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ISSN 1110 - 7278 EJSIE

Personal non-commercial use only. Egypt. J. of Schistosomiasis and Infect. and Endem. Dis. Copyright 2009. All rights reserved.

INTRODUCTION

The history of schistosomiasis in Egypt is longstanding,

with reports of Schistosoma eggs in ancient mummies

(Adams, 2006). However, during the last two decades

signicant progress was undeniably made in the control

of schistosomiasis after the introduction of praziquantelin the national disease control programs (El-Khoby et

al. 1998). Another important cause of infectious chronic

liver disease in the country is Hepatitis C Virus (HCV).

Egypt has the highest countrywide prevalence of HCV

in the world(Frank et al. 2000).The national prevalence

rate of HCV antibody positivity has been estimated to be

between 10-13% (Mohamed, 2004). Early studies have

indicated a frequent association of schistosomiasis and

HCV infection (Koshy et al. 1993; Abdel-Wahab et al.

1994; Waked et al. 1995; Mohamed, 2004). Abdel-Moeti

et al. (2001) reported the detection of HCV-antigen in

the Egyptian laboratory strain of Schistosoma mansonifrom Theodor Bilharz Research Institute (TBRI), Cairo.

They detected the HCV-antigen in some stages of the

trematode life cycle (worms, miracidia, intramolluscan

larvae inBiomphalaria alexandrinaand cercariae) except

eggs w hich showed negative results. Additionally, they

collected six B. alexandrina naturally infected eldsnails from the northwestern coastal region of the Nile

Delta in the Behira Governorate. They detected the virus

antigen in these eld snails and S. mansoni cercariae

shed fromthem. They obtained the same results by using

Real-Time PCR (RT-PCR) technique. Moreover, HCV

RNA quantitation, the third technique they used showed

positive results only in laboratorymiracidia and snails,

and eld snails. After repeating the whole experiments

three times, they concluded that the parasite acts as a

biological carrier of the virus in which the parasiteexists

and replicates (Abdel-Moeti et al. 2001). More recently,

the same results were conrmed by the same group(Soliman et al. 2008).

OriginalA

rticleSchistosoma mansonifrom a Hepatitis C Virus (HCV) Endemic

Region in Egypt Test Negative for HCV

Wael Lotfy1, Abeer Ghazal2and Gamal El-Sawaf2

1Parasitology Department, 2Microbiology Department, Medical Research Institute, Alexandria University

Abstract

Many studies have indicated a frequent epidemiological association of intestinal schistosomiasis and HCV infection in

Egypt. Two previous studies by others reported the role of S. mansonias a carrier of HCV. The present study was done

with the aim to further investigate the role of S. mansonias a carrier of HCV by detection of the 5' UTR region of the virus

in some Egyptian strains of the parasite. Two strains were investigated; one laboratory strain and one eld strain from

Damietta. Two pooled samples of cercariae or worms from each strain were examined by HCV RT-PCR. The laboratory

strain samples were composed of a pool of 10,000 cercariae and a pool of 100 adult worms. The eld strain samplesconsisted of a pool of 70,370 cercariae and a pool of 183 adult worms. In addition, two positive controls were made by

adding pooled human sera positive for HCV to a pool of 10,000 cercariae and a pool of 100 adult worms of the laboratory

strain. Although the two positive controls and the internal controls were positive, the used technique did not detect any

traces of HCV RNA in any of the studied four experimental samples. It seems that the high epidemiological association

between S. mansoniand HCV in Egypt is not due to the parasite acting as a carrier of the virus. Instead, this association

could be attributed to the transmission of the HCV via sharing contaminated syringes, which were used to inject tartar

emetic in the past. Additionally, this could be attributed to the association between chronic S. mansoni infection and

impaired HCV-specic immune responses.

Key Words: Schistosoma mansoni, hepatitis C virus.

Corresponding Author: Wael M. Lotfy

E-mail :[email protected]

Address:Medical Research Institute, 165 El-Horreya

Avenue, Alexandria, Egypt. P.O. Box 21561.

Egypt. J. Schistosomiasis Infect. Endem. Dis. Vol. 31 Jan. 2009: 79-84

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Schistosoma mansoni from a Hepatitis C Virus (HCV) Endemic Region in Egypt Test Negative for HCV

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The present study was done with the aim to further

investigate the role of S. mansonias a carrier of HCV by

detection of the 5' UTR region of the virus in the Egyptian

laboratory stain of S. mansoni from TBRI and in some

eld isolates of the parasite from the northeastern coastal

region of the Nile Delta in the Damietta Governorate.

MATERIAL AND METHODS

Schistosoma mansoni Strains and Samples:

The laboratory strain of S. mansoniwas obtained from

TBRI. A total of 10,000 viable cercariae and 100 viable

adult S. mansoni worms were obtained and stored in

RNAlater (Ambion, Applied Biosystems, CA, USA)

at -20oC till be used for RNA extraction thenHCV RT-

PCR.

In summer 2009, a snail survey was carried out in a

focus of S. mansoni transmission recently discovered

by us in El-Riyad Village, Kafr Saad, the DamiettaGovernorate (N 31.402583, E 31.7041). The prevalence

of theHCV infection in this Governorate is up to 16%

(S.Kamal, personal communication). All the collected

B. alexandrina snails were examined for infection in

the laboratory. Snails were isolated individuallyin wells

of 24-well cell culture plates placed underlight for two

hours and microscopically examined for shedding of S.

mansonicercariae. All shedded cercariae were pooled and

some of them were used immediately for mice infection

and the remainder was stored in RNAlaterat -20oC until

RNA extraction.

Mice Infection and Collection of Worms:

Some of the eld cercariae were used to infect ten male

Swiss albino mice of matching age (8 weeks) and weight

(202g). Mice were obtained from TBRI. Each mouse was

infected with 100 S. mansonicercariae by using the body

tail immersion technique (Oliver and Stirewalt, 1952).

Seven weeks after infection, mice were sacriced and

adult S. mansoniworms were recovered from the hepatic

portal system and the liver by the perfusion technique

(Smithers and Terry, 1965). Schistosoma mansoniworms

were collected, pooled and stored in RNAlaterat -20oC

to be used for RNA extractionthen HCV RT-PCR.

Positive Controls:

As there are no S. mansonicercariae or worms conrmed

positive for HCV, we prepared positive controls using

human serum samples positive for HCV that were mixed

and added to a pool of 10,000 cercariae and a pool of 100

adult worms of theTBRI laboratory strain. The volume

of the added sera wascalculated to give HCV RNA nal

concentration of 1000 IU/mL after RNA extraction of the

two pooled controls (Castelain et al. 2004; Germer et al.

2005).

RNA Extraction:Cercariae and adult worms of S. mansoni were

homogenized and subjected to total RNA extraction by

using Qiagen QIAampRNA Blood Mini Kit (Qiagen,

Hilden, Germany). Briey, cercariae or worms of each

strain were pooled, homogenized and lysed together with

4L internal control (RNA segment amplied with a

differentset of primers and probe, all provided by the kit)

using Buffer RLT--mercaptoethanol. Then they were

centrifuged and 350L of 70% ethanol were added to the

supernatant mixed well and transferred to QIAamp spin

column, then centrifuged for 15 sec at 8000 g. The column

was washed with 700L Buffer RW1, then centrifuged

for 15 sec at 8000g followed by two washing steps using

500L of Buffer RPE. Finally, 40L of RNase free water

were used to elute RNA.

Detection of HCV RNA by RT-PCR:

Ten L of extracted RNA were amplied using TaqMan

probe technique and primers encoding 5' UTR of HCV

(Applied Biosystems, CA, USA). RNA-free water

was used as anegative control. Briey, 0.5L of each

primer and probe labeled with 5' FAM uorescent dyeand 3' TAMRA quencher dye for both HCV and internal

control, 0.25L reverse transcriptase enzyme, 0.25L

AVE buffer and 0.5L RNAse inhibitor were added to

12.5L TaqMan universal PCR master mix (2 folds)

bringing the reaction to a nal volume of 25L. The

amplication prole started by incubation at 48C for 30

min to transcribe viral RNA to cDNA by RT. This was

followed by AmpliTaq Goldactivation at 95C for 10

min, followed by 40 cycles of two-step amplication:

denaturation at 95C for 15 sec, followed by annealing

and extension at 60C for 1min with end point detection.

Software provided in the computer system connected tothe apparatus allows real-time amplication plots to be

viewed and analyzed during the PCR run (Bustin, 2002).

RESULTS

During the present study, a snail survey was conducted

in a focus of S. mansoni recently discovered by us in

Damietta. Among a total of 843 B. alexandrina snails

collected, we found 48 infected snails (5.69% infection

rate). About 71,370 cercariae were shed from the infected

snails. All cercariae were pooled. About 1000 cercariae

were used immediately for mice infection, and about

70,370 cercariae were stored in RNAlaterat -20oC untilRNA extraction. After mice perfusion, a total of 183

adult (94 females and 89 males) S. mansoniworms were

collected, pooled and stored in RNAlaterat -20oC until

RNA extraction.

During the present study, two strains of S. mansoniwere

examined by RT-PCR for detection of the HCV 5' UTR

region. Two pooled samples of cercariae or worms from

each strain were investigated. The laboratory strain

samples were composed of a pool of 10,000 cercariae

and a pool of 100 adult worms. The eld strain samples

consisted of a pool of 70,370 cercariae and a pool of183 adult worms. According to the present results, the

technique used could not detect any traces of HCV RNA

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Lotfy et al.

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in any of the studied four experimental samples. However,

the two positive controls were positive for HCV, and the

two negative controls were negative for HCV. Also, in

all experimental samples and controls the internal control

gave positive results (Table 1).

Table 1:Results of HCV RNA detection in the studied experimental samples and controls.

Schistosoma mansonistrain HCV RNA Control

TBRI (laboratory) Damietta (feld) Positive Negative

Stage

Cercariae Negative(*) Negative(*) Positive(*) Negative(*)

Adult worms Negative(*) Negative(*) Positive(*) Negative(*)

(*) In all experiments the internal control gave positive results.

DISCUSSION

The present results could not prove that S. mansoni

cercariae and worms act as biological vehicles for direct

transmission of HCV. The present results are not inaccordance with the two previous reports (Abdel-Moeti

et al. 2001; Soliman et al. 2008). The reported positive

results by the previous studies may be due to some kind

of contamination. Future investigations may further

resolve this issue.

By literature review it was found that a virus infection

was morphologically demonstrated in a rhabdocoele

turbellaria, Gyratrix hermaphroditis (Reuter, 1975;

Tinsley and Harrap, 1978). Rhabdovirus-like particles

were also morphologically detected in the sporocyst

stage of the trematode Brachylaimus fuscatus fromthe hepatopancreas of the terrestrial snail Ponsadenia

duplocincta. It is not known how and where the sporocysts

get the viral infection and what is the ontogenesis of

the virus. The virus has never been morphologically

demonstrated in the cercariae, which leave the sporocyst

and continually turn into subsequent developmental

stages (metacercaria and hermaphroditic adult). The

virus was not even found in the host tissues surrounding

the sporocyst (Zdarska et al. 1986). Therefore, this

nding could not demonstrate the role of B. fuscatus

in transmission of this virus. It may be reasonable to

believe that the epidemiological association between

S. mansoni and HCV is something other than that theformer is working as a biological carrier of the latter.

This high epidemiologicalassociation between S. mansoni

and HCV in Egypt could be attributed to the transmission

of the HCV via sharing contaminated syringes, which

were used to inject tartar emetic in systematictreatment

of the population in the 1960s and 1970s (Frank et al.

2000; Lavanchy and McMahon, 2000). This hypothesis

could be supported by the ndings that the prevalence

of anti-HCV is higher in Lower Egypt (>15%), where

tartar emetic was used more extensively and several

years longer to treat S. mansoniinfection, than it was inUpper Egypt (

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Financial Support:

This work was partially supported by the U.S.-Egypt

Joint Science and Technology Fund, grant no. BIO9-005-

002.

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