Analysis of the cox1 gene in Echinococcus granulosus from sheep in northeast Iran using PCR high-resolution melting (qPCR-HRM) curve analysis.

Echinococcus granulosus, the etiologic agent of echinococcosis, is one of the most important zoonotic helminthes worldwide. Knowledge of E. granulosus species and genotypes has important implications for epidemiology, control, and prevention of diseases as well as future vaccine and drug designs. There are many molecular methods developed to define genotypes of E. granulosus, among them high resolution melting (HRM) analysis, as a new approach, is a single step and closed tube method. It is appropriate for fast screening of large number of isolates. This technique is an accurate, user friendly, cost-effective, fast and simple method, which does not need post-PCR processes. Between March and lst august 2016, of 726 sheep examined in abattoirs in Razavi Khorasan province, Northeast Iran, 109 harboured cystic echincoccosis lesions (liver samples= 65 and lung samples= 44) which were collected for analysis. Total genomic DNA was extracted from each sample and amplified for the presence of polymorphism in the mitochondrial cox1 gene of Echinococcus granulosus using a high resolution melting curve (HRM) method. A total of 109 hydatid cyst samples analyzed by PCR high-resolution melting (qPCR-HRM) curve of the cox1 gene, all isolates were identified as G1 genotype (sheep strain). G1 is the predominant genotype in sheep in northeast of Iran. The high incidence of the G1 genotype (known to be the predominant E. granulosus genotype infecting humans globally) in sheep has considerable implications for hydatid disease control programs in this area.

Surgical treatment of hepatic cystic echinococcosis in patients co-infected with HIV/AIDS.

Co-infections of cystic echinococcosis (CE) and HIV/AIDS is rare. We report four CE cases that were HIV positive. Three out of the four patients underwent a surgical operation to remove the hydatid cysts in their livers. The operation confirmed that in two of the cases their cysts had ruptured. These patients were given 3 months of albendazole after the operation. Follow-up showed they were remarkably improved in term of their health, although they were still HIV antibody positive 6 months after surgical treatment. Interestingly, the treatment remarkably increased their CD4+ cell population. We showed that surgery is suitable for treating hepatic cystic echinococcosis with HIV/AIDS co-infection.

Immunodiagnosis of sheep infections with Echinococcus granulosus: in 35 years where have we come?

W. K. Yong and D. D. Heath published in 1979 a seminal paper in the first issue of Parasite immunology describing their efforts to determine whether the arc 5 precipitin band, formed when test human serum is reacted against electrophoresed hydatid cyst fluid antigen, would be a suitable immunodiagnostic test for the identification of sheep infected with Echinococcus granulosus. Although they found antibodies to arc 5 in the sera of hydatid-infected sheep, the sera of some sheep harbouring Taenia ovis and T. hydatigena also precipitated the hydatid cyst fluid arc 5 antigen, so they concluded arc 5 antibodies were not suitable for the specific immunodiagnosis of E. granulosus infection in sheep in New Zealand. Subsequent work has shown that the existence of multiple infections with different taeniid species, antigenic cross-reactivity between these related parasites and the low level of specific antibody response to infection continue to hinder efforts to improve the diagnosis of hydatid infection in sheep and other natural intermediate hosts, thereby preventing the development of any practical test. In particular, the poor antibody response of ruminants to naturally acquired hydatid infection may prove an insurmountable barrier in future efforts to develop a reliable and accurate immunological test.

Current status of the genetics and molecular taxonomy of Echinococcus species.

The taxonomy of Echinococcus has long been controversial. Based mainly on differences in morphology and host-parasite specificity characteristics, 16 species and 13 subspecies were originally described. Subsequently, most of these taxa were regarded as synonyms for Echinococcus granulosus and only 4 valid species were recognised: E. granulosus; E. multilocularis; E. oligarthrus and E. vogeli. But, over the past 50 years, laboratory and field observations have revealed considerable phenotypic variability between isolates of Echinococcus, particularly those of E. granulosus, which include differences in: morphology in both larval and adult stages, development in vitro and in vivo, host infectivity and specificity, chemical composition, metabolism, proteins and enzymes, pathogenicity and antigenicity. The application of molecular tools has revealed differences in nucleic acid sequences that reflect this phenotypic variation and the genetic and phenotypic characteristics complement the previous observations made by the descriptive parasitologists many years ago. The fact that some of these variants or strains are poorly or not infective to humans has resulted in a reappraisal of the public health significance of Echinococcus in areas where such variants occur. A revised taxonomy for species in the Echinococcus genus has been proposed that is generally accepted, and is based on the new molecular data and the biological and epidemiological characteristics of host-adapted species and strains.

SerpinB2 deficiency modulates Th1/Th2 responses after schistosome infection.

SerpinB2, also known as plasminogen activator inhibitor type-2, is a major product of macrophages and is upregulated during many infections. Although SerpinB2 inhibits urokinase plasminogen activator in vitro, evidence that this represents its physiological role in vivo is not compelling. We have recently shown that SerpinB2-/-mice generate enhanced Th1 responses after immunization with a Th1 immunogen. Herein,we show that Schistosoma japonicum granulomas induced liver SerpinB2 mRNA expression by >600-fold in wild-type mice. In SerpinB2-/- mice, worm and egg burden, and granuloma number and volume were unaffected. However, granulomas in these mice were associated with reduced fibrosis (as determined by Sirius red staining and image analysis) and increased iNOS, IL-6, IL-10 and TNFa and decreased Arg 1 and IL-13 mRNA expression. SerpinB2-/- mice immunized with soluble egg antigen (SEA) also showed reduced levels of SEA-specific IgG1. SerpinB2 deficiency thus promoted certain Th1 and reduced certain Th2 responses in response to this Th2 immunogen.

Familial aggregation of human helminth infection in the Poyang lake area of China with a focus on genetic susceptibility to schistosomiasis japonica and associated markers of disease.

Human helminthiases are common in China, especially in rural areas where sanitation conditions are poor. Soil-transmitted helminths (STHs) are predominantly found in the southern provinces. Schistosoma japonicum is also endemic to southern China. Here we review the prevalence of helminth infections and polyparasitism in China, and discuss the interactions between helminth parasites in the co-infected host. It is clear that STHs are more prevalent in rural China than previously suggested emphasizing the need for systematic control of STHs. Further, the need for improved sanitation and hygiene conditions to prevent parasite transmission is highlighted. We provide supporting evidence for human genetic susceptibility to both single helminth infection and polyparasitism, and suggest that susceptibility to helminths infections may not be independent of one or the other. We demonstrate an association between single nucleotide polymorphism (SNP) variants in IL-5 and symptomatic S. japonicum infection and discuss the potential role of IL-5 in other helminth infections. Fundamental to disease and morbidity control is adequate and effective diagnosis and surveillance of disease. We discuss the role of sICAM-1 and TNFR-I and -II as candidate markers for schistosome-induced hepatomegaly and fibrosis, and their potential for assessing disease stage and progression in schistosomiasis.

Immunopathogenesis of human schistosomiasis.

Schistosomiasis continues to be a significant cause of parasitic morbidity and mortality worldwide. This review considers the basic features of the pathology and clinical outcomes of hepatointestinal and genitourinary schistosomiasis, presents an overview of the numerous studies on animal models that have clarified many of the immunopathological features, and provides insight into our current understanding of the immunopathogenesis and genetic control of human schistosomiasis. In murine schistosomiasis, pathology is induced by a CD4(+) Th2 driven granulomatous response directed against schistosome eggs lodged in the host liver. The Th2 cytokines IL-4 and IL-13 drive this response, whereas IL-10, IL13Ralpha2, IFN-gamma and a subset of regulatory T-cells act to limit schistosome induced pathology. A variety of cell types including hepatic stellate cells, alternatively activated macrophages and regulatory T-cells have also been implicated in the pathogenesis of schistosomiasis. Current knowledge suggests the immunopathogenic mechanisms underlying human schistosomiasis are likely to be similar. The review also considers the future development of anti-pathology schistosome vaccines. As fibrosis is an important feature of many other diseases such as Crohn's disease and sarcoidosis, a comprehensive understanding of the cellular and molecular mechanisms involved in schistosomiasis may also ultimately contribute to the development an effective disease intervention strategy for other granulofibrotic diseases.

Reflections on the biochemistry of Echinococcus: past, present and future.

This review discusses 5 of my earliest papers on the biochemistry of larval Echinococcus published in Parasitology in the 1970s and 1980s. Two of the publications consider aspects of the basic biochemistry, intermediary metabolism and the regulation of respiratory pathways in E. granulosus and E. multilocularis, and emphasize the existence of inter- and intra-species variation in their general metabolism. The third reports on the detailed biochemical analysis of the tegumental surface of the protoscolex of E. granulosus, and the final 2 papers describe the genomic cloning of Echinococcus DNA fragments and their use, along with other DNA markers, in molecular identification of E. granulosus isolates collected worldwide from areas endemic for hydatid disease. A number of years have elapsed since these publications in Parasitology and, in this Centenary Issue article, I reflect briefly on some of the subsequent studies undertaken in these research areas that have advanced the field. As well, I provide brief insight on new research directions, emphasizing the impact of molecular biology and associated techniques on future studies of Echinococcus and hydatid disease.

Identification of a diagnostic antibody-binding region on the immunogenic protein EpC1 from Echinococcus granulosus and its application in population screening for cystic echinococcosis.

An Echinococcus granulosus cDNA sequence coding for EpC1, a proven serodiagnostic marker for cystic echinococcosis (CE, hydatid disease), has high amino acid sequence identity to a paralogue from Taenia solium, the cause of neurocysticercosis (NCC). To determine diagnostic antibody-binding regions on EpC1 recognized specifically by CE sera, 10 truncated regions (P1-10) of the immunogenic protein were expressed in Escherichia coli and subjected to immunoblotting. One peptide, designated peptide 5 [P5, fused with glutathione-S-transferase (GST)] was positively recognized by sera from mice experimentally infected with oncospheres of E. granulosus and sera from surgically confirmed CE patients. Sera from NCC patients did not react with any of the peptides used. There are four amino acid substitutions in P5 compared with the T. solium sequence and these may form part of the epitope inducing CE-specific antibody. Ninety-seven per cent (58 of 60) of sera from confirmed CE patients recognized P5-GST, which was higher than the parent EpC1 fused with GST which reacted with 92% (55 of 60) of the sera. A population screening survey showed that 424 human sera collected from communities in Xinjiang, an area in China endemic for CE, exhibited 4.5% and 3.3% positivity in immunoblotting analysis to EpC1 and P5, respectively; 19.8% of these sera reacted positively against hydatid cyst fluid (HCF) antigen B. Low numbers of surgical CE cases have been reported from this population, suggesting that HCF-based serology lacks specificity and that EpC1 or its contained P5 peptide may prove more accurate for seroepidemiological surveys of CE.

A correlative study of ultrasound with serology in an area in China co-endemic for human alveolar and cystic echinococcosis.

We correlated ultrasound (US) imaging classifications for human alveolar echinococcosis (AE) and cystic echinococcosis (CE) with serology (ELISA and immunoblotting (IB) incorporating native and recombinant/purified echinococcal antigens) in community surveys (2001-2003) and follow-up (2002 and 2003) of US-confirmed cases in Ningxia, China. One hundred and seventy-one cases (96 with AE, 75 with CE) were identified; of these, US classification and serological data were obtained for 142 and 112 cases, respectively. Seropositive-rates increased in CE patients with highly viable unilocular cyst lesions (Types CL, CE 1 or CE 2) to degenerating primary lesions (CE 3), but then decreased in subjects with inactive (CE 4) or dead (CE 5) cysts. In contrast, there was a constant increase in seropositivity from the early (P1, P2) to the advanced stages (P3, P4) with AE cases. For US-confirmed cases, follow-up by US combined with serology is invaluable for studying the clinical progression of echinococcosis and for detecting recurrent cysts or reinfection post-treatment.

Hospital and community surveys reveal the severe public health problem and socio-economic impact of human echinococcosis in Ningxia Hui Autonomous Region, China.

A comprehensive study of human echinococcosis (caused by Echinococcus granulosus or E. multilocularis), including assessment of hospital records, community surveys and patient follow-up, was conducted in Ningxia Hui Autonomous Region (NHAR), China. In contrast to hospital records that showed 96% of echinococcosis cases were caused by cystic echinococcosis (CE), 56% of cases detected in active community surveys were caused by alveolar echinococcosis (AE). The AE and CE cases co-existed frequently in the same village, even occurring in the same patient. A serious public health problem caused by echinococcosis was evident in southern NHAR, typified by: a long diagnostic history for both AE and CE (7.5 years) compared with a shorter treatment history (4.7 years); a significant mortality rate (39%) caused by AE in one surveyed village, where patients had no previous access to treatment; family aggregation of CE and AE cases; a high proportion of both AE (62.5%) and CE (58%) in females; a high rate of recurrent surgery (30%) for CE demonstrated by surgical records; and frequent symptomatic recurrences (51%) because of discontinuous or sporadic access to chemotherapy for AE. The disease burden for both human AE and CE is thus very severe among these rural communities in NHAR, and this study provides the first attempt to determine the costs of morbidity and surgical intervention of human CE and AE cases both at the hospital and community level in this setting. This information may be useful for assessing the cost effectiveness of designing effective public health programs to control echinococcosis in this and other endemic areas in China and elsewhere.

Molecular confirmation of a case of multiorgan cystic echinococcosis.

We report on the results of radical surgery performed on a 10-yr-old Chinese female with multiple echinococcosis lesions and the diagnosis of the infection by imaging, histology, serology, and DNA analysis. Molecular genotyping provided unequivocal proof that the patient was infected with Echinococcus granulosus, the cause of cystic echinococcosis.

Molecular study of Echinococcus in west-central China.

West-central China is an important endemic focus of both alveolar and cystic echinococcosis where several species of intermediate host are commonly infected with Echinococcus granulosus and E. multilocularis . Isolates of E. granulosus were collected from humans and other animals from different geographical areas of Qinghai, Ningxia, Gansu and Sichuan, and genotyped using the mitochondrial DNA marker ATP synthase subunit 6 gene (atp6). The sheep strain (G1 genotype) of E. granulosus was shown to be the only genotype present in sheep, cattle, goats, yaks and humans in the study areas. However, some heterogeneity in the atp6 sequence was evident in a number of the isolates with the most frequent change being a silent substitution (G/A) at position 360 compared with the G1 reference sequence representing isolates collected from the majority of hosts except humans. Two E. multilocularis isolates examined also had sequences that varied from each other and from the reference E. multilocularis atp6 sequence. The genotypic variation we report may reflect phenotypic differences with important consequences in terms of increased host infectivity for hosts by local Echinococcus strains, possibly impacting on the epidemiology and control of echinococcosis. Such adaptations may also result in different sensitivity to drugs or increased virulence for hosts that will impede control efforts and even affect vaccination strategies against Echinococcus.

Brain metastasis of alveolar echinococcosis in a hyperendemic focus of Echinococcus multilocularis infection.

An unusual female case, with alveolar echinococcosis (AE) disseminated from the primary hepatic lesion to the brain by metastasis formation, was retrospectively identified during a community survey in Ningxia Hui Autonomous Region, northwest China in 2003. Among possible metastases of hepatic AE, locations to the brain are rare and usually fatal; and they have especially been assigned to concomitant immune suppression. An enhancing role of pregnancy, which may be suspected in this case, the favourable outcome after surgery and chemotherapy, and also a mental disability in a child following long-term intrauterine exposure to mebendazole, make the report particularly unique.

Transmission dynamics of the Echinococcus granulosus sheep-dog strain (G1 genotype) in camels in Tunisia.

Cystic echinococcosis, caused by Echinococcus granulosus, is highly endemic in North Africa and the Middle East. This paper examines the abundance and prevalence of infection of E. granulosus in camels in Tunisia. No cysts were found in 103 camels from Kébili, whilst 19 of 188 camels from Benguerden (10.1%) were infected. Of the cysts found 95% were considered fertile with the presence of protoscolices and 80% of protoscolices were considered viable by their ability to exclude aqueous eosin. Molecular techniques were used on cyst material from camels and this demonstrated that the study animals were infected with the G1 sheep strain of E. granulosus. Observed data were fitted to a mathematical model by maximum likelihood techniques to define the parameters and their confidence limits and the negative binomial distribution was used to define the error variance in the observed data. The infection pressure to camels was somewhat lower in comparison to sheep reported in an earlier study. However, because camels are much longer-lived animals, the results of the model fit suggested that older camels have a relatively high prevalence rate, reaching a most likely value of 32% at age 15 years. This could represent an important source of transmission to dogs and hence indirectly to man of this zonotic strain. In common with similar studies on other species, there was no evidence of parasite-induced immunity in camels.

Epidemiological and morbidity assessment of Schistosoma japonicum infection in a migrant fisherman community, the Dongting Lake region, China.

We assessed the epidemiology and morbidity related to Schistosoma japonicum infection by both parasitological (Kato-Katz method and miracidium hatching test) and ultrasound examination in a migrant fisherman community (n = 106) from the Dongting Lake region in China in 2001. A prevalence of 69.8% and a mean infection intensity of 66.5 eggs per gram (epg) were recorded for this group. Males had a higher level of infection (77%) and intensity (92.2 epg) compared with females (58% and 28.6 epg, respectively). Subjects aged 11-20 years had the highest prevalence of infection (91%) and the heaviest infection intensity (85.3 epg) among all age groups. Twenty-six percent of the subjects had not been treated previously for schistosomiasis. Parenchymal fibrosis (> or = stage II) detected by ultrasound and spleen enlargement (or splenectomy) occurred in 37% and 9% of subjects, respectively. Portal vein dilation was detected in 18% subjects, and 58% (11/19) of those patients with portal vein enlargement were shown to have parenchymal fibrosis (> or = stage II). These results indicate that S. japonicum infection and related morbidity in mobile fisherman is still unacceptably high despite the fact that China has implemented a Schistosomiasis Control Project financed by a World Bank Loan for a period of 8 years since 1992. Clearly, new approaches for schistosomiasis control, especially in communities like these mobile fishermen, should be explored.

Molecular epidemiology of cystic echinococcosis.

Echinococcus granulosus exhibits substantial genetic diversity that has important implications for the design and development of vaccines, diagnostic reagents and drugs effective against this parasite. DNA approaches that have been used for accurate identification of these genetic variants are presented here as is a description of their application in molecular epidemiological surveys of cystic echinococcosis in different geographical settings and host assemblages. The recent publication of the complete sequences of the mitochondrial (mt) genomes of the horse and sheep strains of E. granulosus and of E. multilocularis, and the availability of mt DNA sequences for a number of other E. granulosus genotypes, has provided additional genetic information that can be used for more in depth strain characterization and taxonomic studies of these parasites. This very rich sequence information has provided a solid molecular basis, along with a range of different biological, epidemiological, biochemical and other molecular-genetic criteria, for revising the taxonomy of the genus Echinococcus. This has been a controversial issue for some time. Furthermore, the accumulating genetic data may allow insight to several other unresolved questions such as confirming the occurrence and precise nature of the E. granulosus G9 genotype and its reservoir in Poland, whether it is present elsewhere, why the camel strain (G6 genotype) appears to affect humans in certain geographical areas but not others, more precise delineation of the host and geographic ranges of the genotypes characterised to date, and whether additional genotypes of E. granulosus remain to be identified.

Five-year impact of repeated praziquantel treatment on subclinical morbidity due to Schistosoma japonicum in China.

We report the 5-year impact (1996-2001) of repeated praziquantel chemotherapy on subclinical morbidity related to Schistosoma japonicum infection. We repeated stool examinations and hepatosplenic ultrasonography in a cohort of 120 individuals living on an island with endemic infection in Dongting Lake, China. Prevalence of schistosome infection fell by 43% and intensity (geometric mean eggs per gram) declined by 80% over the 5 years. However, transmission persisted at a dangerously high rate of 13% per year for re-infection or new infection in the cohort. The prevalence of left-lobe enlargement and dilated portal vein fell significantly (P < 0.01) to about half initial levels although a few patients progressed during the study period. At study endpoint, infection was nearly twice as common if the portal vein was dilated (23% versus 13%, respectively), but this association was not statistically significant (P > 0.05). However, endpoint infection was even more strongly associated with left-lobe enlargement (57% versus 15%, P < 0.01). The proportions of subjects with improved parenchymal and periportal fibrosis were much higher than the proportions of subjects that progressed (P < 0.05). Reduction of prevalence and intensity of infection, and improvement of subclinical morbidity, were benefits of repeated treatments. Further research is needed to understand why some patients developed fibrosis despite substantial reductions in egg counts and to evaluate the functional importance of residual subclinical morbidity after chemotherapy-based control in the lake and marshland area of China.

Epidemiology and strain characteristics of Echinococcus granulosus in the Benghazi area of eastern Libya.

The incidence of surgically confirmed cystic echinococcosis in eastern Libya was estimated to be at least 4.2 cases/100,000, with significantly more female cases than male. The prevalences of infection with Echinococcus granulosus among 1087 sheep, 881 goats, 428 camels and 614 cattle from the same region, determined postmortem in abattoirs, were 20%, 3.4%, 13.6% and 11%, respectively. Infection in the livestock was age-dependent and, generally, the female animals were more often infected than the male. The measurements of rostellar hooks on protoscoleces collected from sheep and cattle were similar but significantly different from the corresponding measurements of parasites of human or camel origin. However, when a portion of the cytochrome c-oxidase subunit I (cox1) gene from each of 30 protoscolex samples from Libya (12 from cattle, three from humans, five from camels and 10 from sheep) was sequenced, the sequences were all found to be identical to that published for the common sheep strain of E. granulosus.

The molecular epidemiology of Echinococcus granulosus and cystic hydatid disease.

It is now generally recognized that the dog tapeworm Echinococcus granulosus, the cause of cystic hydatid disease, exhibits substantial genetic diversity. This variability has important implications for the design and development of vaccines, diagnostic reagents and drugs effective against this parasite. The paper describes various deoxyribonucleic acid-based approaches that have been used for accurate identification of these genetic variants and their application in molecular epidemiological surveys of cystic hydatid disease in different geographical settings.