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

Molecular Identification and Differentiation of Fasciola Isolates Using PCR- RFLP Method Based on Internal Transcribed Spacer (ITS1, 5.8S rDNA, ITS2)

Background: In this study, we used both ITS1 and ITS2 for molecular identification of Fasciola species.Methods: The region between 18S and 28S of ribosomal DNA was used in PCR-RFLP method for molecular identification of Fasciola species. Ninety trematodes of Fasciola were collected during abattoir inspection from livers of naturally infected sheep and cattle from Khorasan, East Azerbaijan, and Fars provinces in Iran. After DNA extraction, PCR was performed to amplify region ITS1, 5.8S rDNA, ITS2. To select a suitable restriction enzyme, we sequenced and ana-lyzed the PCR products of F. hepatica and F. gigantica samples from sheep and cattle. Tsp509I fast digest restriction enzyme was selected for RFLP method that caused the separation specifi-cally of Fasciola species. Results: The fragment approximately 1000bp in all of the Fasciola samples was amplified and then digested with the Tsp509I restriction endonuclease. Seventy F. hepatica and 20 F. gigantica were identified of total 90 Fasciola isolates.Conclusion: The new PCR-RFLP assay using Tsp509I restriction enzyme provides a simple, practical, fast, low cost, and reliable method for identification and differentiation of Fasciola isolates

Molecular Diagnosis of Strongyloides stercoralis Infection by PCR Detection of Specific DNA in Human Stool Samples

Background: Strongyloidiasis is mostly an asymptomatic infection and diagnosis of latent infec­tions is difficult due to limitations of current parasitological and serological methods. This study was conducted to set up a PCR-based method for molecular diagnosis of Strongyloides stercor­alis infection by detection of copro-DNA in stool samples.Methods: A total of 782 fresh stool samples were collected and examined by agar plate culture. Among those sixteen stool samples, which confirmed to be infected with S. stercoralis were exam­ined as positive control to set up each single and nested PCR, using two primer sets design­ing to amplify partial ribosomal DNA of S. stercoralis genome. Since, single PCR method yielded higher efficacy in detecting positive samples, in the second step, 30 stool samples, which found negative for S. stercoralis by agar plate culture of single stool sample, were examined by sin­gle PCR. Data analysis was performed using McNemar's χ2 test, with consideration of a P-value of <0.05 as indication of significant difference.Results: In amplification of DNA extracted from stool samples, single PCR detected S. stercor­alis DNA target in all 16 positive samples, while nested PCR amplified DNA in only 75% of sam­ples. In the second step, single PCR amplified S. stercoralis extracted DNA in 5 out of 30 sam­ples which were negative by coproculture.Conclusion: Single PCR method amplifying a short (100bp) target represented more efficacies for detection of S. stercoralis in faecal examination compared to agar plate culture and nested PCR, which amplified longer target

Ocular Dirofilariasis, a Case Report

Accidental infection with animal filarial worms in humans is a dilemma for clinicians and parasitologists throughout the world. To date a variety of such rare parasitoses have been reported mostly in tropics and subtropics. Human dirofilariasis is among those unusual zoonotic infections that occasionally have been observed in the eye and in subcutaneous areas exhibiting with nodule formation. Filarial worms are transmitted to humans through invertebrate biological vectors such as certain species of mosquitoes. The present report describes a peculiar case of ocular dirofilariasis in a 49-year-old man resident in Iran

Establishment of Hymenolepis diminuta Life Cycle to Provide Parasite Mass Production

Background: The main object of this experimental work was to practise laboratory production both adult and the larval stage of Hymenolepis diminuta with conventional modification to make further studies easier.Materials & Method: Adults H. diminuta were collected from urban rats in Tehran, Iran. The beetles became infected using blended gravid segments with flour as bait. Cysticercoids have been saved after precise dissection of invertebrate hosts. The exposure of infected beetles to labora­tory rats was performed to establish the life cycle. Result: Out of 57 collected rats, three rats were infected with H. diminuta. Almost all exposed beetles found infected with the larval stage of parasite. About one-month later H. diminuta eggs were seen in stool examination of laboratory rats.Conclusion: Rare human occurrence of H. diminuta along with light level of clinical manifesta­tion of this parasite, underestimate the concerns toward its public health importance. Nowadays, various field of studies, such as biochemistry with special focuses on the capability of H. di­minuta tegument absorption have performed apart from parasitological views alone. In the pre­sent study, establishment of this parasite life cycle has practically provided the access of adult and cysticercoid stages of the tapeworm in further researches

Serological Evaluation of EgAgB16 kDa, a Recombinant Antigen from Echinococcus granulosus for Diagnosis of Human Hydatidosis

Background: Regarding that accurate diagnosis of human hydatidosis still needs more investigations, the present study was conducted to clone, express, and evaluate the gene encoding AgB subunits (EgAgB16 kDa) from Echinococcus granulosus (Iranian G1 strain) and its evaluation by ELISA test.Methods: DNA was extracted from protoscoleces and was utilized by PCR for strain identification. Total RNA was prepared with RNeasy protect mini kit from E. granulosus (Iranian G1 strain) protoscoleces collected from naturally infected sheep with hydatid cyst. Recombinant AgB16 kDa was produced using pETDuet as vector and evaluated by ELISA method. A panel of sera including hydatid cyst-infected individu­als (n=72), healthy individual (n=48), toxoplasmosis (n=4), strongyloidosis (n=4), kala-azar (n=5) and tuberculosis (n=5) were examined using this recombinant antigen.Results: Recombinant protein was purified by affinity chromatography using His-Tag column. After purifica­tion, recombinant protein was confirmed by western blot analysis using His Tag monoclonal anti­body or hydatid positive human serum. The sensitivity, specificity; positive and negative predictive values were calculated as 93.5%, 95.6%, 96% and 92.9%, in that order. The cut-off point was detected 0.3 for rAgB16. Conclusion: While the produced recombinant AgB16 kDa showed promising results in diagnosing human hy­datidosis, but more investigations should be implemented to reach an accurate gold standard

Seroepidemiological Study of Human Hydatidosis in Meshkinshahr District, Ardabil Province, Iran

Background: The aim of this study was to conduct a sero-epidemiological survey in Meshkinshahr, Arda­bil Province, northwestern Iran to detect the rate of hydatidosis in the city and nearby villages. Literature shows that no such study has been conducted so far.Methods: Overall, 670 serum samples were collected from 194 males and 476 females from patients re­ferred to different health centers of the region. All patients filled out a questionnaire and an informed con­sent. Sera were analyzed using indirect-ELISA test. Ten μg /ml antigens (Antigen B derived from hydatid cyst fluid), 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. 11.5. Results: The seroprevalence of human hydatidosis was 1.79% by ELISA test in the region. This rate for fe­males was 1.68% and males 2.6%, respectively. There was no significant difference as regards all fac­tors studied and the seropositivity. According to job, farmers and ranchmen had the highest rate of infec­tion as 3.17%. The sero-prevalence of infection was 2.6%% in illiterate people which showed the highest rate. As regards residency, urban life showed no significant difference with rural life (1.1% vs. 2.58%). Age group of 69-90 yr old, with 4.62% as prevalence had the highest rate of positivity.Conclusion: Obtained sero-prevalence of hydatidosis shows more or less a resemblance to other cities of Iran, although due to the specific condition of the city we expected more rate of sero-positivity

Detection of Echinococcus multilocularis in Carnivores in Razavi Khorasan Province, Iran Using Mitochondrial DNA

Echinococcus multilocularis causes alveolar echinococcosis, a serious zoonotic disease present in many areas of the world. The parasite is maintained in nature through a life cycle in which adult worms in the intestine of carnivores transmit infection to small mammals, predominantly rodents, via eggs in the feces. Humans may accidentally ingest eggs of E. multilocularis through contact with the definitive host or by direct ingestion of contaminated water or foods, causing development of a multivesicular cyst in the viscera, especially liver and lung. We found adult E. multilocularis in the intestine and/or eggs in feces of all wild carnivores examined and in some stray and domestic dogs in villages of Chenaran region, northeastern Iran. The life cycle of E. multilocularis is being maintained in this area by wild carnivores, and the local population and visitors are at risk of infection with alveolar echinococcosis. Intensive health initiatives for control of the parasite and diagnosis of this potentially fatal disease in humans, in this area of Iran, are needed

Development and Evaluation of a New Lateral Flow Immunoassay for Serodiagnosis of Human Fasciolosis

Fasciolosis is an important plant-borne trematode zoonosis. This disease is of both clinical and veterinary relevance and, according to the WHO, is considered a re-emerging disease that is spreading around the world. Fasciolosis has a serious impact on health because of the large size of the parasite and the effects of the parasite in down-regulating the host immune response. Human fasciolosis can be distinguished by an acute phase, in which the parasite migrates through different tissues, and a chronic phase in which it invades the bile ducts. Here we describe the development of a rapid, simple and inexpensive immunochromatographic diagnostic method, based on the use of a recombinant cathepsin L1 protein, which performs better than other more complex indirect methods, providing similar specificity and higher sensitivity. The simplicity of the method represents a great advantage for the intervention systems applied in different endemic areas by WHO, such as passive case finding (e.g. Vietnam) and selective treatment (e.g. Egypt). Because of its characteristics, the system can be applied to both phases of the disease, and in holo, meso and hyperendemic areas where point-of-care testing is required