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.

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.

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.

Echinococcus multilocularis and Echinococcus granulosus in canines in North-Khorasan Province, northeastern Iran, identified using morphology and genetic characterization of mitochondrial DNA.

BACKGROUND: Canids are definitive hosts of Echinococcus multilocularis and Echinococcus granulosus. This study aimed to survey these two Echinococcus species in canids of North-Khorasan Province, northeastern Iran, using morphological criteria and genetic characterization of mitochondrial DNA. METHODS: The carcasses of 106 canids, namely 61 jackals (Canis aureus), 23 foxes (Vulpes vulpes), 19 dogs (Canis familiaris) and three wolves (Canis lupus) were collected from the study area in 2013-2014 and examined for Echinococcus species. Morphological features were assessed by microscopy of adult worms. For molecular characterization, DNA was extracted, mostly from the adult worms but also from eggs. DNA fragments of the cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) mitochondrial genes were amplified and sequenced. Sequences were aligned and compared with reference sequences. Intraspecific and interspecific diversity were calculated and phylogenetic analysis was performed. RESULTS: Overall, 9.4% of the canids (eight jackals and two foxes) were found infected with E. multilocularis by molecular methods, of which seven cases were also confirmed using morphological description of the adult worms. Echinococcus granulosus was found in 6.6% of the canines (four dogs, two jackals and one wolf) as determined by both molecular methods and adult cestode morphology. All E. granulosus isolates were identified as the G1 genotype. Comparative sequence analysis indicated 0-0.7% and 0% intraspecific divergence within E. granulosus isolates and 0% and 0-0.2% within E. multilocularis isolates for cox1 and nad1, respectively. CONCLUSIONS: This study revealed the presence of E. multilocularis and E. granulosus in canids of North-Khorasan Province of Iran. Jackals were found infected with both E. multilocularis and E. granulosus, but infection with the former species was higher.

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.

Analysis of nad2 and nad5 enables reliable identification of genotypes G6 and G7 within the species complex Echinococcus granulosus sensu lato.

The larval stages of tapeworms in the species complex Echinococcus granulosus sensu lato cause a zoonotic disease known as cystic echinococcosis (CE). Within this species complex, genotypes G6 and G7 are among the most common genotypes associated with human CE cases worldwide. However, our understanding of ecology, biology and epidemiology of G6 and G7 is still limited. An essential first step towards this goal is correct genotype identification, but distinguishing genotypes G6 and G7 has been challenging. A recent analysis based on complete mitogenome data revealed that the conventional sequencing of the cox1 (366 bp) gene fragment mistakenly classified a subset of G7 samples as G6. On the other hand, sequencing complete mitogenomes is not practical if only genotype or haplogroup identification is needed. Therefore, a simpler and less costly method is required to distinguish genotypes G6 and G7. We compared 93 complete mitogenomes of G6 and G7 from a wide geographical range and demonstrate that a combination of nad2 (714 bp) and nad5 (680 bp) gene fragments would be the best option to distinguish G6 and G7. Moreover, this method allows assignment of G7 samples into haplogroups G7a and G7b. However, due to very high genetic variability of G6 and G7, we suggest to construct a phylogenetic network based on the nad2 and nad5 sequences in order to be absolutely sure in genotype assignment. For this we provide a reference dataset of 93 concatenated nad2 and nad5 sequences (1394 bp in total) containing representatives of G6 and G7 (and haplogroups G7a and G7b), which can be used for the reconstruction of phylogenetic networks.

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.

Seroprevalence of Human Cystic Echinococcosis in Alborz Province, Central Iran in 2015.

BACKGROUND: The aim of this study was to conduct a sero-epidemiological survey in Alborz Province, central Iran to detect the rate of hydatidosis in the city and nearby villages. METHODS: Overall, 680 serum samples were collected from 536 male and 127 female subjects referred to different health centers of Karaj, Alborz Province, central Iran and nearby villages in 2014-15. All patients filled out a questionnaire and an informed consent. Sera were analyzed using indirect-ELISA test with AgB. Ten µg/ml antigens (Proceeded hydatid 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: Twenty-three cases (3.4%) were positive for hydatidosis by ELISA test. The prevalence of hydatidosis among females and males was 3.1% and 4.7%, respectively. The rate of the disease was significantly higher in areas where dogs were higher (P<0.05). There was no significant difference as regards age groups, sex, job, residency, and literacy. Regarding occupation, housekeepers had the highest rate of infection as 5.9%. The seroprevalence of infection was 4.2% in bachelors and master people which showed the highest rate. As regards residency, urban life showed no significant difference with rural life (2.8% vs. 4.4%). Age group of 30-39 yr old, with 4.3% as prevalence had the highest rate of positivity (P>0.05). CONCLUSION: Because of the specific situation of Alborz Province, and availability of many stray dogs, obtained rate of hydatidosis shows that the authorities should be cautious to monitor the disease.

Distinguishing Echinococcus granulosus sensu stricto genotypes G1 and G3 with confidence: A practical guide.

Cystic echinococcosis (CE), a zoonotic disease caused by tapeworms of the species complex Echinococcus granulosus sensu lato, represents a substantial global health and economic burden. Within this complex, E. granulosus sensu stricto (genotypes G1 and G3) is the most frequent causative agent of human CE. Currently, there is no fully reliable method for assigning samples to genotypes G1 and G3, as the commonly used mitochondrial cox1 and nad1 genes are not sufficiently consistent for the identification and differentiation of these genotypes. Thus, a new genetic assay is required for the accurate assignment of G1 and G3. Here we use a large dataset of near-complete mtDNA sequences (n = 303) to reveal the extent of genetic variation of G1 and G3 on a broad geographical scale and to identify reliable informative positions for G1 and G3. Based on extensive sampling and sequencing data, we developed a new method, that is simple and cost-effective, to designate samples to genotypes G1 and G3. We found that the nad5 is the best gene in mtDNA to differentiate between G1 and G3, and developed new primers for the analysis. Our results also highlight problems related to the commonly used cox1 and nad1. To guarantee consistent identification of G1 and G3, we suggest using the sequencing of the nad5 gene region (680 bp). This region contains six informative positions within a relatively short fragment of the mtDNA, allowing the differentiation of G1 and G3 with confidence. Our method offers clear advantages over the previous ones, providing a significantly more consistent means to distinguish G1 and G3 than the commonly used cox1 and nad1.

Global phylogeography and genetic diversity of the zoonotic tapeworm Echinococcus granulosus sensu stricto genotype G1.

Echinococcus granulosus sensu stricto (s.s.) is the major cause of human cystic echinococcosis worldwide and is listed among the most severe parasitic diseases of humans. To date, numerous studies have investigated the genetic diversity and population structure of E. granulosus s.s. in various geographic regions. However, there has been no global study. Recently, using mitochondrial DNA, it was shown that E. granulosus s.s. G1 and G3 are distinct genotypes, but a larger dataset is required to confirm the distinction of these genotypes. The objectives of this study were to: (i) investigate the distinction of genotypes G1 and G3 using a large global dataset; and (ii) analyse the genetic diversity and phylogeography of genotype G1 on a global scale using near-complete mitogenome sequences. For this study, 222 globally distributed E. granulosus s.s. samples were used, of which 212 belonged to genotype G1 and 10 to G3. Using a total sequence length of 11,682 bp, we inferred phylogenetic networks for three datasets: E. granulosus s.s. (n = 222), G1 (n = 212) and human G1 samples (n = 41). In addition, the Bayesian phylogenetic and phylogeographic analyses were performed. The latter yielded several strongly supported diffusion routes of genotype G1 originating from Turkey, Tunisia and Argentina. We conclude that: (i) using a considerably larger dataset than employed previously, E. granulosus s.s. G1 and G3 are indeed distinct mitochondrial genotypes; (ii) the genetic diversity of E. granulosus s.s. G1 is high globally, with lower values in South America; and (iii) the complex phylogeographic patterns emerging from the phylogenetic and geographic analyses suggest that the current distribution of genotype G1 has been shaped by intensive animal trade.

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.

Wild Rodent Ectoparasites Collected from Northwestern Iran.

BACKGROUND: Rodents play an important role as reservoir of some pathogens, and the host of some ectoparasites as well. These ectoparasites can transmit rodents' pathogens to human or animals. The aim of this study was to assess the distribution and infestation load of ectoparasites on rodents in Meshkin-Shahr District, northwestern Iran. METHODS: Rodents were captured using baited live traps in spring 2014 from Meshkin-Shahr District and were transferred to the laboratory for identification to the species level. Their ectoparasites were collected, mounted and identified. RESULTS: Three rodent species including Meriones persicus (74%), Mus musculus (16.9%) and Cricetulus migratorius (9%) were identified. Among all rodents, 185 specimens (90.69%) were infested with a total of 521 ectoparasites. Overall, 10 arthropods species were collected, including fleas (97.6%), one mite (1.6%) and one louse species (0.6%) as follows: Xenopsylla nubica, X. astia, X. buxtoni, X. cheopis, Nosopsyllus fasciatus, N. iranus, Ctenocephalides felis, Ctenophthalmus rettigismiti, Ornithonyssus sp and one species of genus Polyplax. The most prevalent ectoparasites species was X. nubica (89%). CONCLUSION: Nearly all rodent species were infested with Xenopsylla species. Monitoring of ectoparasites on infested rodents is very important for awareness and early warning towards control of arthropod-borne diseases.

New mitogenome and nuclear evidence on the phylogeny and taxonomy of the highly zoonotic tapeworm Echinococcus granulosus sensu stricto.

Cystic echinococcosis, a zoonotic disease caused by Echinococcus granulosus sensu lato (s. l.), is a significant global public health concern. Echinococcus granulosus s. l. is currently divided into numerous genotypes (G1-G8 and G10) of which G1-G3 are the most frequently implicated genotypes in human infections. Although it has been suggested that G1-G3 could be regarded as a distinct species E. granulosus sensu stricto (s. s.), the evidence to support this is inconclusive. Most importantly, data from nuclear DNA that provide means to investigate the exchange of genetic material between G1-G3 is lacking as none of the published nuclear DNA studies have explicitly included G2 or G3. Moreover, the commonly used relatively short mtDNA sequences, including the complete cox1 gene, have not allowed unequivocal differentiation of genotypes G1-G3. Therefore, significantly longer mtDNA sequences are required to distinguish these genotypes with confidence. The main aim of this study was to evaluate the phylogenetic relations and taxonomy of genotypes G1-G3 using sequences of nearly complete mitogenomes (11,443bp) and three nuclear loci (2984bp). A total of 23 G1-G3 samples were analysed, originating from 5 intermediate host species in 10 countries. The mtDNA data demonstrate that genotypes G1 and G3 are distinct mitochondrial genotypes (separated by 37 mutations), whereas G2 is not a separate genotype or even a monophyletic cluster, but belongs to G3. Nuclear data revealed no genetic separation of G1 and G3, suggesting that these genotypes form a single species due to ongoing gene flow. We conclude that: (a) in the taxonomic sense, genotypes G1 and G3 can be treated as a single species E. granulosus s. s.; (b) genotypes G1 and G3 should be regarded as distinct genotypes only in the context of mitochondrial data; (c) we recommend excluding G2 from the genotype list.

Helminth Infections of Meriones persicus (Persian Jird), Mus musculus (House Mice) and Cricetulus migratorius (Grey Hamster): A Cross-Sectional Study in Meshkin-Shahr District, Northwest Iran.

BACKGROUND: Rodents have important role as reservoirs of different parasites. The aim of this study was to determine helminth parasites of abundant rodents in Meshkin-Shahr, Ardabil Province northwest Iran. METHODS: From April 2014 to March 2015; 205 rodents including 118 Meriones persicus, 63 Mus musculus and 24 Cricetulus migratorius were collected, using live traps. All rodents were dissected and their different tissues examined for infectivity with helminth parasites. RESULTS: Overall, 74.2% of rodents were infected with helminth parasites. The rate of infectivity in M. persicus, M. musculus and C. migratorius was 82.2%, 61.9%, 66.7%, respectively. In general, among all 205 rodents, the species and infection rates of helminthes were as follows: Nematoda: Trichuris sp. (46.8%), Capillaria hepatica (18.1%), Syphacia frederici (14.2%), Aspicularis tetraptera (3.4%), Trichuris rhombomidis (2%), Heligmosomom sp. (2%), Streptopharagus kuntzi (0.5%), Spiruridae gen. sp. (0.5%); Cestoda: Hymenolepis nana fraterna (16.6%) Hymenolepis diminuta (7.3%) tetratiridium of Mesocestoides sp. (1%), Paranoplocephala sp. (0.5%), Cysticercus fasciolaris (0.5%), Taenia endothoracicus larva (0.5%), and Acanthocephala: Moniliformis moniliformis (18.5%). CONCLUSIONS: Variable species of helminthes circulate in the rodents of the study area. Presence of several zoonotic species highlights the potential risk of infections for public health.

Liver condemnation and economic losses due to parasitic infections in slaughtered animals in Iran.

The prevalences of parasitic infections responsible for the condemnation of liver during meat inspection, and their economic implication were estimated in slaughtered animals in Iran. The liver organ was examined for the presence of parasitic lesions during meat inspection in cattle, camel, buffalo, sheep and goat. The parasitic agents observed in the condemned livers of these animals were Fasciola spp., Dicrocoelium dendriticum, Cysticercus tenuicollis and hydatid cyst. The average percentages of liver condemnation for three years period by Fasciola spp., D. dendriticum, Cysticerci and hydatid cyst were 2.12, 2.71, 0.04, and 2.88 %, respectively. The mean prevalence of Fasciola spp. in cattle, sheep, goat, camel and buffalo was 4.32, 1.85, 1.56, 1.31 and 9.31 %, respectively and the mean prevalence of D. dendriticum in those animals were 3.65, 2.66, 2.19, 5.09 and 3.90 %, respectively. Also, the mean prevalence of Cysticerci and hydatid cyst were 0.13 and 3.72 % in cattle, 0.04 and 2.85 % in sheep, 0.05 and 2.40 % in goat, 0.02 and 8.22 % in camel and 0.001 and 5.48 % in buffalo, respectively. The most contributing parasites to marketable liver condemnation were hydatid cyst in sheep, goat and camel and Fasciola spp. in cattle and buffalo, and the average annual cost for condemned livers was 8.2 million USD.

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.

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.

Severe diarrhea due to Isospora belli in a patient with thymoma.

Opportunistic isosporidial infection of the gastrointestinal tract is frequently encountered in patients with acquired immunodeficiency syndrome (AIDS) and is considered to be an AIDS-defining illness. Chronic severe watery diarrhea due to Isospora belli has also been reported in other immunodeficiency states. This report describes severe chronic debilitating diarrhea due to isosporiasis in a patient with mediastinal thymoma, a common tumor of the anterior mediastinum, originating from the epithelial cells of the thymus. Numerous oocysts of I. belli were detected in direct smear preparation of the diarrheic stool sample of the patient, who had an 8-month history of recurrent diarrhea. Duodenal and colonic mucosal biopsies revealed slight degrees of atrophic changes associated with infiltration of the lamina propria by an appreciable number of eosinophiles and the presence of unizoit tissue cysts of I. belli in the surface epithelium of the duodenal mucosa. The patient was first treated with trimethoprim-sulfamethoxazole and subsequently underwent complete thymectomy. Later, due to recurrence of the diarrhea, he was treated with ciprofloxacin.

A case of fatal strongyloidiasis in a patient with chronic lymphocytic leukemia and molecular characterization of the isolate.

Strongyloides stercoralis is a human intestinal parasite which may lead to complicated strongyloidiasis in immunocompromised. Here, a case of complicated strongyloidiasis in a patient with chronic lymphocytic leukemia is reported. Presence of numerous S. stercoralis larvae in feces and sputum confirmed the diagnosis of hyperinfection syndrome in this patient. Following recovery of filariform larvae from agar plate culture of the stool, the isolate was characterized for the ITS1 region of ribosomal DNA gene by nested-PCR and sequencing. Albendazole therapy did not have cure effects; and just at the beginning of taking ivermectin, the patient died. The most important clue to prevent such fatal consequences is early diagnosis and proper treatment.

Immunoperoxidase staining of alveolar hydatid cyst from an experimentally infected gerbil.

Echinococcus multilocularis, the small fox tapeworm, has an extensive geographical range in the northern hemisphere where foxes and small rodents represent natural hosts. The larval stage of this parasite, alveolar echinococcosis (AE), is an emerging zoonosis of increasing importance. It is a serious human illness which is often misdiagnosed as hepatic cancer. If not identified at an early stage of parasite development it can lead to the death of patients. Histological examination of biopsies is one of the classical methods of diagnosis. In this study, in order to gain unequivocal histopathological diagnosis of AE, the immunoperoxidase staining technique was performed on routinely processed histological sections of an experimentally infected gerbil, using rabbit anti-E. multilocularis protoscolex IgG labelled with horseradish peroxidase. Demonstration of AE antigen was achieved by dark brown stain of cyst membranes against a blue background of the host liver cells stained with hematoxylin.