How can schistosome circulating antigen assays be best applied for diagnosing male genital schistosomiasis (MGS): an appraisal using exemplar MGS cases from a longitudinal cohort study among fishermen on the south shoreline of Lake Malawi.

We provide an update on diagnostic methods for the detection of urogenital schistosomiasis (UGS) in men and highlight that satisfactory urine-antigen diagnostics for UGS lag much behind that for intestinal schistosomiasis, where application of a urine-based point-of-care strip assay, the circulating cathodic antigen (CCA) test, is now advocated. Making specific reference to male genital schistosomiasis (MGS), we place greater emphasis on parasitological detection methods and clinical assessment of internal genitalia with ultrasonography. Unlike the advances made in defining a clinical standard protocol for female genital schistosomiasis, MGS remains inadequately defined. Whilst urine filtration with microscopic examination for ova of Schistosoma haematobium is a convenient but error-prone proxy of MGS, we describe a novel low-cost sampling and direct visualization method for the enumeration of ova in semen. Using exemplar clinical cases of MGS from our longitudinal cohort study among fishermen along the shoreline of Lake Malawi, the portfolio of diagnostic needs is appraised including: the use of symptomatology questionnaires, urine analysis (egg count and CCA measurement), semen analysis (egg count, circulating anodic antigen measurement and real-time polymerase chain reaction analysis) alongside clinical assessment with portable ultrasonography.

In-depth proteomic characterization of Schistosoma haematobium: Towards the development of new tools for elimination.

BACKGROUND: Schistosomiasis is a neglected disease affecting hundreds of millions worldwide. Of the three main species affecting humans, Schistosoma haematobium is the most common, and is the leading cause of urogenital schistosomiasis. S. haematobium infection can cause different urogenital clinical complications, particularly in the bladder, and furthermore, this parasite has been strongly linked with squamous cell carcinoma. A comprehensive analysis of the molecular composition of its different proteomes will contribute to developing new tools against this devastating disease. METHODS AND FINDINGS: By combining a comprehensive protein fractionation approach consisting of OFFGEL electrophoresis with high-throughput mass spectrometry, we have performed the first in-depth characterisation of the different discrete proteomes of S. haematobium that are predicted to interact with human host tissues, including the secreted and tegumental proteomes of adult flukes and secreted and soluble egg proteomes. A total of 662, 239, 210 and 138 proteins were found in the adult tegument, adult secreted, soluble egg and secreted egg proteomes, respectively. In addition, we probed these distinct proteomes with urine to assess urinary antibody responses from naturally infected human subjects with different infection intensities, and identified adult fluke secreted and tegument extracts as being the best predictors of infection. CONCLUSION: We provide a comprehensive dataset of proteins from the adult and egg stages of S. haematobium and highlight their utility as diagnostic markers of infection intensity. Protein composition was markedly different between the different extracts, highlighting the distinct subsets of proteins that different development stages present in their different niches. Furthermore, we have identified adult fluke ES and tegument extracts as best predictors of infection using urine antibodies of naturally infected people. This study provides the first steps towards the development of novel tools to control this important neglected tropical disease.

Lung Involvement in Chronic Schistosomiasis with Bladder Squamous Cell Carcinoma.

We report a case of chronic Schistosoma haematobium infection with pseudometastatic pulmonary nodules and high-grade squamous cell carcinoma in a 30-year-old man in Mali. Lung biopsies revealed chronic pulmonary involvement of S. haematobium and ruled out lung metastases.

The substructure of three repetitive DNA regions of Schistosoma haematobium group species as a potential marker for species recognition and interbreeding detection.

BACKGROUND: Schistosoma haematobium is the causative agent of human urogenital schistosomiasis affecting ~112 million people in Africa and the Middle East. The parasite is transmitted by snails of the genus Bulinus, which also transmit other closely related human and animal schistosomes. The accurate discrimination of S. haematobium from species infecting animals will aid effective control and elimination programs. Previously we have shown the utility of different repetitive nuclear DNA sequences (DraI, sh73bp, and sh77bp) for the identification of S. haematobium-group species and inter-repeat sequences for discriminating S. haematobium from S. bovis. RESULTS: In this current study we clarify the structural arrangement and association between the three repetitive sequences (DraI, sh73bp, and sh77bp) in both S. haematobium and S. bovis, with a unique repeat linker being found in S. haematobium (Sh64bp repeat linker) and in S. bovis (Sb30bp repeat linker). Sequence data showed that the 3'-end of the repeat linker was connected to the DraI repetitive sequence array, and at the 5'-end of the repeat linker sh73bp and sh77bp were arranged in an alternating manner. Species-specific oligonucleotides were designed targeting the species-specific repeat linkers and used in a reverse line blot (RLB) hybridization assay enabling differentiation between S. haematobium and S. bovis. The assay was used to discriminate natural infections in wild caught Bulinus globosus. CONCLUSION: This research enabled the characterisation of species-specific DNA regions that enabled the design of species-specific oligonucleotides that can be used to rapidly differentiate between S. haematobium and S. bovis and also have the potential to aid the detection of natural hybridization between these two species.

SCHISTOSOMA-SPECIFIC 26 KDA PROTEIN FOR A DIAGNOSIS OF ACUTE AND CHRONIC SCHISTOSOMIASIS.

One of the world wide major public health problems is the schistosomiasis that is caused by Schistosoma (S.) heamatobium. It is also one of the main concerns for the public health community in Egypt. There are several immunodiagnostic methods used for that diagnosis of such disease, but some are more sensitive and specific than others. The purified 26 kDa Schistosoma-specific protein (PSPA-26) detection in serum samples is found out to be more valuable in diagnosis; it also helps in the early diagnosis which will lead to the early treatment before the irreversible damage takes place. PSPA-26 was purified from whole worms by DEAE-Sephadex G-75 ion exchange chromatography and then was injected into rabbits to produce specific polyclonal antibodies (anti-PSPA-26 pAb) which were then used as a primary capture in the indirect ELISA technique to reveal its reactivity using infected human sera. The anti-PSPA-26 was then labeled with horse-radish peroxidase (HRP) and used as a secondary capture. Sandwich ELISA was done for serum samples of human and hamsters infected with S. heamatobium. The results revealed a sensitivity of 85% for human and 80% for hamster's samples, and a specificity of 95% for human and 91.1% for hamsters samples by comparing them with those infected with other parasites and control samples. Data obtained concluded that PSPA-26 antigen can be used as a diagnostic marker for S. haematobium infection using the sandwich ELISA which is cost effective and applicable technique.

Three-dimensional structure of a schistosome serpin revealing an unusual configuration of the helical subdomain.

Parasitic organisms are constantly challenged by the defence mechanisms of their respective hosts, which often depend on serine protease activities. Consequently, protease inhibitors such as those belonging to the serpin superfamily have emerged as protective elements that support the survival of the parasites. This report describes the crystal structure of ShSPI, a serpin from the trematode Schistosoma haematobium. The protein is exposed on the surface of invading cercaria as well as of adult worms, suggesting its involvement in the parasite-host interaction. While generally conforming to the well established serpin fold, the structure reveals several distinctive features, mostly concerning the helical subdomain of the protein. It is proposed that these peculiarities are related to the unique biological properties of a small serpin subfamily which is conserved among pathogenic schistosomes.

Targeting molecular signaling pathways of Schistosoma haemotobium infection in bladder cancer.

Since 1911 epidemiological evidence indicates that S. haematobium is associated with squamous cell carcinoma of the bladder. However, the mechanisms of this interaction are not clearly defined. Using normal epithelial cells, S. haematobium parasite extracts were able to induce cancer-like phenotypes such as proliferation, apoptosis, migration, invasion and tumorigenesis. The parasite extracts on normal urothelium also presented carcinogenic and mutagenic ability. To further elucidate the biological effects of this parasite, new estrogenic molecules were identified in its extracts. These estrogens are also present in the sera of Schistosoma-infected patients, and they have the ability to repress ER transcriptional activity both in estrogen-responsive MCF7 cells and normal urothelial HCV29 cells. This review will present some of the recent studies of mass spectrometry of S. haematobium extracts and sequence analysis of bladder tissue treated with the same extracts. Finally the molecular and cellular events that might be responsible for schistosomiasis-related bladder cancer will be discussed.

Enzymatic activity and immunolocalization of Schistosoma mansoni and Schistosoma haematobium neutral sphingomyelinase.

We predicted, and provided evidence for, the existence of a schistosome tegument-associated Mg(2+)-dependent neutral sphingomyelinase (nSMase), which controls hydrolysis of surface membrane sphingomyelin molecules, thus allowing nutrients, but not host antibodies, to access proteins at the host-parasite interface. While a putative nSMase was identified in a recent Schistosoma mansoni genome sequencing and analysis study, our report is the first to measure nSMase enzymatic activity in Triton X-100-solubilized surface membrane (Sup 1) and whole worm soluble (SWAP) molecules of male and female S. mansoni and Schistosoma haematobium. Neutral, but no acidic, sphingomyelinase activity was readily detectable by the amplex red sphingomyelinase assay, and increased with incubation time and protein amount. Like nSMase family members, the schistosome nSMase activity was significantly (P<0.05 to <0.0001) enhanced by unsaturated fatty acids and phosphatidyl serine and significantly (P<0.01) decreased following exposure to the nSMase specific inhibitor GW4869. Peptides based on the published sequence of S. mansoni putative nSMase and used in a multiple antigen peptide form induced the generation of specific antibodies, which readily bound to the immunogen and to the cognate protein in Sup 1 and SWAP. Immunofluorescence studies suggested the parasite nSMase is located in the worm tegument and gut lining. Studies using RNA interference are in progress to define nSMase role in larval and adult worm surface membrane antigen exposure and unsaturated fatty acid-mediated attrition.

ShAR1alpha and ShAR1beta: novel putative nicotinic acetylcholine receptor subunits from the platyhelminth blood fluke Schistosoma.

The cDNAs for two novel neuronal-type nicotinic acetylcholine receptor (nAChR) subunits have been cloned and characterised from the parasitic trematode blood fluke Schistosoma haematobium. One of these encodes a putative nAChR alpha-subunit named ShAR1alpha, whilst the second encodes a potential non-alpha subunit, ShAR1beta. These ShARs possess the key structural features common to all nAChRs, but they are unusual in that they have very large cytoplasmic domains spanning M3 and M4. Overall, the ShAR1alpha and ShAR1beta proteins share 37% identity and 53% similarity, but excluding the residues of the M3-M4 domain this rises to 52% identity and 71% similarity. Sequence comparisons with other nAChR polypeptides indicate that both ShARs are most similar to the invertebrate alpha7-like subunits identified in insects and nematodes, and to the vertebrate subunits alpha7 and alpha8. Outside of the M3-M4 domain, 45% and 40%, respectively, of the ShAR1alpha and ShAR1beta residues are conserved in the ACR-16 subunit from Caenorhabditis elegans. Phylogenetic analysis suggests that the ShARs share a common lineage with members of the ACR-16 group as well as alpha7 and alpha8. Immunolocalisation studies revealed distinct and non-overlapping patterns of distribution for ShAR1alpha and ShAR1beta within the parasite. ShAR1beta was localised within the musculature and on discrete cell bodies within the connective parenchyma. In contrast, ShAR1alpha was localised exclusively to the surface membranes, suggesting it may contribute to the regulatory nAChR we have characterised previously. In Xenopus oocyte expression studies, ShAR1alpha did not form functional channels on its own or in combination with ShAR1beta or the chick beta2 subunit. Furthermore, a chimera in which the M3-M4 domain of ShAR1alpha was replaced with that of chick alpha7 was also non-functional. We discuss our findings in the context of the proposed role for surface nAChRs in the regulation of glucose uptake in the parasite, and the potential exploitation of these receptors as targets for cholinergic schistosomicides.

Evidence for the presence of active cytochrome P450 systems in Schistosoma mansoni and Schistosoma haematobium adult worms.

Extracts of the adult worms of both Schistosoma mansoni and Schistosoma haematobium can metabolise some typical P450 substrates but to differing degrees. S. mansoni worm extracts displayed a approximately 12-fold higher specific activity for an aminopyrine substrate than rat liver microsomes. At 4 mM substrate concentration the demethylation reaction with N-nitrosodimethylamine (NDMA) (5 nmol HCHO/mg protein/min) was only half that of rat liver microsomes, whereas in extracts of S. haematobium, no detectable activity was found towards NDMA. Using ethylmorphine as substrate the demethylation activity of S. mansoni extracts (1.82 nmol HCHO/mg protein/min) was 5.5-fold lower than that of rat liver microsomes. Benzphetamine demethylase activity was also readily detectable in S. mansoni worm extracts at 6.79 nmol HCHO/mg protein/min compared with 10.20 nmol HCHO/mg protein/min in the case of rat liver microsomes. When aniline was used as substrate, surprisingly, no activity was found in worm extracts of either S. mansoni or S. haematobium, whereas rat liver microsomes showed high activity towards this amine. The anti-P450 2E1 and 2B1/2 cross-reacted with both worm homogenates and gave a specific band corresponding to a protein of molecular weight of approximately 50.0 kDa. A study with anti-P450 IVA antibody revealed that while this protein was strongly expressed in S. haematobium worm extracts, no immunoreactivity was observed with extracts of S. mansoni. Immunoblotting analyses with anti-P450 IIIA and P450 1A1 did not detect immunoreactive protein in either S. mansoni or S. haematobium.

Horizontal and vertical transfer of mouse endogenous retroviral DNA sequences in schistosomes.

The in situ polymerase chain reaction (PCR) results revealed that mouse type A and type C retroviral sequences were transmitted horizontally from the host to schistosomes. The signals to these retroviral sequences were observed in the nuclei of the mesenchymal and reproductive cells of 8-week Schistosoma japonicum. These signals were also detected in the nuclei of the mesenchymal and reproductive cells and in the cytoplasm of the tegumental tubercles of 24-week S. mansoni. Furthermore, mouse type A retroviral sequence was detected in the DNA extracted from the cercariae of both species. However, mouse type C retroviral sequence and mouse type 2 Alu sequence (B2) were difficult to detect in the cercarial DNA of either species. These findings may indicate that some host sequences are propagated in the schistosome progeny, that is to say, not only horizontal but also vertical transfer of the host gene may occur in schistosomes.

Purification and crystallization of a novel membrane-anchored protein: the Schistosoma haematobium serpin.

A unique serine-protease inhibitor (serpin) of the blood fluke S. haematobium has been crystallized. It is an antitrypsin with an unusual residue (phenylalanine) at its reactive center. Unlike any known member of this gene family, it is a membrane-anchored protein on the surface of the parasite. The location of this serpin and immunological response to the protein indicate that it may play a important role in host-parasite interaction. The crystals belong to the trigonal space group P3221 or P3121 with unit-cell parameters a = b = 64.7, c = 186.7 A, alpha = 90.0, beta = 90.0, gamma = 120.0 degrees. There is one molecule per asymmetric unit and the crystals diffracted to 2.2 A.

Proteoglycans from adult worms of Schistosoma haematobium.

Different types of proteoglycans (PGs) from adult worms of Schistosoma haematobium, were sequentially extracted using chaotropic agents under associative conditions (0.5 M GnCl), dissociative conditions (4 M GnCl) and detergents (Triton X-100 and SDS). The extracts were designated F1, F2, F3 and F4, respectively. The highest amount of uronic acid and carbohydrate was detected in the associative extract (F1) while the highest amount of protein was detected in the SDS extract (F4). Agarose polyacrylamide gel electrophoresis (A-PAGE) indicated the presence of a different PG in each extract with different electrophoretic mobilities. Agarose gel electrophoresis of glycosaminoglycan (GAG) separated from GnCl, associative and dissociative extracts, and the residue suggested the presence of dermatan sulphate in the two extracts and the residue, in addition to a GAG-like material found in the associative extract only. This glycosaminoglycan showed resistance to digestion with all mucopolysaccharidases and nitrous acid treatment. Gel filtration chromatography of associative extract on Sepharose CL-6B indicated the presence of three main uronic acid peaks (P1, P2 and P3). Chondroitin sulphate was the main GAG that could be detected in peak one (P1). Peak two (P2) contains carbohydrate and uronic acid but has no protein or absorbance at 280 nm. P2 has two types of GAGs: dermatan sulphate and a GAG-like material. The role of this PG in helping the adult schistosomes in evading immobilization by the host blood clotting cascade is discussed. Antibodies to peak one and peak two were detected in hamster sera infected with S. haematobium and S. mansoni using the ELISA test. The specificity of peak two was found to be evident in its low cross-reactivity (18.9%) when confronted with S. mansoni infected sera.

Biological and biochemical comparative studies on Schistosoma mansoni from two localities in Egypt where S. haematobium is endemic.

The changing pattern of Schistosoma mansoni and S. haematobium distribution in Egypt is generally attributed to ecological changes caused by the construction of the Aswan High Dam. Although S. mansoni was previously restricted to Lower Egypt, it is now found at certain foci in Upper Egypt. In areas of Lower Egypt where S. mansoni and S. haematobium are sympatric, S. mansoni eggs are shed almost exclusively in the stools of patients, whereas in Upper Egypt they are more frequently shed in the urine. In spite of this difference, the eggs and adult worms obtained from hamsters infected with S. mansoni strains from each of these areas proved to be morphologically identical. Protein patterns and isoenzyme profiles of male or female adult worms of each of the two isolates, obtained from infected hamsters, also proved virtually identical. In hamsters with mixed infections of S. mansoni and S. haematobium, some S. mansoni females cross-mated with S. haematobium males and they then developed ovaries and laid eggs which were typical of S. mansoni and which were excreted from the urinary bladder. In Upper Egypt, which is predominantly a S. haematobium area, patients with established infections may have a preponderance of S. haematobium males associated with S. mansoni females. These females may then migrate to the vesicular plexus and deposit S. mansoni eggs in the urinary bladder, to be shed subsequently in the urine. The observations appear to be better explained by the phenomenon of parthenogenesis than by the production of true genetic hybrids.

Nicotinic acetylcholine receptors on the surface of the blood fluke Schistosoma.

Blood dwelling stages of schistosomes have acetylcholinesterase (AChE) on their teguments. As an initial step towards understanding the function of tegumental AChE, we have used specific ligand-binding assays to identify nicotinic acetylcholine receptors (nAChR) on the schistosome surface. AChR could not be detected on migratory stages using fluoroscein isothiocyanate-alpha-bungarotoxin binding but the amount of specific labelling increased on sexual pairing and as the parasites matured into egg-producing adults. Both AChE and nAChR were concentrated on the dorsal surface of the adult male. These results indicate a role for AChE and AChR associated with the transporting function of this membrane.