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The American Journal of Tropical Medicine and Hygiene logoLink to The American Journal of Tropical Medicine and Hygiene
. 2014 May 7;90(5):823–829. doi: 10.4269/ajtmh.13-0703

Schistosomiasis Does Not Affect the Outcome of HCV Infection in Genotype 4-Infected Patients

Walaa R Allam 1, Ahmed Barakat 1, Zainab Zakaria 1, Gehan Galal 1, Tamer S Abdel-Ghafar 1, Mohamed El-Tabbakh 1, Nabiel Mikhail 1, Imam Waked 1, Sayed F Abdelwahab 1,*
PMCID: PMC4015572  PMID: 24615138

Abstract

Although reports suggest that Schistosoma mansoni increases hepatitis C virus (HCV) morbidity and chronicity, its impact on HCV spontaneous resolution is not clear. HCV genotype, viral load, abdominal ultrasonographic findings, and HCV-specific cell-mediated immunity (CMI) were examined among 141 healthcare workers infected with HCV (68 workers with and 73 workers without S. mansoni). HCV genotype 4 was dominate, and viral loads were 2.62 ± 0.69 × 106 and 4.24 ± 1.4 × 106 IU/mL among patients with and without coinfection, respectively (P = 0.309); 23.5% with and 32.9% without coinfection had spontaneously resolved HCV infection (P = 0.297). Interferon-γ spot-forming cells/106 peripheral blood mononuclear cells among responding viremic patients with and without coinfection were 716 ± 194 and 587 ± 162, whereas among aviremic patients, it was 794 ± 272 and 365 ± 36 (P > 0.05), respectively. In conclusion, there was no statistical difference in HCV spontaneous resolution, viral load, liver pathology, or CMI in patients with or without S. mansoni coinfection, suggesting that it did not impact the outcome of HCV infection.

Introduction

Hepatitis C virus (HCV) is a major causative agent of chronic hepatitis. Approximately 70% of infected persons develop chronic hepatitis C, and 20–30% may progress to cirrhosis and hepatocellular carcinoma.1–3 The prevalence of HCV infection in Egypt is the highest in the world, where ∼15% of the general population have HCV antibodies and two-thirds of those individuals have active HCV infection or viremia.4 The progression of liver disease in HCV-infected patients may be affected by viral factors (e.g., viral load, viral genotype, and quasispecies diversity).5 Also, many host factors have been observed to increase the risk of progression of liver disease, including race, male sex, older age at infection, and immunosuppressed states.6 Other complicating factors include schistosomiasis, human immunodeficiency virus (HIV) coinfections, and alcohol intake.2,7

Concomitant schistosomiasis and HCV infections are common in Egypt and many developing countries, where schistosomiasis is endemic,8 especially among persons in contact with river water and individuals working in agriculture. Several reports suggest that Schistosoma mansoni increases HCV morbidity and chronicity, leading to viral persistence and accelerated progression of hepatic complications. In this regard, compared with patients infected with HCV alone, the majority of HCV and Schistosoma coinfected patients failed to clear HCV infection and progressed to chronic hepatitis, with a more severe clinical course,9 higher HCV-RNA titers,10,11 higher incidence of cirrhosis and hepatocellular carcinoma,12–15 poor response to interferon (IFN) therapy,16,17 a higher treatment relapse rate,17 and higher mortality rates.11,18

Little is known about the mechanisms of HCV and S. mansoni interactions, and the immunological changes that occur during such a coinfection are not clearly defined,19 mainly because of the lack of a small animal models that can support both infections. Spontaneous resolution of HCV monoinfection is associated with strong CD4+ and CD8+ T-cell responses.20–25 Chronic infection with schistosomiasis induces a T-helper 2 (Th-2) cell-mediated immune (CMI) response while suppressing Th-1 response, which may be the cause for a poor prognosis of HCV infection.26 This immunosuppression caused by S. mansoni coinfection may even persist beyond having active schistosomiasis (i.e., having viable adult worms).27 How coinfection impacts the clearance or persistence of HCV infection is not clear and understudied. In this study, the effect of S. mansoni infection on HCV clearance, viral load, liver pathology, and HCV-specific CMI response was examined in patients with and without coinfection in a cross-sectional survey of 141 Egyptian healthcare workers (HCWs) at the National Liver Institute (NLI) in the Nile Delta.

Subjects and Methods

Study subjects.

We previously reported the prevalence28 and incidence29 of HCV markers among 859 HCWs at the NLI at Menoufiya University; 141 (16.6%) HCWs were positive for HCV antibodies, 13 (1.5%) HCWs were positive for hepatitis B virus (HBV) surface antigen (HBsAg), and 2 (0.02%) HCWs were positive for both viruses. Antibodies to S. mansoni were detected in 297 (34.6%) of these HCWs. In this study, we examine the impact of schistosomiasis on 141 HCV antibody-positive HCWs. They were 92 males and 49 females, with a mean age of 41.2 ± 10.2 years. At the time of this study, none of the HCV-infected subjects were aware of HCV status and did not receive any standard of care treatment for the infection. According to S. mansoni antibody status, these 141 HCWs were classified into two categories. The first category included 68 coinfected patients who were positive for both HCV and S. mansoni antibodies, and the second category included 73 patients infected with HCV alone who were positive for HCV antibodies but not S. mansoni antibodies. Data regarding age, sex, residence, education, job category, and clinical history were obtained using standard demographic questionnaires. Blood samples were collected for laboratory testing and immunological studies. The study protocol was approved by the ethical committee of the NLI, and each participant signed a consent form before enrollment.

Detection of HCV, S. mansoni, and other biomarkers.

Serum samples were collected in plain vacutainer tubes, and serum alanine aminotransferase (ALT) levels were measured using routine clinical test kits (HUMAN Gesellschaft für Biochemica und Diagnostica mbH, Wiesbaden, Germany). Anti-HCV was tested using a third generation enzyme immunoassay (EIA; Murex Anti-HCV, version 4.0, Abbott Park, IL) according to the manufacturer's instructions. HBsAg (Murex) and HBV core antibody (HBc; total anti-HBc immunoglobulin M [IgM] and IgG; Adaltis, Milan, Italy) EIAs were performed according to the manufacturers' instructions and as previously described.28 Detection and quantification of HCV-RNA was performed on subjects' sera after extraction of RNA using the Qiagen viral RNA extraction kit (QIAgen, Limburg, Netherlands) and a quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) by strand-specific AgPath-ID one-step assay according to the manufacturer's instructions (Applied Biosystems-Life Technologies Corporation, Foster City, CA). The kit uses HCV-specific primers and probes as well as internal controls. Detection of S. mansoni-specific IgG was performed by EIA according to the manufacturer's instructions (Novatec Immunodiagnostica GmbH, Diezenbach, Germany).

HCV genotyping was conducted by restriction fragment-length polymorphism (RFLP) analysis of the 5′ non-coding region using two sets of restriction endonucleases, MvaI/HinfI and RsaI/HaeIII, as previously described.30 The genotyping data were confirmed by conventional PCR using genotype-specific primers as previously described.31

Abdominal ultrasonography.

Routine abdominal ultrasound was performed for all subjects included in the study.

Synthetic HCV antigens and other control antigens.

HCV genotype 4a isolate ED43 peptide antigens composed of 15 amino acids (15mer) and overlapped by 11 amino acids were used in this study. These synthetic peptides were obtained from the National Institute of Allergy and Infectious Diseases' Biodefense and Emerging Infections Research Resources Repository and were at least 80% pure. There were 585 peptides that were combined in seven pools and labeled with alphabetical letters as follows: E2”, representing the viral envelope protein E2 (92 peptides); F”, comprising the N-terminal one-half of the NS3 protein (78 peptides); G”, comprising the remaining one-half of NS3 (78 peptides); H”, comprising the NS4a and NS4b proteins (79 peptides); I”, comprising the NS5a protein (111 peptides); L”, representing the first one-half of NS5b (75 peptides); M”, covering the remainder of NS5b protein (72 peptides). Negative control cultures included cells stimulated with culture medium alone but containing the solvent used for the preparation of the peptides (dimethyl sulfoxide). Cytomegalovirus lysate (Virusys Corporation) and Cytomegalovirus, Epstein-Barr Virus and Influenza Virus (CEF) peptide pool (Pantecs GmbH, Germany) were used as positive controls for antigen-specific responses, whereas staphylocoocal enterotoxin B (SEB; Sigma, MO) was used as a polyclonal positive control.

IFN-γ ELISpot assay.

To examine if there is a difference in HCV-specific CMI responses among those subjects with HCV alone and those subjects with concomitant S. mansoni infection (active or inactive; because antibodies cannot differentiate current from past infection), we performed an IFN-γ enzyme linked immunospot (Elispot) assay to assess HCV-specific CMI response. A convenience sample of 56 patients was examined for HCV-specific CMI responses. These subjects were 27 coinfected patients (20 patients with and 7 patients without HCV viremia) and 29 subjects who were positive for HCV infection alone (14 patients with and 15 patients without HCV viremia). Approximately 15 mL whole blood were collected into ethylene diamine tetraacetic acid (EDTA) vacutainer tubes (Becton Dickinson Biosciences, NJ). Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation, and viability was determined by the trypan blue exclusion method. The ELISpot assay is described elsewhere.32 Briefly, PBMCs (2 × 105/well) were incubated in triplicate cultures in the ELISpot plates (Whatman Unifilter, Pittsburgh, PA) coated with anti-human IFN-γ antibody (Mabtech, Stockholm, Sweden) and incubated for ∼16 hours with or without recombinant HCV antigens at 3 μg/mL for each single peptide in complete RMPI-1640 medium. Negative and positive controls included medium containing dimethyl sulfoxide alone and 0.1 μg/mL SEB, respectively. At the end of the incubation period, the assay was developed until the appearance of spots, and then, the wells were rinsed with tap water to stop the reaction. The number of spots per well was counted using an automated ELISpot reader (Cellular Technology Ltd., Cleveland, OH). Mean numbers of IFN-γ spot-forming cells (SFCs) in control wells were subtracted from antigen-stimulated wells to correct for background cytokine production and are expressed as SFCs per 1 million PBMCs. A positive HCV antigen-specific response was considered if the SFCs in the presence of antigen were at least threefold the number of SFCs in the medium control and if there was > 55 SFCs/million PBMCs as we previously reported.33

Statistical analysis.

All data were entered into a Microsoft Access database (Redmond, WA). Duplicate data entry was performed to ensure quality control. Analysis was done on SPSS package version 17.0. (SPSS Inc., IL). Fisher exact test was performed for categorical data, whereas Student's t test (or Kruskal–Wallis U test, when appropriate) was performed for comparison of continuous data.

Results

Characterization of the study population.

This study was conducted on 141 HCWs from the NLI at Menoufiya University and included 68 patients who were coinfected with HCV and S. mansoni, of whom 16 (23.5%) patients had spontaneously resolved HCV infection (i.e., negative HCV-RNA), and 73 patients who were positive for HCV infection alone, of whom 24 (32.9%) patients had spontaneously resolved HCV infection. There was no significant difference in the percentage of subjects who spontaneously resolved HCV infection in the presence or absence of S. mansoni (P = 0.297). The subjects have an overall mean age of 41.2 ± 10.2 years. Among them, 92 (65.2%) patients were males (P = 0.081), and 69.8% of patients were rural residents (P = 0.793). The job category and detailed demographic characteristics of the study subjects with different HCV and S. mansoni infection states are shown in Table 1 .

Table 1.

Demographic characteristics of patients infected with HCV and/or S. mansoni

Characteristic HCV and S. mansoni coinfection HCV alone P value
Chronic (N = 52; %) Resolved (N = 16; %) Chronic (N = 49; %) Resolved (N = 24; %)
Male sex 38 (73) 12 (75) 25 (52) 17 (71) 0.081
Mean age (years) 42 ± 9.6 39 ± 10 40 ± 10 43 ± 9 0.335
Rural residence 41(79) 13 (81) 35 (71) 18 (75) 0.793
Occupational category
 Physician/medical staff/student 1 (1.9) 1 (6.3) 1 (2.0) 3 (12.5) 0.147
 Senior nurse/nurse/nursing student 4 (7.7) 4 (25) 13 (26.5) 6 (25)
 Housekeeping 30 (57.7) 8 (50.0) 18 (36.7) 8 (33.3)
 Administrative and pharmacy staff 15 (28.8) 3 (18.8) 15 (30.6) 5 (20.8)
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Age is shown as mean ± SD.

HCV-RNA loads are not significantly different between those subjects infected with HCV alone or combined with S. mansoni infection.

All subjects positive for HCV antibodies were tested for HCV current infection and viral load by RT-PCR, and 101 of 141 HCV-antibody positive HCWs (71.6%) were also HCV-RNA–positive. Unexpectedly, the average HCV-RNA loads among 52 viremic patients infected with both HCV and schistosomiasis (2.62 ± 0.69 × 106; range = 2.29–2,360 × 103 IU/mL) were slightly lower than the loads measured in 49 viremic patients with HCV infection alone (4.24 ± 1.4 × 106; range = 0.357–4,770 × 103 IU/mL) (Figure 1 ). The difference in HCV viral loads was not statistically significant (P = 0.309) (Table 2 ). No correlation could be established between HCV RNA titers and serum ALT levels (below).

Figure 1.

Figure 1.

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Serum levels of HCV RNA in HCV-infected patients without and with S. mansoni coinfection. Data are expressed as scatter plot and the bars represent the means ± SEMs.

Table 2.

Laboratory data of patients infected with HCV or S. mansoni alone or combined

Characteristic HCV and S. mansoni coinfection HCV alone P value
Chronic (N = 52; %) Resolved (N = 16; %) Chronic (N = 49; %) Resolved (N = 24; %)
RNA titer (× 106 IU/mL)* 2.6 ± 0.69 N/A 4.2 ± 1.45 N/A 0.309
HCV genotype
 Genotype 4 47 (90%) N/A 39 (80) N/A 0.905
 Genotype 1 1 (2%) N/A 0 N/A
 Mixed genotypes 1 and 4 0 N/A 3 (6.0) N/A
 Mixed genotypes 2 and 4 1 (2%) N/A 5 (10) N/A
 Non-typable 3 (6%) N/A 2 (4) N/A
Serum ALT level (U/L) 45 ± 24 27 ± 12 50 ± 40 27 ± 17 0.0001*
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Data are shown as mean ± SEM unless otherwise indicated. N/A = not applicable.

*

Statistically significant.

HCV genotyping showed that five HCV-RNA samples (5%) were non-typable by our method. The great majority of 96 typed samples (95%) belonged to HCV genotype 4. HCV genotype 4 was found in almost all typed samples either as a single genotype in 89 (93%) of 96 subjects (96% in HCV and S. mansoni coinfected subjects and 83% in HCV monoinfected subjects) or with other HCV genotypes. Genotype 1 was found combined with genotype 4 in three (6.4%) of the HCV monoinfected subjects but not in the HCV coinfected subjects. Also, genotype 2 was combined with genotype 4 in two (4%) of the coinfected subjects and five (10.2%) of the monoinfected subjects (Table 2).

Liver inflammation as measured by ALT levels and ultrasound in the study subjects.

As shown in Table 2, we did not find a significant difference in ALT levels between the viremic subjects infected with HCV alone and those subjects coinfected with S. mansoni (P = 0.456), and this finding was also true for those subjects without HCV-RNA (P > 0.05). These results are consistent with the HCV viral load data. There were significantly higher ALT levels in chronic subjects with and without S. mansoni coinfection compared with those subjects who resolved HCV infection (P < 0.05) (Table 2).

Additional examination of the liver of viremic subjects by ultrasound imaging showed that 27% and 39% of those patients with and without S. mansoni coinfection, respectively, had normal abdominal ultrasound findings. The abnormal findings among those viremic subjects with and without S. mansoni coinfection ranged from coarse liver and splenomegaly (29% versus 25%, respectively) to liver cirrhosis and splenomegaly (19% versus 11%, respectively). Also, periportal fibrosis was found in 25% of the viremic subjects with S. mansoni coinfection, whereas echogenic liver was found in 25% of those subjects infected with HCV alone.

However, 58% and 62% of those subjects who had resolved HCV infection with and without S. mansoni coinfection had normal abdominal ultrasound findings, respectively. The abnormal findings among those aviremic subjects with and without S. mansoni coinfection were echogenic liver (25% also associated with periportal fibrosis versus 38%, respectively). Also, periportal fibrosis was found in 17% of those aviremic coinfected subjects. No significant differences were found in the ultrasound findings between those subjects with and without S. mansoni coinfection among the total, viremic, or aviremic subjects (P > 0.05).

HCV-specific CMI responses among the study subjects.

A convenience sample of the study subjects was tested for HCV-specific CMI and included 27 coinfected patients (20 patients with and 7 patients without HCV-RNA) and 29 subjects who were positive for HCV monoinfection (14 subjects with and 15 subjects without HCV-RNA). Only 8 of 20 (40%) and 11 of 14 (79%) viremic patients with and without Schistosoma, respectively, had a positive HCV-specific CMI response to at least one of seven HCV antigen pools tested (P = 0.026). However, 4 of 7 (57%) and 9 of 15 (60%) aviremic patients with and without Schistosoma, respectively, had a positive HCV-specific CMI response to at least one of seven HCV antigen pools tested (P = 0.327) (Figure 2). The magnitude of the HCV-specific CMI response measured by comparing the total mean (± SEM) of SFCs among viremic (chronic) patients with and without Schistosoma coinfection was 716 ± 194 and 587 ± 162 (P = 0.617), respectively, whereas among aviremic (resolved) patients, it was 794 ± 272 and 365 ± 36 (P = 0.036), respectively (Figure 2), showing higher CMI response among coinfected patients. No significant differences (P > 0.05) were found in the breadth or quality of the response (number of responding antigen pools in each subject) to seven antigen pools tested among the coinfected and HCV moninfected subjects with or without HCV RNA (data not shown).

Figure 2.

Figure 2.

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IFN-γ ELISPOT responses in the study subjects. PBMCs were stimulated in triplicates with seven sets of pooled overlapping 15mer HCV genotype 4a peptide antigens for 16–18 hours as described. The subjects were classified into four groups: HCV and schistosoma coninfected patients with and without HCV-RNA and HCV monoinfected patients with and without RNA. The total cumulative number of HCV-specific IFN-γ SFCs for the seven pools per 106 PBMCs is shown for the responding subjects only. The lines represent the means ± SEMs.

Discussion

This cross-sectional survey examined the effect of S. mansoni infection on HCV clearance, viral load, liver inflammation, and HCV-specific CMI responses in coinfected patients and compared it with the effect in those patients infected with HCV alone among 141 Egyptian HCWs at the NLI in the Nile Delta, where HCV antibody prevalence reaches 24% in the rural areas.34,35 HCV genotype 4 existed in almost all HCV infections, with about 11% coinfections with other HCV genotypes (mainly genotypes 1 and 2). We show that there was no statistical difference in HCV spontaneous resolution, viral load, liver pathology, or CMI in patients with or without S. mansoni coinfection, suggesting that it did not impact the outcome of HCV infection. Several aspects of these data warrant additional discussion.

First, the subjects included in this study were 68 HCWs infected with both HCV and S. mansoni and 73 HCV-infected HCWs without evidence of S. mansoni infection. Notably, there was no statistical difference in HCV spontaneous clearance in patients infected with HCV alone or coinfected with S. mansoni. In this regard, 16 (23.5%) of the coinfected subjects and 24 (32.9%) of the HCV-infected patients spontaneously resolved HCV infection (P = 0.297), suggesting no impact of schistosomiasis on HCV spontaneous resolution. Also, the average RNA load in coinfected and HCV monoinfected patients was statistically non-significant. In addition, there were no significant differences in the mean IFN-γ SFCs/106 PBMCs among viremic patients coinfected with S. mansoni and those patients infected with HCV alone. Unexpectedly, among aviremic subjects, there was a statistically higher frequency of HCV-specific IFN-γ SFCs in coinfected patients among the total responding subjects (P = 0.036). These data suggest that S. mansoni does not impact the outcome of HCV infection.

Second, it was reported that patients coinfected with HCV and schistosomiasis have higher viral loads, higher incidence of cirrhosis, hepatocellular carcinoma, poor response to IFN therapy, and higher mortality rates because of liver-related causes compared with patients infected with HCV alone.9,11,36 Also, immunosuppression was sometimes observed in Schistosoma and HCV coinfection.37,38 This immunosuppression was explained by a shift in T-cell response by down-regulating the Th-1 cytokines while favoring a Th-2 environment and response caused by chronic Schistosoma infection in experimental mouse model.39,40 In this regard, our data did not show any significant increase in the spontaneous resolution of HCV infection among HCV monoinfected patients compared with those patients coninfected with S. mansoni. However, because the liver is the main target for both pathogens, it was suggested that coinfection with S. mansoni might cause localized suppression of CMI in the liver, inducing viral persistence and more severe hepatic complications.11 Our data did not indicate an increase in viral load in HCV and S. mansoni coinfected patients compared with HCV monoinfected patients. Actually, HCV-RNA titers were higher in subjects infected with HCV alone compared with coinfected patients. However, this increase was not significant.

Third, ALT data did not reflect any more severe liver inflammation caused by Schistosoma coinfection. ALT levels were not significantly higher in coinfected subjects, whether viremic or aviremic, compared with subjects with HCV infection alone. However, ALT levels were significantly higher in viremic subjects with and without concomitant S. mansoni infection compared with patients who resolved HCV infection. In this regard, a study by Derbala and others41 suggested a lack of association of ALT levels with schistosomiasis. Consistent with ALT results and similar to our ultrasound findings of comparable liver pathology and cirrhosis proportions among subjects with and without Schistosoma infection (whether viremic or aviremic), Derbala and others41 also found that chronic HCV patients with or without schistosomiasis had similar fibrosis and response to treatment rates and that schistosomiasis coinfection is not a surrogate of poor response. Data by Kamal and others11 contradict our findings, because higher ALT levels were found among coinfected subjects. This contradiction could be attributed to proven active schistosomal infection in their study,11 which was based on long-term follow-up, whereas our study was a cross-sectional survey. Our findings were confirmed with comparable abdominal ultrasound findings among coinfected patients and those patients infected with HCV alone.

Fourth, the CMI data presented in this study show that there is no decrease in the frequency of IFN-γ SFCs by cells isolated from patients coinfected with S. mansoni compared with those cells of patients infected with HCV alone. This finding was true for the magnitude and breadth of HCV-specific CMI responses (Figure 2) (data not shown). Others have reported dysfunctional HCV-specific T cells that were unable to secret IFN-γ in HCV and S. mansoni coinfected patients.21,26,42 However, another study had findings43 similar to ours where no significant differences in CMI responses were found between those infected with HCV alone and those infected with HCV combined with schistosomiasis. Of note, in this study, there was a significant increase in the number of subjects responding to HCV antigens by IFN-γ secretion in HCV monoinfection patients compared with coinfected subjects with viremia (79% versus 40%; P = 0.026), which may explain reports of decreased response in coinfected subjects compared with HCV monoinfected subjects. This difference could be attributed to the limited number of subjects tested in this report. Importantly, there was no significant difference in the quality (breadth) of the CMI responses among the responding subjects examined in this study.

Fifth, we measured S. mansoni infection using IgG EIA, which does not differentiate between current and previous infection. Although it is considered a limitation in this study, the objective of our study was to see any difference in the spontaneous resolution in coinfected subjects compared with subjects infected with HCV alone, regardless of active S. mansoni infection, which is not clear and understudied. In this regard, the immunosuppression caused by S. mansoni coinfection may not be limited to active infections only (i.e., having viable adult worms).27 Another limitation of this study is that it is a cross-sectional study, which could explain the differences in the outcomes compared with other prospective or longitudinal studies.11,44,45 Also, we evaluated liver damage in both groups using ultrasonography and not the more accurate fibrotest/fibroscan (they were not available) or the more invasive liver biopsy, which would have been refused by most subjects. Finally, the measurement of HCV-specific CMI responses to pooled HCV peptides and not fine mapping of the responses to individual peptides is another limitation. It could be examined in future studies. Notably, among those subjects without viremia, there was a statistically higher frequency of HCV-specific IFN-γ SFCs in coinfected patients among the total subjects. We speculate that coinfection with S. mansoni may actually increase the lifespan of HCV-specific memory cells.

In conclusion, HCV genotype 4-infected Egyptian HCWs previously or currently infected with S. mansoni did not have suppressed viral clearance, increased RNA levels, or liver inflammation, suggesting that the clearance of HCV is not impaired by the presence of S. mansoni. Also, Schistosoma infection did not suppress HCV-specific CMI in either magnitude or breadth of the response compared with HCV infection alone.

ACKNOWLEDGMENTS

The authors thank Hoayda M. Ahmed, Rehab M. El-Sayed, Dr. Mohamed A. Mahmoud, and Prof. Eman Rewasha (National Liver Institute) for their assistance with sample collection and enrollment of the healthcare workers. Enas S. Aziz (Egyblood) performed data entry. We appreciate the assistance that we received during this study from Dr. Mohamed Hashem, Dr. Samer S. El-Kamry, and Prof. G. T. Strickland (University of Maryland). We particularly thank Mr. Mohamed K. Mohamed and Dr. Nabeil Khoury (previous Egyptian Company for Blood Transfusion Services [Egyblood] CEOs) for their support throughout the conduct of the study. We also thank the HEPACIVAC project (New preventative and therapeutic Hepatitis C vaccines: from pre-clinical to phase 1) consortium members led by Prof. R. Cortese for their helpful comments during the conduct of the study. The hepatitis C virus peptides were provided by Prof. A. Nicosia and his team at Okairos, Italy.

Footnotes

Financial support: This study was supported by European Union 6th Framework Program Contract no. 0374435 to the HEPACIVAC Consortium and the Egyptian Company for Blood Transfusion Services (Egyblood).

Authors' addresses: Walaa R. Allam, Zainab Zakaria, and Gehan Galal, Egyptian Company for Blood Transfusion Services (Egyblood), Agouza, Giza, Egypt, E-mails: walaaramadan@gmail.com, zainab_ali52@hotmail.com, and gehan_galal@hotmail.com. Ahmed Barakat, Department of Microbiology, Faculty of Science, Ain Shams University, Cairo, Egypt, E-mail: barakat.ainshams@gmail.com. Tamer S. Abdel-Ghafar, Mohamed El-Tabbakh, and Imam Waked, Department of Hepatology, National Liver Institute, Menoufiya University, Menoufiya, Egypt, E-mails: tamerghfar@yahoo.com, meltabbakh@liver-eg.org, and iwaked@liver-eg.org. Nabeil Mikhail, South Egypt Cancer Institute, Assuit, Egypt, E-mail: nabiel.mikhail@gmail.com. Sayed F. Abdelwahab, Department of Microbiology and Immunology, Faculty of Medicine, Minia University, Minia, Egypt, E-mail: sayed.awahab@mu.edu.eg.

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