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Abstract Cystic echinococcosis (CE) caused by the larval stage of Echinococcus granulosus is a re-emerging problem with great economic and medical impact in developing countries including Egypt and it was listed by WHO as one of the neglected tropical diseases. E. granulosus has a number of intraspecific variants or strains which have been characterized by morphological, biochemical, biological, developmental and molecular approaches. They were identified and classified by mitochondrial and nuclear markers into ten genotypes (G1 to G10) namely E. granulosus sensu stricto (s.s.) (G1–G3), Echinococcus equinus (G4), Echinococcus ortleppi (G5) and Echinococcus canadensis (G6–G10) and the lion strain (Echinococcus felidis). These strains shows differences in geographical distribution, host specificity, ecology, transmission dynamics, development rate, infectivity to human, antigenicity and sensitivity to chemotherapeutic agents. Camels have been involved in the transmission cycle and the epidemiology of E. granulosus especially in rural communities where dogs (definitive host) are infected by ingestion of infected camel carcasses containing the hydatid cysts. However the most common strain infecting human was known to be G1, recent molecular characterization of human and animal isolates demonstrated the involvement of the camel G6 strain in causing human infection. To detect the specific strain in any endemic locality is a must to apply the most suitable control programmes. CE is diagnosed by different methods; immunological and serological tests, radiological and by molecular techniques as PCR which showed high sensitivity and specificity. Detection of circulating E. granulosus antibodies in patients’ sera remains the method of choice and showed greater sensitivity than detection of circulating antigens in diagnosis of CE. Standardization of techniques of antigen preparation and characterization of new antigens still needed in order to improve the immunodiagnosis of CE. The present work aimed to study and compare the protein profile of HCF obtained from different Egyptian CE patients as well as different livestock reservoir animals with identification of antigenic proteins. Moreover, probing HCF and patients’ sera for the detection of the G1 or G6 genotype. This aims to identify new antigens, which may provide information about parasite survival strategies in the host and thus improving CE diagnosis, treatment and control. The study was conducted on 90 subjects of both sexes; 70 patients and 20 healthy individuals after taking informed consent as well as animal isolates after permission. Human subjects were classified into two groups: group I (CE patients): -group I-A: including cases of hepatic CE -group I-B: including cases of pulmonary CE. -group I-C: including cases of CE with multiple organs affection. -group I-D: including cases of CE after medical and/or surgical treatment. They were selected from Abdominal Ultrasonographic Unit of Tropical Medicine Department,Kasr El-Aini Hospital, Department of Cardiothoracic Surgery, Faculty of Medicine, Ain Shams University and Diagnostic and Research Laboratory of Parasitic Diseases in the Department of Medical Parasitology, Faculty of Medicine, Ain Shams University. group II (Control group): -group II-A: including cases of other parasitic infections; schistosomiasis, fascioliasis, taeniasis and amoebic liver abscess. -group II-B: including cases of patients with other mass occupying lesions; hepatocellular carcinoma, simple liver cyst and bronchogenic carcinoma. -group II-C: including cases of apparently healthy individuals with no history of parasitic infections, negative urine and stool examination and negative IHAT for hydatid disease. They were selected from Department of Tropical Medicine, Chest Medicine (pulmonary diseases and tuberculosis), Faculty of Medicine, Ain Shams University and the Diagnostic and Research Laboratory of Parasitic Diseases in the Department of Medical Parasitology, Faculty of Medicine, Ain Shams University. Animal samples (isolates): Cyst from animals (camel and sheep) were obtained from Cairo abattoirs and the fluid was examined for protoscolices. No cysts from Pigs were available. The present study had two parts; molecular and immunological.The Descriptive analysis of the CE cases showed that CE was more common among females (65%) and most of the cases represented in the middle age (the mean is 33.9 years). The molecular part was carried out by purification of HCF of human and animal’s isolates. Deposited protoscolices underwent DNA extraction with subsequent PCR using specific primers for detection of either G6 or G1 genotype. Then DNA sequencing and comparing the results on GenBank was done. The human cases’ HCF were collected by percutanuous aspiration injection reaspiration (PAIR) technique. Using conventional PCR, all the CE cases under the study; hepatic, pulmonary and multiple organ, were of G6 genotype (camel strain); showed band at 254 bp. Regarding the animal isolates, the examined camel cysts were fertile and they were all of the G6 genotype (band at 254 using G6 primers). All examined sheep cysts (30 were negative either microscopically (no protoscolices) and by PCR using G6 or G1 primers. This suggests that the camels are the main animal reservoir of CE in Egypt and the cause of the presented human infections. Also sera of the studied human groups were collected to detect the G1 or G6 genotype, 27 out of 30 samples were G6 genotype showed characteristic band at 254 bp using G6 primers and were all negative using G1. Results of PCR in sera was compared to microscopic examination of the HCF, showed relatively low sensitivity 90% against 100%, however it still represent a reliable tool for CE diagnosis more safe than PAIR technique and microscopic examination of HCF. Moreover, PCR for G6and G1 of CE was negative in all control subjects, giving it a 100% specificity. The results of PCR were confirmed and verified by bidirectional DNA sequencing. The tested isolates showed 100% homology with G6 Japanese strain (Camel) by Nakao et al. (2007) in the GenBank. Also, alignment of the isolates with the Egyptian G6 strain, showed 100% identities and (0) gabs which indicate homology. The immunological part of the study, crude HCF antigens were prepared from the supernatant of HCF of human and animal isolates. Their protein profiles were analyzed by using 12% SDS-PAGE under reducing conditions. After Standardization of protein concentration, 25 µg protein showed the best band resolution. The protein profiles of human and camel isolates were similar composed of 11 bands (164, 130, 110, 80, 75, 48, 35, 25, 20, 18 and 12 KDa) with some differences at the high molecular weight bands; the164, 130 KDa were absent in all camel isolates. Using human crude HCF antigen pool, EITB revealed that anti E. granulosus total IgG of the hyperimmune rat serum reacted with 9 antigenic bands, varying in molecular weight from 110 kDa to 12 kDa (110, 80, 75, 65, 56, 48, 35, 25 and 12). Statistical analysis of these molecular weights bands recognized by total IgG of the studied groups’ sera showed that the 48-kDa and 12-kDa protein bands reacted against 30 (100%) of the patient’s sera IgG. None of the sera from control groups (GpΠ) recognized the 12 KDa band indicating a highly significant difference but the 48 KDaband was only recognized by 2 sera of control group of other parasitic infections. Also, 35 kDa bands reacted against 20 (66.7%) of CE cases versus 2 (4%) of schistosomal sera (GpΠ-A). Moreover, 75, 56 and 25 KDa bands reacted with 7 (23.3%), 3(10%) and 10 (33.3%) of the CE patients’ sera, respectively and none of the control sera recognized these bands which indicating a highly significant difference (p<0.01). On contrast, 110, 80 and 65 KDa bands were not recognized by both CE cases and control groups. Also, EITB was used in the present study to follow up patients after medical or surgical treatment, sera of 10 CE cases 6-12 months after treatment were blotted against human crude HCF pool antigen. It showed absence of all antigenic bands except 35 KDa band which was recognized by 4 (40%) patients (P value: <0.01) indicating a highly significant difference. On statistical analysis of the recognition of each protein band by anti E. granulosus IgG using human crude antigen pool showed that 12 kDa gave the highest sensitivity (100%) and specificity (100%). Moreover, it wasn’t detected in any sera of patient after treatment making it sensitive to be used in the assessment and follow up of treatment. On conclusion, G6 genotype was detected in all human CE cases as well as camel isolates. Moreover, EITB was used to identify antigenic proteins of human crude HCF to be used in diagnosis and post- treatment follow up of CE patient. The test showed high sensitivity and specificity in the identification of potent antigens. The 48 and 12 KDa antigenic proteins were detected in 100% of CE cases’ sera and disappeared 6-12 months after treatment making them sensitive for the use in diagnosis and post treatment follow up. |