Excretory- Secretory Antigens of Fasciola hepatica in Diagnosis

of Human Fasciolosis

In the name of God

Fasciola hepatica

• The common name of this parasite, the "sheep liver fluke "

• It has been estimated that up to 17 million people are infected and that another 180 million are at risk

• Fasciola hepatica, a trematode parasite of ruminant and human

• The disease is established by eating contaminated plants with infective metacercaria which are derived from an intermediate molluscan host .

Early diagnosis of this disease is very important in order to treat patients successfully.

Diagnosis

• Parasitological diagnosis

• Serological diagnosis

Parasitological diagnosis

• Using this method eggs are not detected until the latent period of infection when much of the liver damage has already occurred.

• In addition, eggs are released sporadically from the bile ducts and hence stool samples of infected patients can contain no eggs

At present bellow methods are being considered in diagnosing of the human fascioliasis

• haemaglutination (HA)

• indirect fluorescence antibody test (IFAT)

• counterelectrophoresis (CEP)

• enzyme–linked immunosorbent assay (ELISA)

• EITB or Western blotting

Serological diagnosis

Preparation of antigens

F. hepatica were collected from the bile ducts and washed 6 times in 0.01 M phosphate buffered saline (PBS) at 37 ¡C. The flukes were then left for 2-3 h in PBS at 37 ¡C to allow regurgitation of the cecal contents. The viability of flukes after this incubation was normally 100%. The flukes were then harvested and used to prepare excretory/secretory (E/S) extracts as follows. E/S product isolated parasites were washed 6 times with 0.01 M (PBS), pH 7.2, and a further 6 times with RPMI- 1640 medium. The worms were then incubated in cellcultured flasks in RPMI-1640 medium with 100 IU of penicillin and 100 µg of streptomycin per milliliter of medium (1 fluke per 3 ml) at 37 ¡C in a 5% CO2 incubator for 24 h. After the incubation, the supernatant was collected and centrifuged at 5000 g for 30 min at 4 ¡C. The samples were aliquoted and stored at – 70 ‘C.

Refrence : Dalton JP, Heffernan M (1989). Thiol protease released in vitro by Fasciola hepatica. Mol Bio Para, 35:161-66.

ES antigen showed less cross-reaction cases than somatic one

In similar immunodiagnostic studies on this parasite, it was demonstrated that ES antigen was more specific than other somatic and surface antigens .

In Western blotting studies using excretory/secretory antigens in humans infected with F. hepatica, it was determined that bands 12, 17, 25, 27, 29, and 49.5 kDa were specific for humans .

Hammami demonstrated that two bands of 29 and 57 kDa could be recognized by infected sera.

29 and 57 kDa

Refrence :Hammami H, Ayadi A, Camus d, Dutoit E (1997). Diagnostic value of the demonstration of specific antigen of Fasciola hepatica by western blot technique. J Parasit, 4(3): 291-95.

sensitivity specificity

29 kDa 93 % 100 %

57 kDa 57 % 100 %

Intapan accounted the sensitivity, specificity, positive and negative predictive values for 27 kDa protein band yielded from the ES antigen as 100%, 98%, 66.7% and 100%.

Refrence : Pew pan M Intapan, Wanchai Maleewong, Wongkham C, Tomana karn k, Leamviteevanic k, Pipitgool V, Sukolapong V (1998). Excretory-secretory antigenic components of adult Fasciola hepatica recognized by infected human sera. Southest Asian J Trop med Public health(29).

27 kDa

sensitivity specificitynegative

predictive values

Pasitive predictive

values

27 kDa 100 % 98 % 66.7 % 100 %

17 & 23 kDa

sensitivity specificitynegative

predictive values

Pasitive predictive

values

ES Ag 100 % 98 % 66.7 % 100 %

Refrence : WESTERN BLOT TECHNIQUE STANDARDIZATION OF THE DIAGNOSIS OF HUMAN FASCIOLOSIS USING Fasciola hepatica EXCRETED-SECRETED ANTIGENS

sensitivity specificity

negative predictive

values

Pasitive predictive

values

ES Ag 95.2% 98% 96.2% 97.5%

29 and 27 kDa

Refrence : *MB Rokni, A Baghernejad, M Mohebali, EB Kia(2004) Enzyme-Linked Immunotransfer Blot Analysis of Somatic and Excretory- Secretory Antigens of Fasciola hepatica in Diagnosis of Human Fasciolosis

60-66 kDa

Ortiz et al are reported that antibody responses were developed against 60-66 kDa in E/S and SU antigens and 17 kDa in SO antigen.

Refrence : Ortiez PL, Claxton JR, Clarkson MJ, McGarry J, Williams DJL (2000). The specificity of antibody responses in cattle naturally exposed to Fasciola hepatica. Vet Parasit, 93; 121-34.

Using E/S antigens, recounted that anapproximately 15 kDa F. hepatica E/S antigenscan be used for species diagnosis in cattle.

Qureshi et al

Qureshi T, Wagner G, Lynn Drawe D, Davis D, Craig T (1995). Enzyme linked Immuno transfer blot analysis of excretory-secretory proteins of Fasioloides magna & Fasciola hepatica. Uet parasit, 58 :357-63.

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