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human CE and AE

3.1 Introduction

Echinococcosis is a worldwide zoonosis caused by the larval stages of tapeworms

(cestodes) belonging to the genus Echinococcus (family Taeniidae).

Echinococcus granulosus and Echinococcus multilocularis, which cause human

cystic echinococcosis (CE) and alveolar echinococcosis (AE) respectively, are highly endemic in China (Wen and Yang, 1997; Craig, 2004). Both cause serious and potentially life-threatening diseases, the latter especially with high fatality rates and poor prognosis if not diagnosed and treated in the early stages (Zhou et al., 2000; WHO/OIE 2001; Craig et al., 2003). Mixed CE and AE cases are rare but have also been reported in China (Wen et al., 1992; Yang et al., 2006a; Yang et al., 2006b). Currently, mortality for human CE may vary between 0.2% and 4.5%, and for human AE 10-15% (Wen and Yang, 1997; WHO/OIE 2001; McManus et al., 2003; Zhang et al., 2003).

Early diagnosis of human echinococcosis is difficult because CE and AE patients usually have no signs or symptoms during the first few years of infection. Human echinococcosis commonly comes to the attention of clinicians because of non-specific clinical signs (e.g. upper abdominal pain, jaundice, allergic reactions) or due to incidental image findings of echinococcal cysts or lesions, or after specific mass-screening surveys by ultrasound and/or serology (WHO 1996; WHO/OIE 2001; Zhang and McManus, 2006).

The frequent difficulty in obtaining a definitive diagnosis is one reason why immunological methods have played an important role of diagnosis of human echinococcosis (Wen et al., 1995, Rogan and Craig 1997, 2002; WHO/OIE 2001). Almost all traditional immunodiagnostic methods (e.g. Casoni intradermal test, complement fixation test, indirect haemagglutination test, indirect immunofluorescence antibody test, immunoelectrophoresis (IEP), and latex agglutination test), have now been replaced by the enzyme-linked immunosorbent assay (ELISA) and/or immunoblotting (IB) which are commonly performed in routine laboratory diagnosis of human echinococcosis (Rogan and Craig, 2002;

Craig et al., 2003). Hydatid cyst fluid lipoprotein antigen B (AgB) from E. granulosus, and Em2/Em2plus, and/or Em18 antigens from E. multilocularis, are

currently considered to be the most specific native or recombinant antigens for immunodiagnosis of human CE and AE respectively (Gottstein et al., 1987; Ito, 2002; Zhang et al., 2003; Carmena et al., 2005, 2006).

Although ELISA and immunoblot are very useful laboratory tests for human echinococcosis, a rapid immunological method that can be used for initial diagnosis of clinically suspected CE or AE, and that could be applied in community screening, would be extremely convenient. Rapid serological test formats such as dot-ELISA have been previously assessed for both human CE and AE, and although useful in conjunction with mass ultrasound screening, they were temperamental and difficult to use and interpret (Zheng et al., 1986; Rogan et al., 1991; Eliades et al., 1998; Qiao et al., 1999; Craig et al., 2000). Dot immuno-gold filtration assay (DIGFA) is a rapid immunodiagnostic test similar to a ‘pregnancy’ test that uses colloidal gold conjugated antibody or antigen instead of enzyme or fluorescence conjugates (Faulk and Taylor 1971; Horisberger et al., 1975; May 1991; Chun and Chu 1989; Xiao et al., 1995). Antigens are attached on a nitrocellulose membrane, and serum or whole blood applied, followed by colloidal gold conjugated anti-human antibodies to give a desired color change to indicate a positive or negative reaction.

In the current study a rapid DIGFA has been developed for human echinococcosis and assessed with four different native antigen-preparations including, E. granulosus crude hydatid cyst fluid antigen (EgCF), hydatid cyst fluid native

antigen B (AgB), an E. granulosus protoscolex antigen extract (EgP), and an E. multilocularis metacestode laminated layerextract (Em2). The test was assessed

in Xinjiang Medical University Hospital (Urumqi, northwestern China), which has treated over 6000 human echinococcosis cases in the last 40 years (Wen and Yang, 1997). The current study showed that the major advantages of DIGFA were rapidity, convenience, and ability to provide initial diagnosis and even differentiation of cystic and alveolar echinococcosis in approximately 80% of cases either in clinical or community screening settings.

3.2 Methods and Approaches

3.2.1 Serum samples and echinococcosis patients 3.2.1.1. Hospitalized hydatid patients

Archived serum panels used in the initial laboratory development and standardization of the DIGFA, were available from 108 post-operative hepatic CE cases, 34 post-operative hepatic AE cases, and 101 healthy controls collected from Xinjiang Medical University Hospital (XMUH) during 1998–2000. In addition 25 sera from cysticercosis (Taenia solium) patients were a gift from Prof. Y.H. Liu, Chongqing Medical University, P. R. China. (Table 3.1).

A serum panel was also available to assess hospital-based diagnosis of DIGFA and compared with standard ELISA. It consisted of 857 CE sera including 717 hepatic CE cases: among them, 516 ultrasound and/or surgery confirmed patients with less than 2 years post-surgery, 64 lung CE cases (diagnosed by X-ray or computerized tomography (CT)), 11 abdominal CE (diagnosed by ultrasound or CT), 18 multi-organ CE (diagnosed by ultrasound and CT) and 47 non-liver/lung CE cases (diagnosed by ultrasound, CT or magnetic resonance imaging (MRI)) (Table 3.2 and Fig. 3.1). In addition, sera from 42 liver AE cases and 1 mixed AE/CE case were assessed. In total 702 serum samples from non-hydatid disease patients were used as negative controls: non-parasite simple cystic disease 153, carcinoma 85, tuberculosis 28, solid or complicated space-occupying lesions (non-echinococcosis by imaging) 266, cirrhosis 6, abscess 13, cysticercosis 3, cholecystitis/gallstones 12, other patients treated in internal medicine (for hypertension, diabetes, and other clinical conditions) 88 and healthy individuals 5 (Table 3.3). All samples were collected and tested in XMUH during the period 1999–2006. For non-endemic controls, 35 sera from healthy people were collected from a hospital in Greater Manchester, UK, which is a non-endemic area.

Table 3.1: Hospital serum samples for developing and application of DIGFA serodiagnosis of human echinococcosis

Duration Where CE AE Mixed

CE/AE