479

Neotrop. Helminthol., 8(2), 2014

2014 Asociación Peruana de Helmintología e Invertebrados Afines (APHIA)

ISSN: 2218-6425 impreso / ISSN: 1995-1043 on line

RESEARCH NOTE / NOTA CIENTÍFICA

SEROLOGICAL PROFILE OF CYSTIC ECHINOCOCCOSIS IN SERA FROM INDIVIDUALS IN

SOUTHERN BRAZIL BY IMMUNOBLOT

PERFIL SEROLÓGICO DE EQUINOCOCOSIS QUÍSTICA EN LOS SUEROS DE INDIVIDUOS

DEL SUR DE BRASIL POR IMMUNOBLOT

1 1 1

Daniel Daipert-Garcia , Leandro Batista das Neves , Simone de Oliveira Mendes ,

1 2 1

Fernanda Barbosa de Almeida , José Roberto Machado-Silva , Rosângela Rodrigues-Silva .

Abstract

Resumen

Keywords: cistic echinococcosis - Echinococcus granulosus - immunoblot - laboratorial diagnosis.

Palabras claves: equinococosis quística - Echinococcus granulosus - immunoblot - diagnosis de laboratorio.

Suggested citation: Daipert-Garcia, D, das Neves, LB, Mendes, SO, de Almeida, FB, Machado-Silva, JR & Rodrigues-Silva, R.

2014. Serological profile of cystic echinococcosis in sera from individuals from southern Brazil by immunoblot. Neotropical

Helminthology, vol. 8, n°2, jul-dec, pp. 479-485.

1Serviço de Referência Nacional em Hidatidose- Laboratório de Helmintos Parasitos de Vertebrados-Instituto Oswaldo Cruz- Fundação Oswaldo Cruz.

2Laboratório de Helmintologia Romero Lascasas Porto- Departamento de Microbiologia, Imunologia e Parasitologia- Faculdade de Ciências Médicas-

Universidade do Estado do Rio de Janeiro.

Rosângela Rodrigues e Silva – tel +55 21 25621485 – Avenida Brasil 4365, Pavilhão Cardoso Fontes, 3ª andar, sala 56.

The cystic echinococcosis, caused by Echinococcus granulosus, is endemic in southern Brazil,

especially in the state of Rio Grande do Sul (RS). Recent advances in imaging technology and in

immunological tests provide powerful and non-invasive tools for its diagnosis based on the

identification of cysts and detection of antibodies. We analyzed the serological profile of the

genus Echinococcus by immunoblot in sera from patients from southern of Brazil who had been

referred to the National reference service in hydatid disease between January 2010 and July 2013.

Among 160 sera samples, 16 were reactive for at least one of the proteins (10, 18, 28, and 40 kDa),

indicating that the pattern of reactivity can vary among the patients.

La equinococosis quística es causada por Echinococcus granulosus, es endémica en el sur de

Brasil, especialmente en Rio Grande do Sul (RS). Los recientes avances tecnológicos de imágenes

y en pruebas inmunológicas proporcionan herramientas potentes y no invasivas para el

diagnóstico con base en la identificación de los quistes y la detección de anticuerpos. En este

trabajo se analizó el perfil serológico para el género Echinococcus por immunoblot en sueros de

pacientes del sur de Brasil, que fueron enviados para el Servicio Nacional de Referencia en

Hidatidosis entre enero de 2010 y julio de 2013. De las 160 muestras de suero, 16 fueron reactivas

durante al menos una de las proteínas (10, 18, 28, y 40 kDa), lo que indica que el patrón de

reactividad puede variar entre los pacientes.

Daipert-Garcia et al.

Serological profile of cystic Echinococcosis

The genus Echinococcus, Rudolphi 1801,

comprises tapeworms belonging to the

Taeniidae family. Based on light microscopy

studies, four species that affect human are

regarded as valid taxa within this genus (Eckert

& Deplazes, 2004). All species present

heteroxenous life-cycle, in which carnivores

have a role as the definitive host by possessing

adult worms in the small intestine, while

herbivores are the intermediate hosts, in which

the larval stage (metacestode) develops, mainly

in the liver. Echinococcus transmission is

principally maintained in a carnivore-herbivore-

carnivore cycle in rural areas (Joshi et al., 1997).

Echinococcosis or hydatid disease is a zoonosis

considered by the World Health Organization

(WHO) as an emerging or re-emerging disease

of increasing concern (Moro & Schantz, 2009).

In South America, the disease is endemic in the

southern cone of the continent, including the

south of Brazil, and in the Andean region

(Gavidia et al., 2008), where sheep farming is

the main economic activity (De La Rue, 2008).

Because humans and carnivores share the same

environment, people can become infected with

Echinococcus eggs eliminated with carnivore

(Lamberti et al., 2014). Humans are aberrant

hosts and infection can result in life-threatening

illness (Eckert & Deplazes, 2004). Herbivores

become infected by a similar mechanism. The

disease caused in humans and herbivores is

named echinococcosis. Animal infection causes

economic losses through the unsuitability of

infected livers for marketing (Valiyeva et al.,

2013; Cardona & Carmena, 2013).

The larval stage, commonly called aqueous

vesicle or blister, consists of few or numerous

spherical to sub-spherical isolated or contiguous

vesicles macroscopically characterized as clear

fluid-filled spheres. Histological examination

reveals that these cysts contain a thicker outer

laminated non-cellular layer (adventitious layer)

composed of inflammatory cells and collagen

deposited by host cells (Lewall & McCorkell,

1986). This layer is composed of carbohydrates

and exhibits physiological and immunological

activities, delivering antigens to the host and

acting as a barrier against the immunological

response (Gottstein & Hemphill, 1997). In

general, it is treated with anthelmintics and, in

many cases, surgery becomes necessary to

remove the cyst (Eckert et al., 2000).

Humans produce a significant immune response

during chronic cystic echinococcosis infection

(Siracusano et al., 2012). Serological tests use

whole-crude fluid isolated from metacestodes

from Echinococcus granulosus infection (Moro

& Schantz, 2009). The serological profile in

individuals living in an endemic area of Uruguay

analyzed by the enzyme linked immunosorbent

assay (ELISA) has been reported (Hernández et

al., 2005). However, the situation in other

endemic regions of South America is poorly

understood. In Brazil there are few data on the

occurrence of the disease. Early detection and

use of an accurate method is fundamental

(Pawlowski et al., 2001).

Echinococcus species share common antigens

that can be used to diagnose all types of

echinococcosis infections (Moro & Schantz,

2009). In this study, we applied an immunoblot

technique using the total crude antigen extracted

from E. granulosus hydatid fluid obtained from

sheep to detect infection by E. granulosus in

human serum samples from southern Brazil

received by the National reference service in

hydatid disease between January 2010 and July

2013.

Serum samples collected during January 2010

and July 2013 from patients with suspected

cystic echinococcosis by Central Public Health

Laboratory were submitted to National

Reference Service in Hydatid Disease (Instituto

Oswaldo Cruz, Fundação Oswaldo Cruz,

Brazil). This work was approved by the IPEC-

Fiocruz institutional review board (nº

0002.1.000.009-04).

A total of 160 human serum samples were tested

INTRODUCTION

MATERIAL AND METHODS

480

Neotrop. Helminthol., 8(2), 2014

for the presence of echinococcosis IgG

antibodies using an immunoblot assay. In brief,

four reactive bands were considered markers of

a positive reaction (Pereira, 2014). These bands

corresponded to molecular weights of 10, 18, 28,

and 40 kDa and were present in sera from

patients with surgically confirmed disease.

Of the total of 160 samples, 153 were from

patients living in the state of Rio Grande do Sul

(RS) and 7 from Santa Catarina (SC). Sera were

considered reactive when they had IgG

antibodies recognizing at least one of the four

antigenic proteins used as reference.

Of the 160 samples, all were processed and 16

were considered positive by presenting an

immune response to at least one of the proteins

of interest (10, 18, 28, and 40 kDa). Of these 16

patients, eight were female and eight were male

and the age range was wide, from 2 to 74 years.

All the reactive patients were from

municipalities in Rio Grande do Sul (Fig. 1).

Among reactive samples, nine were positive for

all four proteins, one sample showed reactivity

to three proteins (10, 18 and 40kDa) and six

showed reactivity to only two proteins, five of

which reacted with proteins 28 and 40 kDa, and

only one reacting with proteins 10 and 18 kDa

(Fig.2).

Cystic echinococcosis is a zoonosis of

increasing concern worldwide. In the human

host, accidental ingestion of eggs is followed by

long-term growth of metacestode, resulting in

chronic cyst-forming disease. Although the

initial phase of the primary infection is always

asymptomatic (Eckert & Deplazes, 2004), a

significant immune response is produced

(Siracusano et al., 2012). During this phase,

clinical signs are not pathognomonic and vary

with the rate of parasite growth.

Like other tissue tapeworms, laboratory

diagnosis based on direct parasite visualization

is not available. Therefore, imaging techniques

such as computed tomography (CT), ultrasound

examination (US) and magnetic resonance

imaging (MRI) play a role in diagnosing and

staging the larval parasite (Stojkovic et al.,

2012). Serological tests are useful both for

RESULTS

Figure 1. Origin of samples reactive in the immunoblot test received from southern Brazil. Illustration by Heloisa Diniz, Serviço

de produção e tratamento de imagem, IOC, FIOCRUZ.

DISCUSSION

481

Daipert-Garcia et al.

Serological profile of cystic Echinococcosis

Figure 2. Reactive samples in immunoblot. PC – Positive control, NC – Negative control, 1- Reactive sample for 4 bands, 2 -

Reactive sample for 3 bands, 3/4 - Reactive sample for 2 bands.

confirmation of suspected cases and

epidemiological investigation (Zhang et al.,

2012).

Here, we utilized an immunoblot assay using

crude antigens from bovine hydatid cyst fluid for

the diagnosis of cystic echinococcosis. This

approach deserves comments.

First, this tool was successfully used in Peru

(Moro et al., 2005) and Uruguay (Barbieri et al.,

1998).

Second, although our aim was not to answer

epidemiological questions, the results confirm

that human infection is not limited to Santana do

Livramento (Larrieu & Zanini, 2012) or the

border with Argentina (Monteiro et al., 2014).

Third, it is known that species of Echinococcus

share antigens. Thus, in those areas where more

than one species is present, the cross-reactivity

between them could be a problem for the correct

diagnosis using immunological tests (Al-Yaman

& Knobloch, 1989). However, the geographical

distribution of E. granulosus and E. vogeli

(species with occurrence in Brazil) are quite

different (D'Alessandro, 1997). The latter occurs

in the northeast region and causes polycystic

echinococcosis, while the former is distributed

in the southeast and cause cystic echinococcosis.

Fourth, some researchers have made efforts to

purify and identify specific antigens from

Echinococcus sp., as well as to construct

recombinant antigens, for use in ELISA and

immunoblot assays (Liance et al., 2000; Virginio

et al., 2003) to diagnose echinococcosis

infections. However, these tests are not yet

routinely used and have not been tested for all

species of the genus.

Fifth, in other helminth infections heterologous

antigens have been useful for immunodiagnosis.

Due to the similarity shared between Taenia

crassiceps (Zeder, 1800) and Taenia solium

(Linnaeus, 1758) molecules, obtaining antigen

extracts from T. crassiceps has been a good

alternative source of antigens for

immunodiagnosis of neurocysticercosis (Peralta

et al., 2010). Obtaining pigs naturally infected

with T. solium larvae is often difficult, but the

larval form of the species T. crassiceps

reproduces asexually by intraperitoneal passage

through Balb / c mice (Gomes et al., 2007;

Peralta et al., 2002, 2010).

Another successful use of heterologous antigens

occurs in the diagnosis of strongyloidiasis

(Machado et al., 2001). The most important

limitation of immunodiagnosis is the difficulty

482

Neotrop. Helminthol., 8(2), 2014

of obtaining infective larvae of Strongyloides

stercoralis (Bavay, 1876), for use in the

preparation of antigens. For this reason, studies

have been performed using antigens of infective

larvae of Strongyloides ratti (Sandground, 1925)

and S. venezuelensis (Brumpt, 1934) to develop

serological methods (Rodrigues et al., 2004).

This study confirms that crude antigens of E.

granulosus can be a useful source of antigens for

immunological reactions to detect specific

antibodies in patients with hydatid disease, as

well as to show that the pattern of the proteins

can be different from patient to patient.

It is important to remember that although being

an important approach to help diagnosis of

echinococcosis, the reactivity observed in

immunoblot analysis using crude antigens of E.

granulosus cannot serve as a definitive

diagnosis, since it does not differentiate simple

contact with the parasite (Liance et al., 2000), as

well as past infections from current infections.

However, evaluation of all the tools that have the

potential to improve identification of this

zoonotic disease is important. Because of cross-

reactivity, epidemiological information,

working practices of the patient, as well as

complementary image examination are

important to determine a specific diagnosis (Li

et al., 2010). The characterization of antigenic

proteins that are reactive against reportedly

positive Echinococcus sera is needed to improve

the quality of diagnosis. Also, the use of

recombinant proteins is promising. The

possibility of this approach has been proven by

previous studies (Virginio et al., 2003; Li et al.,

2004), but further research is necessary to

evaluate their sensitivity and specificity within

the valid taxa of Echinococcus.

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Received September 11, 2014.

Accepted October 25, 2014.

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