified HCV RNA in clinical samples. The assay is based on inducing aggregation of citrate AuNPs decorated with a specific nucleic acid probe. Two types of cationic AuNPs, cysteamine and CTAB capped, were compared to achieve maximum assay performance. The technology is simple, rapid, cost effective and quantitative with 93.3% sensitivity, high specificity and detection limit of 4.57 IU/µl. Finally, our data suggest that RNA folding impact the aggregation behavior of the functionalized AuNPs, with broader applications in other nucleic acid detection technologies.

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.

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.

It is challenging to find genes with stable transcripts for use as reference genes for quantitative realtime polymerase chain reaction (qRT-PCR) during viral infection. Autographa californica nucleopolyhedrovirus (AcMNPV) is known to globally shut off host gene transcription in Sf21 cells and to modify their cytoskeletons. In this study, seven host genes were selected for validation as references for gene expression experiments using qRT-PCR. Two of them, ecdysoneless (ECD) and myosin showed stable RNA levels in our previous microarray study at 6, 12, and 24 hpi for both genes and 48 hpi for ECD. The others, actin, tubulin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and 28S ribosome (28S), are commonly employed as reference genes for qRT-PCR. Ribosomal protein L35 (L35) gene was selected to test if ribosomal protein genes show stable RNA transcript levels similar to 28S and 18S rRNA and to validate the microarray data. In addition to 28S, previously known to have stable transcript levels, qRT-PCR showed that ECD transcript levels remained constant throughout the time course of AcMNPV infection. Transcripts of cytoskeleton genes such as actin, tubulin, and myosin declined dramatically as the infection progressed. GAPDH and L35 transcripts also declined over time. These results indicate that ECD is a reliable reference gene for qRT-PCR experiments during AcMNPV infection of Spodoptera frugiperda cells. Although 28S could be used as a reference gene for these experiments, it is less useful than ECD because of its abundance, which might make it difficult to establish an accurate baseline value for data analysis.

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.

BackgroundHepatitis C Virus (HCV) infection is a global health burden particularly in Egypt, where HCV genotype 4a (GT-4a) predominates. The prevention and control of HCV infection will remain a challenge until the development of an effective vaccine that protects against different genotypes. Several HCV GT-1-based vaccines are in different stages of clinical trials, but antigenic differences could make protection against other genotypes problematic. In this regard, data comparing the cell-mediated immune (CMI) response to different HCV genotypes are limited. We aimed to ex vivo investigate whether GT-1-based vaccine may protect against HCV GT-4 infections. This was carried out on samples collected from genotype 4 infected/exposed subjects.Methods/Principal FindingsThe CMI responses of 35 subjects; infected with HCV GT-4/or who had spontaneously-resolved the infection and 10 healthy control subjects; to two sets of seven HCV overlapping 15-mer peptide pools derived from both genotypes; and covering most of the viral proteins; were evaluated. This was carried out using an interferon gamma (IFNγ) enzyme-linked immunospot (ELISpot) assay. Peripheral blood mononuclear cells (PBMC) from 17 subjects (48%) responded to at least one peptide pool derived from GT-1b/GT-4a with 13 subjects responding to peptide pools from both genotypes. A strong correlation was found in the responses to both genotypes (r = 0.82, p<0.001; 95% confidence interval = 0.562–0.933). The average IFNγ total spot forming cells (SFC)/106 PBMC (±SE) from the responding subjects for GT-1b and GT-4a was 216±56 and 199±55, respectively (p = 0.833). Also, there were no significant differences between those who cleared their HCV infection or who remained HCV-RNA positive (p = 0.8).Conclusion/SignificanceOur data suggest that an effective GT-1b vaccine could protect from GT-4a infection. These data could help in HCV rationale vaccine design and efficacy studies and further our understanding of HCV cross protection against different genotypes.

Mixed Cryoglobulinemic Vasculitis (MCV) is a prominent extra-hepatic manifestation of Hepatitis C virus (HCV) infection. HCV has been reported to cause B-cell disorders and genomic instability. Here, we investigated B-cell activation and genome stability in HCV-MCV patients receiving the direct antiviral agent, Sofosbuvir, at multiple centers in Egypt. Clinical manifestations in HCV-MCV patients were improved at the end of treatment (EOT), such as purpura (100%), articular manifestations (75%) and neuropathy (68%). Eighteen patients (56%) showed vasculitis relapse after EOT. BAFF and APRIL were higher at EOT and continued to increase one year following treatment onset. Chromosomal breaks were elevated at EOT compared to baseline levels and were sustained at 3 and 6 months post treatment. We report increased expression of DNA genome stability transcripts such as topoisomerase 1 and TDP1 in HCV-MCV patients after treatment, which continued to increase at 12 months from treatment onset. This data suggest that B-cell activation and DNA damage are important determinants of HCV-MCV treatment outcomes.