Background: Schistosomiasis is an infection caused by blood flukes of the genus Schistosoma. Schistosomes are successful parasites, apparently as a result of prolonged co-evolution with their hosts. Studies done nationwide in Egypt found the highest risk of hepatitis C virus (HCV) co-infection is in those infested with schistosome without history of blood transfusions. However, the association between schistosomiasis and HCV infection is incompletely understood. Aims: The overall aim of this study was to assess whether or not a genomic association between schistosomal infestation and HCV infection exists. Materials and Methods: Oligonucleotide specific primers of HCV-polymerase chain reaction (PCR) diagnostics were used to screen the genomic DNA and cDNA library pool of Schistosoma mansoni as templates based on the end-point PCR approach. Results: Screening of schistosome DNA by PCR, lead to the detection of sequences similar to HCV. PCR products were obtained when adult worms genomic DNA were used as templates while no PCR products were amplified from S. mansoni λZAPII cDNA library pool. The resulting PCR products were sequenced and compared with the other closely related HCV sequence database at the website < http://hcv.lanl.gov>. Conclusions: This work demonstrates the existence of HCV and its replication in the genomic DNA of S. mansoni. In addition, it highlights the fact that the parasite can carry the virus genome and therefore, is considered as a nonhuman vector for the transmission of HCV infections.

INTRODUCTION

Schistosomes are blood flukes mainly belonging to three species of the trematode parasite Schistosoma: S. hematobium, S. mansoni and S. japonicum. Less widely distributed members are S. intercalatum and S. mekongi. S. hematobium is the most prevalent of the species and is endemic in Africa and the Middle East. S. mansoni infection is endemic in Africa, South America, Caribbean and areas of the Middle East, while S. japonicum exists in parts of East Asia. [1] In Egypt, S. mansoni occurs intensively in the Nile Delta, while S. hematobium is endemic in the entire Nile Valley. [2],[3] The World Health Organization (W.H.O.) estimated > 200 million persons in 74 countries are infected of which 85% live in Africa, South of Sahara. [4],[5] In the Middle East including Egypt, the most important risk factor of squamous cell carcinoma and bladder tumors is infestation of the bladder by S. hematobium. [6],[7] Schistosomes become successful parasites as a result of prolonged co-evolution with their hosts. They have evolved several ways to evade the host immune system and other host defenses. [8]

Hepatitis C virus (HCV) belongs to the Flaviviridae family and the genus Hepacivirus. It is a spherical, enveloped single stranded positive sense RNA virus. The single positive-strand RNA genome contains a large open reading frame flanked by highly conserved untranslated regions at both the 5` and the 3` termini. [9] The virus genome is about 9600 nucleotides. Due to the lack of proofreading by the HCV RNA polymerase, HCV has an exceptionally high mutation rate and therefore a high genetic variability. [10]

W.H.O. estimates that approximately 170-200 million people are infected with HCV worldwide and around 3-4 million are newly infected each year. [11] Egypt has the highest HCV countrywide prevalence ranging from 10% to > 40% among different regions and demographic groups. [12],[13],[14] HCV infection is an important public health concern because the majority of infections do not resolve, but lead to chronic hepatitis C, one of the most frequent causes of liver diseases. [2],[15],[16]

Co-infection with schistosomiasis and HCV is verified by HCV-RNA in schistosomiasis positive individuals, and the detection of the HCV-specific antibody (Ab) (anti-HCV) in the serum of patients co-infected with the two. [17],[18] The epidemiological situation of schistosomiasis and HCV co-infection among Egyptians nationwide is not clear. Few reports showed that in Egypt schistosomiasis positive individuals are co-infected with HCV as well as demonstrating mortality higher rates than patients with single infection. [18],[19],[20] The prevalence of HCV-Ab was reported to be 70% in adults suffering from schistosomiasis and had no prior history of blood transfusion. [2],[13] Moreover, the prevalence of HCV within the schistosomiasis infected population is far higher compared to the general population of HCV mono-infected. This is supported by the high HCV loads in the Schistosoma infected patients suggesting that HCV could positively be transmitted by Schistosoma.

The area of schistosomiasis/HCV co-infection is intriguing and presents many challenges for the future global health. The connection between schistosomiasis and the occurrence of HCV was suggested by Bahgat et al., [21] and also in this work. Several reports attempted to reveal the underlying mechanisms of the schistosomiasis/HCV co-infection. The majority of these have focused on the injections of humans with poorly sterilized syringes and blood transfusions. [2],[13]

This study aims to investigate the possibility of the simultaneous schistosomal infestation and HCV infection and the genomic relationship between them, by exploring whether the parasite could be a vector of transmission of the virus to human.

MATERIALS AND METHODS

DNA extraction from adult worms

Adult worms of S. mansoni were collected from the Schistosoma Biology Supply Center at Theodor Bilharz Research Institute (PO Box 30 Imbaba, Giza, Egypt) and kept in 70% ethanol at 4°C. Genomic DNA of adult worms was extracted by standard procedures. Briefly, the adult worms were kept in lysis buffer (0.1 M EDTA, pH 8.0, 0.1 M Tris-HCl, pH 7.5, 0.2 M NaCl, 1% sodium dodecyl sulfate, 0.2% β-mercaptoethanol, and 100 μg of proteinase K), at 65°C for 2 h. Lysis was followed by using the DNA preparation mini Kit (Jena Bioscience, Egypt) according to the manufacturer′s instructions. The DNA white pellets were taken and kept at 4°C until used as templates in polymerase chain reaction (PCR). DNA concentration and purity was measured using a quantitative NanoDrop 1000 spectrophotometer according to the manufacturer′s protocol.

Polymerase chain reaction

The DNA templates for PCR assay were the purified genomic DNA sequence extracted from the adult worms (described in this work) and the λZAPII cDNA library pool extracted from S. mansoni (Wu et al., [22],[23] and were kindly provided by Dr. Philip LoVerde, SUNYAB, USA). The HCV-genome universal specific primer sequence was used. Forward primer (F; 5`-CGCAGAAAGCGTCTAGCCAT-3`) and reverse primer (R; 5`-CTCGCAAGCACCCTATCAGG-3`) were obtained from published sequences, [24] which were designed to specifically anneal to conserved regions within the HCV-5`UTR. [9],[21] Other primers sequences described by Ohno et al., [25] designed for highly conservative 5`UTR of the corresponding HCV genome were used. These specific primers were 20 nt sequences str F (5`-AGACCGTGCACCATGAGCAC-3`) and str R (5′-TGTTGCATAATTGATCCCGT-3`). Primers synthesis were performed and ordered from Thermo Hybrid Premier Biosoft International (www.Premier.Biosoft.com).

All PCR amplifications were made in final volumes of 50 μL in a nuclease-free Eppendorf tube with 40 μL of PCR Universal TaqMan Master Mix (Applied Biosystems) containing 0.5 μL each of the two amplification primers at a final concentration of 30 pmoles for each primer and 4 μL of DNA templates (0.078 μg/1 μL). The PCR tubes were placed and carried out in a DNA Thermal Cycler apparatus (Perkin-Elmer). When the temperature in the block reached 95°C and kept at that temperature for 4 min for strand separation. After that, 35 cycles of amplification were performed. The cycle of denaturation at 95°C for 0.5 min, annealing at 58°C for 1 min and nucleotide addition (extension) at 72°C for 1 min. When the last cycle was completed, the reactions were kept for another 8 min at 72°C for a final extension step followed by cooling at 4°C until the tubes were removed from the machine. The amplified PCR products were mixed with a 6X gel DNA loading dye (Fermentas, Cat. No. R0611) and examined by electrophoresis on 2% agarose gels stained with 0.5 μg/ml of ethidium bromide (EB) for visualization under UV light. The DNA bands were documented using a Biorad Gel Doc 1000 system and quantified using Quantity One version 4.2.3 software (Bio-Rad) according to the manufacturer′s descriptions. All products obtained were sized using a standard 100-bp DNA Ladder molecular weight (Jena Bioscience GmbH, Cat. No. M214). Positive control PCR reactions were also performed to evaluate the efficiency of the PCR reactions with the same guidelines as mentioned above. Negative controls were included in all PCR-runs to prevent misjudging following the risk of contaminations of the samples.

Direct sequencing of amplified DNA

With the advent of DNA methodologies, like the introduction of PCR and nucleotide sequencing, it has become common practice to sequence the PCR products commercially. We sent the PCR products for sequencing to the Biotechnology Research Center at Suez Canal University, Ismailia 41522, Egypt. Briefly, the amplified PCR products were extracted once with ethanol precipitation after the phenol-chloroform treatment. [26] Direct sequencing of the PCR amplified and purified products was performed with the automatic sequencing assay using the Taq Dye Deoxy Terminator Cycle Sequencing Kit with the ABI 3700 automated DNA sequencer (Applied Biosystems) following the manufacturer′s protocol for the sequencing reaction. One strand of the DNA was sequenced. The sequencing results have been further grouped and analyzed. The nucleotide sequences from this study were multiple aligned and edited manually by visual inspection. The sequences from this study were compared alongside with those of HCV reference sequences retrieved from the Los Alamos HCV database (http://hcv.lanl.gov/content/hcv-index). [27],[28]

RESULTS

0DNA techniques and polymerase chain reaction amplification

Among the total of 21 independent PCR amplification reactions carried out with different primers, only two bands were observed and yielded two sets of the primer sequences including the primer pair str F (5`-CGCAGAAAGCGTCTAGCCAT-3`), str R (5`-CTCGCAAGCACCCTATCAGG-3`), while the other primer set refers to the forward primer (F; 5`-AGACCGTGCACCATGAGCAC-3`) and the reverse primer (R; 5` TGTTGCATAATTGATCCCGT-3`) [Figure 1], [lane 2] and [lane 3].{Figure 1}

Analysis of the agarose gel electrophoresis revealed several interesting findings. The amount of amplified PCR products detected by agarose gel [Figure 1] was almost thick and the extent of amplification nearly reached a peak level after 35 cycles of amplification suggesting that the primers set were specific. The optimal concentration of the PCR DNA products for the samples was quantified using a NanoDrop ND 1000 spectrophotometer instrument (Thermo Scientific). All experiments were repeated at least 2 times with the same conditions, and the mean value was used for further analysis. No differences between the amplified DNA concentrations in each of the repeated PCR products were observed as judged by the NanoDrop and the agarose-stained gel results. These results revealed that PCR amplifications induced by the primers set were specific [Figure 1]. Moreover, the amplified PCR DNA products on agarose gel electrophoresis identified nonidentical mobilities and hence represented different sequences.

The high fidelity PCR products yielded only long band sizes of ~ 240 bp and ~ 350 bp on agarose gel electrophoresis including the PCR primers [Figure 1], [lane 2] and [lane 3]. A negative control sample was done without DNA template in order to check for contamination [Figure 1], [lane 1]. Other primers failed to amplify any specific DNA sequence when S. mansoni genomic DNA or λZAPII cDNA library pool is used as a template. These results indicate that the described PCR system may not be specific for screening of HCV genome or the target sequences were not present when S. mansoni genomic DNA and λZAPII cDNA library pool are used as templates. Obviously, we cannot draw any firm conclusions on this point since our study material is small during the study period.

Direct sequencing of polymerase chain reaction amplified products

Direct sequencing of the PCR amplified genomic DNA was performed to confirm the PCR products. Only one PCR amplified product was successfully sequenced using a dilution of the previously described original PCR primers with the sequence str F (F; 5`-CGCAGAAAGCGTCTAGCCAT-3`) and the sequence str R (R; 5′-CTCGCAAGCACCCTATCAGG-3`). The obtained sequencing data were identified and delivered via the HCV database searchable website < http://hcv.lanl.gov>.

DISCUSSION

The area of schistosomiasis and HCV co-infection is intriguing and presents many challenges. This co-infection, will definitely, has a significant impact on global heath. It is of great importance to explore the possible genomic association between Schistosoma and HCV co-infection and the underlying mechanisms.

Co-infection with schistosomiasis and HCV leads to a more-rapid progression of the known pathology of hepatic schistosomiasis expressed as accelerated fibrosis to decompensated liver cirrhosis, end-stage liver disease and hepatocellular carcinoma. All of these conditions are synergized in Schistosoma-HCV co-infection resulting in a higher incidence of liver-related morbidity and mortality. [21],[29],[30],[31],[32]

The PCR technique showed a remarkable sensitivity and specificity when schistosome [33] and HCV [34] examinations were used as the reference test. It is more sensitive and also renders a more-rapid result mainly than isolation and detection in routine diagnosis. In the current study, we investigated the usefulness of the PCR assay to screen the schistosome DNA by using oligonucleotide specific primers of HCV-genome. Furthermore, this PCR based approach was carried out to clarify the possible genomic association between schistosomiasis and HCV infection, through existence of HCV and its replication in the genomic DNA of Schistosoma parasite.

The molecular weights of the amplified fragments [Figure 1] produced by the PCR assay from adult worms DNA agreed with previously reported detection of the HCV genome using the same primers using the same primers. [9],[24] The absence of any amplification PCR products from negative control sample, also reflects the specificity of the used primers for the detection of HCV-genome when S. mansoni DNA is used as a template.

In this work, evidence for viral genetic material and Schistosoma-HCV genomic association was confirmed by PCR method. However, these findings need to be verified in a larger prospective study.

ACKNOWLEDGMENTS

We thank Dr. Abdullah I. Al-falouji at the Biotechnology Research Center, Suez Canal University for everything that concerns PCR and sequencing works. Further we thank professors Nour EL-Din H. Saleh and Nahla S. El-Shenawy at the Department of Zoology, Faculty of Science, Suez Canal University, for assistance in writing the manuscript.