Excretory- Secretory Antigens of Fasciola hepatica in Diagnosis of Human Fasciolosis In the name of...
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Transcript of Excretory- Secretory Antigens of Fasciola hepatica in Diagnosis of Human Fasciolosis In the name of...
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Excretory- Secretory Antigens of Fasciola hepatica in Diagnosis
of Human Fasciolosis
In the name of God
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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 .
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Early diagnosis of this disease is very important in order to treat patients successfully.
Diagnosis
• Parasitological diagnosis
• Serological diagnosis
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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
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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
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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.
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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 .
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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 %
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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 %
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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
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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
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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.
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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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THE END
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