Evaluation of the Stability of Coated Plates with Antigen at Different Temperatures and Times by ELISA Test to Diagnose Fasciolosis

"nBackground: Considering that ELISA method presently is the test of choice for diagnosis of fasciolo­sis, the present study was undertaken to evaluate the maximum validity of coated plates at dif­ferent temperatures and different times during one year of evaluation."nMethods: Serum samples of patients infected with fasciolosis (n=10), hydatidosis (n=5), toxocaria­sis (n=5), and negative control sera (n=5) were examined. Two series of plates were consid­ered. The first series were coated with Fasciola homogenate Ag 12 ug/ml, and after some steps were blocked with gelatin and preserved at different temperatures as -80 °C , -20 °C, -4 °C and +4°C. The 2nd series were treated under the same criteria but were not blocked with gelatin. Each series were examined by ELISA test from 1st month to 12th month. Sera with 1:125 dilution, and peroxidase-conjugated goat anti-human IgG diluted 1:10000 were considered optimum."nResults: To ease reporting the results and due to many similarities only results related to 1st, 6th and 12th months were analyzed and sensitivity, specificity plus cut-off were determined for each series separately. "nConclusion: Preserving the coated plates, while unblocked at -80°C for 6-8 months is pertinent and functional and in that case, we can be sure the best out put would be applicable

Modulation of the Immune Response to DNA Vaccine Encoding Gene of 8-kDa Subunit of Echinococcus granulosus Antigen B Using Murine Interleukin-12 Plasmid in BALB/c Mice

Abstract Background: The current study was designed to evaluate immune responses induced by DNA vaccines encoding 8-kDa subunit of antigen B (HydI) of Echinococcus granulosus and murine interleukin 12 (IL-12) as genetic adjuvants in BALB/c mice. Methods: Expression plasmid pcDNA3.1 containing HydI (pcHyd1) as vaccine along with the murine interleukin 12 (pcMIL12) as adjuvant were used. Thirty-five mice in the five experimental groups received PBS, empty pcDNA3.1, pcHydІ, pcMIL-12, and pcHydІ+ pcMIL-12 in days zero, 14th and 28th. Two weeks after the last immunization, evaluation of the immune response was performed by evaluating the proliferation of splenic lymphocytes, IFN-γ and IL-4, determination of IgG isotyping titer. Results: Mice that received the pcHydI+pcMIL12 exhibited higher levels of lymphocyte proliferation compared to mice that received the pcHydI alone (P<0.001), and produced significantly more IFN-γ in comparison to other groups (P< 0.001). In addition, they produced significantly less IL-4 than mice receiving the PBS and the empty plasmid (P<0.023). The IgG2a levels were clearly higher in pcHydI+pcMIL12 group in comparison with the groups of pcHydI alone, empty plasmid, and PBS. In contrast, IgG1 was elevated in the group of pcHydI. Conclusion: Co-delivery of IL-12 with DNA encoding 8-kDa subunit of antigen B was effective significantly in inducing the immune response in mice

Specific detection of fasciola hepatica and f. Gigantica in infected domesticated animals using high-resolution melting analysis (HRM)

Background: It is difficult to make an exact morphological distinction between Fasciola hepatica and Fasciola gigantica. We used High Resolution Melting analysis (HRM) method to differentiate the F. hepatica species from F. gigantica in order to differentiate them. Methods: Overall, 80 adult liver flukes were collected from infected slaughtered animals including cattle, sheep and goats from Lorestan Province, western Iran from Sep 2015 to Aug 2017. Genomic DNA was extracted using commercial DNA extraction kit. The multilocus sequences of mDNA including COX1, COX3 and ND6 were amplified employing real-time PCR & HRM analysis. Specific and universal primer pairs were designed for differentiation Fasciola spp. Results: Universal primers cannot be used to distinguish between these two species, but in the contrary, specific primer pairs of each species could differentiate them properly. Molecular identification using specific primer pairs were consistent. Conclusion: HRM is a simple, fast and reliable method for detecting and differentiating F. hepatica from F. gigantica and can be used for diagnostic and epidemiological purposes

Seroprevalence of Human Fascioliasis Using Indirect ELISA in Isfahan District, Central Iran In 2013

Background: The aim of this study was to detect the seroprevalence of human fascioliasis in Isfahan County, central Iran in 2013. Methods: Overall, 471 sera samples were collected from people recalled randomly to 20 health centers in the city of Isfahan and 10 related villages in 2014. Sera were examined using ELISA test. A questionnaire was filled out for each participant. Results: Altogether eight cases (1.7%) were seropositive which had the OD absorbance in ELISA test more than the calculated cutoff of 0.36. All of them were female. One positive subject had a history of consuming Delar (Local dish) and three seropositive cases with history of eating Zeitoon-Parvadeh (Proceeded olive). Conclusion: Isfahan County might be considered as one area apt for fascioliasis.More studies in terms of veterinary investigation and verifying the risk factors are necessary.  

Serological study of Human Fasciolosis in Patients Referring to the School of Public Health, Tehran University of Medical Sciences, Tehran, Iran during 2008-2014

Background: Fascioliasis is a zoonotic disease of livestock and human caused by Fasciola species. Here in, the results of serological evaluation of fascioliasis in peo­ple referring to the School of Public Health, Tehran University of Medical Sci­ences during 2008-2014 are presented. Methods: Demographic characterizations, symptoms and eosinophil rate were registered for every patient. Using somatic antigen of Fasciola, ELISA was per­formed and the results were analyzed. Data of questioners were analyzed as well. Results: Among 206 applicants, 24.8% were seropositive for fascioliasis, included 21% female and 28.3% male. Mean range of age of patients was between 13 to 67 yr. The highest rate of seropositivity was found among 20-30 yr old patients. Most of the patients had hypereosinophilia. All patients had history of eating raw vegeta­bles, or drinking unsafe water. Patients were referring from different provinces of Iran, including Gilan, Mazandaran, Tehran, Ardabil, Khuzestan, Lorestan, North Khorasan, Kermanshah, Azerbaijan, Fars, Kordestan, Hamedan and Markazi. Conclusion: During recent years, variety of provinces in Iran, where patients with fascioliasis are referred, has been increased. Patients coming from Gilan and Mazanda­ran provinces were referred early after the onset of their symptoms. Most probably, physicians in Gilan and Mazandaran are more alert on fascioliasis than other provinces. Previous wrong diagnosis was more common among patients refer­ring from other provinces than Gilan and Mazandaran provinces

Genotyping and Phylogenetic Analysis of Fasciola Spp. Isolated from Sheep and Cattle Using PCR-RFLP in Ardabil Province, Northwestern Iran.

The aim of this study was to detect the genotype of Fasciola spp. in Meshkin-Shahr, Ardabil Province, northwestern Iran in different hosts using PCR-RFLP.The parasite hosts included cattle, and sheep. Overall, 70 adult flukes from livers of slaughtered animals were collected from the abattoirs of aforementioned area. The included 35 samples from infected sheep and 35 samples from 35 infected cattle. PCR-RFLP and sequence analysis of the first nuclear ribosomal internal transcribed spacer (ITS 1) region from Fasciola species were used to conduct the study.The fragment of approximately 700bp in all of the Fasciola samples was amplified. PCR products of ITS 1 were subjected for digestion by restriction enzyme. RsaI restriction enzyme was selected for RFLP method that caused the separation specifically of Fasciola species. Amplicons with the sequences of F. hepatica had a pattern of about 360, 100, and 60 bp band size, whereas F. gigantica worms had a profile of 360, 170, and 60 bp in size, respectively. Results based on PCR-RFLP analysis were confirmed by sequence analysis of representative ITS 1 amplicons. No hybrid forms were detected in the present study. All sheep were infected with F. hepatica but cattle were infected with both species.Both species of Fasciola are present in Ardabil. The method described here can be valuable for identification of Fasciola species in endemic parts for fasciolosis, regions with intermediate species and in that overlapping distribution area

Seroprevalence of Human Fasciolosis in Lorestan Province, Western Iran, in 2015-16

Background: The aim of this study was the seroepidemiological survey for detecting the status of human fasciolosis in Lorestan Province, western Iran. Methods: This cross-sectional study was conducted in 2015-16. Based on statistical estimations, 1256 serum samples were collected from different parts of Lorestan Province, western Iran, and stored at -20 °C until use. The collected serum samples were analyzed at Tehran University of Medical Sciences, Tehran, Iran using indirect ELISA method. Results: Anti-Fasciola antibodies were detected in 16 (1.3%) individuals. Regarding the seropositivity to fasciolosis, no significant differences were found between age groups, sex, level of education and occupation; however significant differences were observed regarding location, consuming local freshwater plants and water resources (P<0.02.) Conclusion: Local freshwater plants and unfiltered water resources were probably the main sources of the infection. Health education by local health centers to elevate awareness of people, and providing facilities for safer drinking water, especially in rural areas may help decrease the risk of fasciolosis infection in this region

Seroprevalence of Human Fascioliasis in Meshkin-Shahr District, Ardabil Province, Northwestern Iran in 2012

Background: The aim of this study was to conduct a seroprevalence survey in Meshkin-Shahr, Ardabil Province, north western Iran to detect the rate of human fascioliasis in the city and nearby villages. Literature shows that no such study has been conducted so far. Methods: Overall, 458 serum samples were collected by randomized cluster sam-pling method from 153 males and 305 females referred to different health centers of the region after recalling by staff in those centers in 2012. All cases filled out a questionnaire and an informed consent. Sera were analyzed using indirect-ELISA test. Ten μg /ml antigens (Liver Fluke Homogenate), serum dilutions of 1:500 and conjugate anti-human coombs with 1:10000 dilutions were utilized to perform the test. Data analysis was conducted using SPSS software ver. 18. Results: Nine cases (1.96%) were positive for fascioliasis by ELISA test. The sero-prevalence of fascioliasis among females was 1.63% and 2.6% in males. There was no significant difference as regards age groups, sex, job, residency, literacy and con-suming row vegetable. According to job, unemployment subjects had the highest rate of infection as 5.9%. The seroprevalence of infection was 1.52% in illiterate people. As for residency, urban life showed no significant difference with rural life (2.4% vs. 1.42). Age group of 40-49 yr old, with 3.3% seropositivity had the highest rate. Conclusion: Obtained seroprevalence of fascioliasis shows immediate attention of health authorities to the diseases in the area. The adjacent of Ardabil Province to endemic areas of fasciolosis accentuates this attention

Comparative evaluation of real-time PCR and ELISA for the detection of human fascioliasis

Abstract Fascioliasis is a zoonotic parasitic infection caused by Fasciola species in humans and animals. Despite significant advances in vaccination and new therapeutic agents, little attention has been paid to validating methods for the diagnosis of fascioliasis in humans. Serological techniques are convenient assays that significantly improves the diagnosis of Fasciola infection. However, a more sensitive method is required. The aim of this study was to compare the Real-Time PCR technique with the indirect-ELISA for the detection of Fasciola hepatica in human. Using a panel of sera from patients infected with Fasciola hepatica (n = 51), other parasitic infections (n = 7), and uninfected controls (n = 12), we optimized an ELISA which employs an excretory–secretory antigens from F. hepatica for the detection of human fascioliasis. After DNA extraction from the samples, molecular analysis was done using Real-Time PCR technique based on the Fasciola ribosomal ITS1 sequence. Of 70 patient serum samples, 44 (62.86%) samples were identified as positive F. hepatica infection using ELISA and Real-Time PCR assays. There was no cross-reaction with other parasitic diseases such as toxoplasmosis, leishmaniasis, taeniasis, hydatidosis, trichinosis, toxocariasis, and strongyloidiasis. The significant difference between the agreement and similarity of the results of patients with indirect ELISA and Real-Time PCR was 94.4% and 99.2%, respectively (Cohen’s kappa ≥ 0.7; P = 0.02). Based on the Kappa agreement findings, the significant agreement between the results of ELISA and Real-Time PCR indicates the accuracy and reliability of these tests in the diagnosis of F. hepatica in humans

Emerging cases of fascioliasis in lorestan province, western iran: Case series report

Fascioliasis is a zoonotic disease caused by Fasciola spp. We report five serologically and molecularly confirmed cases in an emerging region in Iran. A retrospective, case series study, performed in Lorestan Province, west of Iran between January 2015 and June 2016. From 1256 patients examined, 16 patients had positive serum ELISA. Five cases were approved as infected with fasciolosis using stool exam and PCR. Age ranged from 24 to 80 yr with mean age of 45 years. All of patients were adults and four of them had abdominal and back pain. Other symptoms included fever and chills, coughing and sore throat, weight loss, cutaneous manifestations. All patients lived in the rural environment, and four reported the ingestion of raw aquatic plants such as watercress. In fecal examination for fluke eggs, four samples were positive for F. hepatica eggs. Conventional PCR analysis showed that five human stools were positive for F. hepatica. All of 5 patients were treated with the usual dose of triclabendazole. A history of recent consumption of raw aquatic plants (in 4 out of 5 patients) is an important finding, but in one patient the source of infection remained unclear. Lorestan should be considered as an emerging region for this disease and further research in this province should be carried out