Evaluation of a novel Echinococcus granulosus recombinant fusion B-EpC1 antigen for the diagnosis of human cystic echinococcosis using indirect ELISA in comparison with a commercial diagnostic ELISA kit.

Cystic echinococcosis (CE) is a zoonotic parasitic disease caused by the metacestode of Echinococcus granulosus sensu lato (s.l.). A large proportion of the patients are asymptomatic at the early and late stages of the disease. CE diagnosis is mainly based on imaging techniques. Laboratory diagnosis including antibody-antigen (recombinant or fusion recombinant) can be used for the diagnosis and follow up of CE and alveolar echinococcosis (AE), but need optimization and standardization. This study aimed to evaluate the efficacy of a recombinant B-EpC1 (rB-EpC1) fusion antigen comprising B1, B2, B4, and EpC1 antigens of E. granulosus using indirect ELISA in comparison with a commercial ELISA kit for the serodiagnosis of CE. The recombinant protein was expressed in the expression host, E. coli BL21, and purified. This recombinant antigen was then evaluated by indirect ELISA and compared to the commercial CE diagnostic kit (Vircell, Spain). The study samples included 124 human sera consisting of 62 sera of patients with CE, and 62 sera of individuals without clinical evidences of CE and specific anti-CE antibodies in routine indirect ELISA. The diagnostic sensitivity and specificity of the indirect rB-EpC1-ELISA test for detection of specific anti-hydatid cyst antibodies in human CE were 95.2% and 96.8%, respectively. Also, the diagnostic sensitivity and specificity of the commercial ELISA test were 96.8% in this study. Initial evaluation of the recombinant fusion antigen (B-EpC1) was promising for the detection of CE by ELISA in clinical settings. Standardization and evaluation of recombinant fusion protein require further studies.

Zoonotic transmission of Teladorsagia circumcincta and Trichostrongylus species in Guilan province, northern Iran: molecular and morphological characterizations.

BACKGROUND: Parasitic trichostrongyloid nematodes have a worldwide distribution in ruminants and frequently have been reported from humans in Middle and Far East, particularly in rural communities with poor personal hygiene and close cohabitation with herbivorous animals. Different species of the genus Trichostrongylus are the most common trichostrongyloids in humans in endemic areas. Also, Ostertagia species are gastrointestinal nematodes that mainly infect cattle, sheep and goats and in rare occasion humans. The aim of the present study was to identify the trichostrongyloid nematodes obtained from a familial infection in Guilan province, northern Iran, using morphological and molecular criteria. METHODS: After anthelmintic treatment, all fecal materials of the patients were collected up to 48 h and male adult worms were isolated. Morphological identification of the adult worms was performed using valid nematode keys. Genomic DNA was extracted from one male worm of each species. PCR amplification of ITS2-rDNA region was carried out, and products were sequenced. Phylogenetic analysis of the nucleotide sequence data was performed using MEGA 6.0 software. RESULTS: Adult worms expelled from the patients were identified as T. colubriformis, T. vitrinus and Teladorsagia circumcincta based on morphological characteristics of the males. Phylogenetic analysis illustrated that each species obtained in current study was placed together with reference sequences submitted to GenBank database. CONCLUSIONS: The finding of current study confirms the zoonotic aspect of Trichostrongylus species and T. circumcincta in inhabitants of Guilan province. The occurrence of natural human infection by T. circumcincta is reported for the first time in Iran and the second time in the world.

Molecular characterization of human isolates of Strongyloides stercoralis and Rhabditis spp. based on mitochondrial cytochrome c oxidase subunit 1 (cox1).

BACKGROUND: Due to the similarity of Strongyloides stercoralis with free-living nematodes of Rhabditis species they might be miss-diagnosed with each other in microscopical examination of stool samples. The aim of this study was molecular characterization and differentiation of human derived isolates of S. stercoralis and Rhabditis species based on the mitochondrial gene of cytochrome c oxidase subunit 1 (cox1) amplification. METHODS: Using parasitological methods, ten isolates of S. stercoralis and three isolates of Rhabditis spp. were obtained from fresh stool samples of patients and the genomic DNA of the samples were extracted. PCR amplification of cox1 gene was carried out for all the isolates and the products were sequenced. RESULTS: The phylogenetic analysis illustrated that S. stercoralis and Rhabditis spp. isolates were placed in two distinguishable separate clades. Inter-species genetic variation between isolates of S. stercoralis and Rhabditis spp. were ranged from 13.5 to 14.5%. CONCLUSIONS: Cox1 gene was a suitable marker for discrimination of S. stercoralis from Rhabditis spp. retrieved from human in the current study. The availability of gene sequence information will be helpful in the future development and validation of discriminatory PCR-based assays of these nematodes.

Genetic diversity and phylogeography of the elusive, but epidemiologically important Echinococcus granulosus sensu stricto genotype G3.

Cystic echinococcosis (CE) is a severe parasitic disease caused by the species complex Echinococcus granulosus sensu lato. Human infections are most commonly associated with E. granulosus sensu stricto (s.s.), comprising genotypes G1 and G3. The objective of the current study was to provide first insight into the genetic diversity and phylogeography of genotype G3. Despite the epidemiological importance of the genotype, it has remained poorly explored due to the ambiguity in the definition of the genotype. However, it was recently demonstrated that long sequences of mitochondrial DNA (mtDNA) provide a reliable method to discriminate G1 and G3 from each other. Therefore, we sequenced near-complete mtDNA of 39 G3 samples, covering most of the known distribution range and host spectra of the genotype. The phylogenetic network revealed high genetic variation within E. granulosus s.s. G3 and while G3 is significantly less prevalent worldwide than G1, the genetic diversity of both of the genotypes is equally high. We also present the results of the Bayesian phylogeographic analysis, which yielded several well-supported diffusion routes of genotype G3 originating from Turkey and Iran, suggesting the Middle East as the origin of the genotype.

Molecular Phylogenetics of Trichostrongylus Species (Nematoda: Trichostrongylidae) from Humans of Mazandaran Province, Iran.

The present study was performed to analyze molecularly the phylogenetic positions of human-infecting Trichostrongylus species in Mazandaran Province, Iran, which is an endemic area for trichostrongyliasis. DNA from 7 Trichostrongylus infected stool samples were extracted by using in-house (IH) method. PCR amplification of ITS2-rDNA region was performed, and products were sequenced. Phylogenetic analysis of the nucleotide sequence data was performed using MEGA 5.0 software. Six out of 7 isolates had high similarity with Trichostrongylus colubriformis, while the other one showed high homology with Trichostrongylus axei registered in GenBank reference sequences. Intra-specific variations within isolates of T. colubriformis and T. axei amounted to 0-1.8% and 0-0.6%, respectively. Trichostrongylus species obtained in the present study were in a cluster with the relevant reference sequences from previous studies. BLAST analysis indicated that there was 100% homology among all 6 ITS2 sequences of T. colubriformis in the present study and most previously registered sequences of T. colubriformis from human, sheep, and goat isolates from Iran and also human isolates from Laos, Thailand, and France. The ITS2 sequence of T. axei exhibited 99.4% homology with the human isolate of T. axei from Thailand, sheep isolates from New Zealand and Iran, and cattle isolate from USA.

Molecular Characterization of Spirometra erinaceieuropaei from Jungle Cat (Felis chaus) in North of Iran.

PURPOSE: The aim of this study is to conduct a molecular characterization of Spirometra tapeworm from jungle cat (Felis chaus) in Guilan Province, north of Iran using DNA sequence analysis of the mitochondrial cytochrome c oxidase subunit 1 (Cox1) and 12S rDNA sequences. METHODS: Morphological features of the adult tapeworm of Spirometra were evaluated using specific staining and light microscopy. The molecular characterization was performed using partial Cox1 and 12S rDNA regions. Genetic diversity was calculated and phylogenetic trees of the obtained sequences were constructed. RESULTS: Morphological features were compatible with previous description of adult Spirometra erinaceieuropaei. The Cox1 sequence of the specimen showed 100% similarity with S. erinaceieuropaei sequences in GenBank from Korea, China and Iran. Also, the 12S rDNA sequence revealed 99.7% similarity with S. erinaceieuropaei isolates from China and Japan. Intra-species variation within isolates of S. erinaceieuropaei was 0-1.4% and 0-4.6% for Cox1 and 12S rDNA genes, respectively. CONCLUSION: This is the first report of molecular characterization of S. erinaceieuropaei in jungle cat, F. chaus in Iran. Jungle cat probably plays a major role as reservoir host in maintaining of this parasite in this area with favorable climate condition. Needs for further assessment on the role of appropriate hosts, especially intermediate/paratenic hosts as well as the potential risk of human infectivity with sparganosis is emphasized.

Application of Nested-qPCR-High Resolution Melting (HRM) Technology on Strongyloides stercoralis Isolates from Iran.

PURPOSE: Strongyloides stercoralis is a parasite with special characteristics presenting it as a unique nematode. Iran is an endemic area for S. stercoralis. In this study, nested-qPCR-high resolution melting (HRM) technology was applied on some human isolates of S. stercoralis from this country by focusing on evolutionary genetics analysis. METHODS: Twelve human isolates of S. stercoralis were collected from four endemic provinces of Iran. Genomic DNA was extracted from a single filariform larva for every isolate. Using specific primers targeting partial regions in cox1 gene, nested-qPCR-HRM was performed and melting-curve profiles were analyzed alongside the evaluation of genetic proximity and phylogenetic analysis using MEGA7 and DnaSP5 software. RESULTS: The melting temperature (Tm) values of the isolates were 77.9 °C-78.3 °C. All isolates from Guilan, Mazandaran, and Khouzestan Provinces shared Tm values of 78.2 °C to 78.3 °C, while the isolates from Hormozgan Province showed Tm values of 77.9 °C, 78.0 °C, and 78.1 °C. The phylogenetic tree illustrated that the sequences of the current study included nine haplotypes. Tajima's D index analyses showed that cox1 gene in S. stercoralis isolates was negative (Tajima's D = - 0.27). CONCLUSION: The isolates were divided into five temperature groups. Although HRM assay compared to PCR sequencing identified more limited genetic changes, it revealed that the mean of Tm of the isolates from Hormozgan Province was lower than those of other provinces and represented specific haplotypes for this geographical region on the phylogenetic tree.

Molecular Characterization of Mesocestoides litteratus (Cestoda: Cyclophyllidea: Mesocestoididae) Tetrathyridium Isolated from Two Species of Rodents from Iran.

PURPOSE: Mesocestoides spp. are Cyclophyllidean tapeworms with zoonotic importance. The current study aimed to investigate the molecular characteristics of Mesocestoides larvae (tetrathyridium) isolated from the abdominal cavity of persion jird, Meriones persicus, and from the liver of grey hamster, Cricetulus migratorius, in Ardabil Province, northwest Iran. METHODS: Genomic DNA of the isolates of Mesocestoides tetrathyridium were extracted, and mitochondrial gene of cytochrome-c oxidase subunit1 (cox1) was amplified. Sequencing of PCR products were performed and phylogenic analysis was run using MEGA 6.0 software. RESULTS: Both isolates were identified as Mesocestoides litteratus, showing high identity with M. litteratus sequences available in GenBank. Also, they had 100% homology to each other. Intra-species variation within isolates of M. litteratus were 0-2.4%. The phylogenetic reconstruction based on the partial sequence of the cox1 gene showed that our sequences of M. litteratus were clustered with M. litteratus isolates from Slovakia, Netherlands, Germany and Italy. CONCLUSION: This is the first molecular description of M. litteratus from M. persicus and C. migratorius. Phylogenetic analysis illustrated that M. litteratus isolates of the current study had very high identities with the isolates of this species from other countries.

First molecular identification of Sarcocystis miescheriana (Protozoa, Apicomplexa) from wild boar (Sus scrofa) in Iran.

Sarcocystis isolate obtained from the thigh muscle of a wild boar (Sus scrofa), captured from Gilan Province, northern Iran, was subjected to molecular analysis. Genomic DNA was obtained using a DNA extraction tissue kit and Polymerase chain reaction (PCR) for amplification of the 18S ribosomal DNA region yielded an 842 bp DNA band on agarose gel. Analysis of DNA sequencing by BLAST confirmed the isolate as Sarcocystis miescheriana and the sequence was deposited in GenBank by Accession No. GU395554. This is the first molecular identification of an isolate of S. miescheriana in Iran.

Sequence analysis of cox1 and nad1 genes in Echinococcus granulosus G3 genotype in camels (Camelus dromedarius) from central Iran.

Nineteen hydatid cyst isolates collected from camels in central Iran were subjected to sequences analysis of mitochondrial cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) genes. A consensus sequence obtained containing 366 nucleotides for cox1 and 471 nucleotides for nad1 genes. Overall, the camel isolates indicated five different sequences in cox1 and nine in nad1 genes. The sequences analysis indicated that 26.3%, 42.1%, and 31.6% of isolates belonging to G1, G3, and G6 genotypes of Echinococcus granulosus, respectively. The isolates with G3 genotype indicated one cox1 sequence having 100% homology with reference G3 sequence (AN: M84663) and two different nad1 sequences, one having 100% homology with reference G3 sequence (AN: AJ237634) and the other with a silent mutation (G to A) in position 279. The presence of G3 genotype (buffalo strain) of E. granulosus as dominant genotype in camels is emphasized. As G3 genotype has formerly been reported in human, the epidemiological role of camels is warranted in future surveys.

Genetic Characterization of Echinococcus granulosus Sensu Lato in Livestock and Human Isolates from North of Iran Indicates the Presence of E. ortleppi in Cattle.

PURPOSE: Identification of different genotypes of echinococcal cyst in various domestic herbivores and humans within the target area was the principal aim of the present study, performed using sequence data of cox1 and nad1 mitochondrial genes. METHODS: A total of 57 cystic echinococcosis (CE) cysts were isolated from indigenous livestock including 45 cattle, 9 sheep and 3 goats from several slaughterhouses in Guilan Province. Moreover, 12 formalin-fixed paraffin-embedded (FFPE) CE cyst tissues from humans were also included, obtained from the archives of several hospitals in Rasht, the capital of Guilan. Genetic sequencing was conducted using mitochondrial cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) genes. RESULTS: Our results found that E. granulosus sensu stricto (s.s.) and E. ortleppi were present in 92.7% and 7.2% isolates, respectively. E. granulosus s.s. (genotypes G1 and G3) and E. ortleppi were isolated from various livestock whereas all CE cysts isolated from humans were E. granulosus s.s. G1 genotype. CONCLUSION: We found that E. granulosus s.s. G1 was the predominant genotype within the study region. This is the first study to report E. ortleppi in cattle in Iran.

Gastrointestinal Helminthic Parasites of Stray Cats (Felis catus) in Northwest Iran.

BACKGROUND: Stray cats are considered an important source of various human and animal diseases, particularly diseases of parasitic helminths. We aimed to investigate the distribution of zoonotic species of gastrointestinal helminths in stray cats in Meshkin-Shahr district in Ardabil Province in the northwest of Iran. METHODS: The gastrointestinal tract of 104 stray cats from villages of Meshkin-Shahr district were provided during 2014-2015. Each gastrointestinal tract was cut into distinct sections, including esophagus, stomach, small intestine, and large intestine, and each section was examined separately for detection of helminths. Helminths were collected and then identified at the species level after clearing and staining. RESULTS: Overall, 88 out of 104 cats (84.6%) were found to be infected with at least one gastrointestinal helminth. The rate of infection for each species was as follows: Toxocara mystax (syn. cati) (49%), Taenia taeniaeformis (44.2%), Joyexiella pasqualei (32.7%), Dipylidium caninum (23.1%), Rictularia cahirensis (4.8%), and Physaloptera praeputialis (4.8%). Among these parasites, only Ph. praeputialis was collected from the stomach, all other helminths were collected from the small intestine. CONCLUSION: The results demonstrate a high infection rate of stray cats with zoonotic helminths. The presence of zoonotic species in stray cats, particularly T. mystax, has public health importance.

In vitro effects of tropisetron and granisetron against Echinococcus granulosus (s.s.) protoscoleces by involvement of calcineurin and calmodulin.

BACKGROUND: Cystic echinococcosis (CE) is a disease caused by the larval stage of Echinococcus granulosus sensu lato  (s.l.). The treatment of CE mainly relies on the use of benzimidazoles, which can commonly cause adverse side effects. Therefore, more efficient treatment options are needed. Drug repurposing is a useful approach for advancing drug development. We have evaluated the in vitro protoscolicidal effects of tropisetron and granisetron in E. granulosus sensu stricto (s.s.) and assessed the expression of the calcineurin (CaN) and calmodulin (CaM) genes, both of which have been linked to cellular signaling activities and thus are potentially promising targets for the development of drugs. METHODS: Protoscoleces (PSC) of E. granulosus (s.s.) (genotype G1) obtained from sheep hepatic hydatid cysts were exposed to tropisetron and granisetron at concentrations of 50, 150 and 250 µM for various periods of time up to 10 days. Cyclosporine A (CsA) and albendazole sulfoxide were used for comparison. Changes in the morphology of PSC were investigated by light microscopy and scanning electron microscopy. Gene expression was assessed using real-time PCR at the mRNA level for E. granulosus calcineurin subunit A (Eg-CaN-A), calcineurin subunit B (Eg-CaN-B) and calmodulin (Eg-CaM) after a 24-h exposure at 50 and 250 µM, respectively. RESULTS: At 150 and 250 µM, tropisetron had the highest protoscolicidal effect, whereas CsA was most effective at 50 µM. Granisetron, however, was less effective than tropisetron at all three concentrations. Examination of morphological alterations revealed that the rate at which PSC were killed increased with increasing rate of PSC evagination, as observed in PSC exposed to tropisetron. Gene expression analysis revealed that tropisetron at 50 µM significantly upregulated Eg-CaN-B and Eg-CaM expression while at 250 µM it significantly downregulated both Eg-CaN-B and Eg-CaM expressions; in comparison, granisetron decreased the expression of all three genes at both concentrations. CONCLUSIONS: Tropisetron exhibited a higher efficacy than granisetron against E. granulosus (s.s.) PSC, which is probably due to the different mechanisms of action of the two drugs. The concentration-dependent effect of tropisetron on calcineurin gene expression might reflect its dual functions, which should stimulate future research into its mechanism of action and evaluation of its potential therapeutical effect in the treatment of CE.

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.

Identification and differentiation of Fasciola hepatica and Fasciola gigantica using a simple PCR-restriction enzyme method.

Accurate morphological differentiation between the liver fluke species Fasciola hepatica and Fasciola gigantica is difficult. We evaluated PCR-restriction enzyme profiles of internal transcribed spacer 1 (ITS1) that could aid in their identification. Fifty F. hepatica and 30 F. gigantica specimens were collected from different hosts in three provinces of Iran. For DNA extraction, we crushed fragments of the worms between two glass slides as a new method to break down the cells. DNA from the crushed materials was then extracted with a conventional phenol-chloroform method and with the newly developed technique, commercial FTA cards. A primer pair was selected to amplify a 463-bp region of the ITS1 sequence. After sequencing 14 samples and in silico analysis, cutting sites of all known enzymes were predicted and TasI was selected as the enzyme that yielded the most informative profile. Crushing produced enough DNA for PCR amplification with both the phenol-chloroform and commercial FTA card method. The DNA extracted from all samples was successfully amplified and yielded a single sharp band of the expected size. Digestion of PCR products with TasI allowed us to distinguish the two species. In all samples, molecular identification was consistent with morphological identification. Our PCR-restriction enzyme profile is a simple, rapid and reliable method for differentiating F. hepatica and F. gigantica, and can be used for diagnostic and epidemiological purposes.

Echinococcus granulosus genotypes in livestock of Iran indicating high frequency of G1 genotype in camels.

In this study, 112 Echinococcus granulosus isolates from different livestock of Iran were genotyped by PCR amplification of ribosomal DNA-internal transcribed spacer 1 (rDNA-ITS1) region followed by restriction fragment length polymorphism (RFLP) with the enzyme RsaI. The possibility of intra-genotype variation was also investigated using randomly amplified polymorphic DNA (RAPD) analysis. Isolates from sheep, goats, cattle and the majority of camels (12 of 18; 66.7%) were identified as the G1 genotype and a few camel isolates (6 of 18; 33.3%) belonged to the G6 genotype. Overall G1 and G6 genotypes were identified in 94.6% (106 of 112) and 5.3% (6 of 112) of all isolates, respectively. RAPD analysis based on 15 separate primers showed 7-14 bands of 200-3000bp for strain G1. Considering each individual primer, no differences observed among isolates from different hosts and between livers and lungs. This study confirmed the existence of G1 and G6 genotypes in Iran. Moreover, G1 is much more prevalent even in camels, indicating the importance of sheep-dog cycle in public health. Studying intra-genotypic variation of E. granulosus warrants more research using other primers and methods.

Seroprevalence of Toxocara Infection in Association with Different Risk Factors among Children of 4-12 Years Old Referred to Some Medical Centers in Aras Free Zone, Northwest Iran.

BACKGROUND: Toxocariosis is a parasitic disease caused by the larval stage of Toxocara species from dog and cat. It has a worldwide distribution with higher prevalence in children. This study aimed to determine seroprevalence of Toxocara infection and its association with some risk factors among children of Aras Free Zone (Jolfa City) in Northwest of Iran. METHODS: Sera were collected from 514 children aged 4-12 yr old attending to some medical centers in the study area from May 2018 to Feb 2019. Anti-Toxocara IgG antibodies assay was performed using commercial ELISA kit (Nova Tec, Germany). The seropositivity rate was determined and its association with different demographic criteria and risk factors were statistically analyzed. RESULTS: The overall seroprevalence was 2.3% (12/514). Risk factors of children's age group and contact with either pet animals (dog and cat) and/or soil were significantly associated with seropositivity. However, there was not any relationship between Toxocara infection and gender of children, place of residency (urban or rural) and their mothers' education level. CONCLUSION: Both girls and boys are at risk of Toxocara infection in the study area. Younger age of childhood and contact with sources of infection were important associated factors. More probably, additional criteria are involved in the initiation of infection.

The Burden of Cystic Echinococcosis in Kenya: A Review Article.

BACKGROUND: Cystic echinococcosis (CE), caused by the larval stage of Echinococcus granulosus sensu lato (s.l), is a zoonotic parasitic disease with a worldwide distribution. Kenya is one of the high endemic countries of CE with the endemic areas in the country being under immense occupation of traditional pastoralists. Turkana area in Kenya, has in the past recorded the highest prevalence of CE in the world. METHODS: The keywords cystic echinococcosis; Prevalence; Diagnosis; Risk-factors; Kenya were searched on google scholar and PubMed and the important literature materials retrieved for further analysis. RESULTS: The most notable infection risk factor for this disease in the country is the close association between man, dogs, and livestock. Successful control of CE in Kenya requires application of innovative interventions achieved after the review of the disease situation in the country. With the emergence and advent of new diagnostic techniques, CE organ-specific infections and transmission pattern in Kenya differ from what is commonly reported in literature. CONCLUSION: A better understanding of CE prevalence of different hosts, its transmission pattern and the pathogenicity might make it possible to set up more effective control programs in future.

Critical diagnosis of complicated strongyloidosis with nested-PCR and High Resolution Melting analysis (HRM)

Diagnosis of strongyloidosis is sometimes problematic and requires novel techniques. Here, critical diagnosis of a complicated case of strongyloidosis using molecular methods is reported. A young woman referred to the Diagnostic Laboratory of Strongyloidiasis in School of Public Health, Tehran University of Medical Sciences. She had taken albendazole before referring to the laboratory. She had cerebral edema, behavior disorders, hypereosinophilia and titer of IgE >2000 IU/mL. The patient had history of intestinal and skin disorders and steroid therapy. For detection of Strongyloides stercoralis infection, parasitological techniques and novel methods of nested-PCR and HRM analysis were applied on stool samples upon admission and during the following up. On the samples provided upon first admission, parasitology showed negative results, while both molecular methods revealed infection with S. stercoralis. After specific treatment, during the following up, the patient general health was much improved and the results of all parasitological and molecular tests were negative for strongyloidosis. Application of novel sensitive diagnostic methods for detection of S. stercoralis is necessary, especially once parasitological techniques have lack of sensitivity.

Design and Construction of a Fusion Peptide Containing B1, B2, B4, and EPC1 Epitopes for Diagnosis of Human Cystic Echinococcosis.

BACKGROUND: Cystic echinococcosis (CE), larval stage of Echinococcus granulosus, immunodiagnostics is still a challenge due to asymptomatic nature of CE during the early phase of infection and imperfection of diagnostic antigens. In silico design and assessments of hydatid cyst antigens provide preeminent information for novel and favorable diagnostic methods. METHODS: This study was performed at the Tehran University of Medical Sciences, Tehran, Iran in 2018. The sequences of B2, EPC1, B1 and B4 antigens were collected and analyzed for sequence conservancy by protein BLAST search and CLUSTALW multiple sequence alignment. The secondary and 3D structures were predicted using ab initio and threading methods. The antigens were analyzed for their B cell epitopic content using linear and conformational B cell epitope prediction tools. The final diagnostic antigen was designed by fusing the selected epitopic determinants form each antigen. RESULTS: Given the conservancy results and B cell epitope predictions, the whole B2 antigen along with amino acids spanning 1-50, 1-30, and 30-81 regions of EPC1, B1 and B4 antigens were selected to design the final antigen. High surface accessibility (75%), protein stability, low free energy and high number of amino acids involved in B cell epitopes were desirable properties for the final antigen to interact with antibodies against CE. CONCLUSION: In silico design of such antigens is useful for better diagnosis of CE, decrease the cost and the time required for antigen design, while avoiding the ethical aspects of in vivo studies.