Polycystic echinococcosis (PE) is a zoonosis endemic in the Neotropical region of the Americas. It is caused by the larval stage of the cestode Echinococcus vogeli, which develops as harmful cysts that slowly grow in the liver, lungs and other organs of humans and other host species. Human PE diagnosis is usually based on clinical and epidemiological aspects and imaging techniques, often requiring confirmation by immunological assays. The currently available serological tests for detecting antibodies against Echinococcus spp. are mostly based on complex, variable and poorly characterized mixtures of native parasite antigens, which impairs specificity and/or sensitivity. In this scenario, the evaluation of well-characterized alternative antigens is urgently needed for the improvement of PE diagnosis. Here, two subunits (AgB8/1 and AgB8/2) of the major secretory antigen from Echinococcus granulosus (antigen B (AgB)), of diagnostic value for cystic echinococcosis, were validated for PE diagnosis. These antigens, produced as pure recombinant proteins (rAgB8/1 and rAgB8/2) in Escherichia coli, allowed detecting specific immunoglobulin G antibodies in sera from PE patients in an enzyme-linked immunosorbent assay, with sensitivities of 83.72% and 81.40%, respectively, and specificities of 83.12% and 80.09%, respectively. The use of recombinant proteins overcomes difficulties to obtain parasite material and reduced non-specific reactions and costs. Our results demonstrated reproducibility and accuracy high enough to be considered valid according to the acceptance criteria for Food and Drug Administration assay validation. This qualifies rAgB8/1 and rAgB8/2 as potential substitutes for the currently used parasite crude or partially purified antigens.

Infection is a significant cause of morbidity and mortality in patients with chronic kidney disease, especially who were under dialysis due to their depressed immunity. Toxoplasma gondii is a ubiquitous parasite that causes severe manifestations in immunocompromised patients. This case-control study was conducted to the immunodiagnosis and molecular validation of T. gondii infection among patients with end-stage renal disease undergoing haemodialysis. The study population consisted of 260 haemodialysis patients and 259 healthy controls referred to the main dialysis centres of Tehran, Iran during 2016. Anti-T. gondii immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies were assessed using enzyme-linked immunosorbent assay. As well, the T. gondii genomic DNA in whole blood samples of IgM-positive patients and healthy controls was evaluated using GRA6-polymerase chain reaction (PCR) and SAG1-loop-mediated isothermal amplification (LAMP) assays. The anti-T. gondii IgG and IgM antibodies were detected in 175 (67.3%) and 18 (7%) of haemodialysis patients and 122 (47%) and 4 (1.5%) of controls, respectively. Two of the 18 blood samples from IgM-positive patients and none of the IgM-positive control subjects were positive by GRA6-PCR. Whereas, nine and two blood samples of IgM-positive patients and controls were positive for Toxoplasma DNA by a SAG1-LAMP technique respectively. The seropositivity of the Toxoplasma IgM antibody was significantly different between haemodialysis patients and healthy controls which was confirmed by PCR and LAMP. The higher prevalence of T. gondii infection in haemodialysis patients compared with the controls proposes that these patients can be a group at risk for toxoplasmosis and screening for toxoplasmosis before dialysis is necessary for the patients.

Human ascariasis is a neglected tropical disease of great relevance to public health and is considered the most frequent helminthiasis in poor regions. Accurately diagnosing this parasite has been challenging due to limitations of current diagnostic methods. Immunoglobulin Y (IgY) technology is a very effective alternative for the production of highly specific and profitable antibodies. This study aimed to produce and apply anti-Ascaris suum IgY antibodies in the immunodiagnosis of human ascariasis. Five immunizations comprising total saline extract from A. suum adult life forms were given at 14-day intervals to Gallus gallus domesticus hens of the Isa Brown line. Eggs and blood samples were collected weekly and fortnightly, respectively, to monitor the production of antibodies. The specificity of antibodies was confirmed by dot-blot, kinetic enzyme-linked immunosorbent assay (ELISA), avidity ELISA, immunoblotting and indirect immunofluorescence antibody tests. The application for disease diagnosis was performed through the detection of immune complexes in human serum samples by sandwich ELISA. Peaks of IgY anti-A. suum production occurred at weeks 6 and 8. IgY showed high avidity levels after the second dose of immunization, ranging from 64% to 93%, with a mean avidity index of 78.30%. Purified IgY recognized 12 bands of proteins from A. suum saline extract. Eggs, the uterine portion and cuticles of A. suum female adult are reactive in immunofluorescence. The detection of immune complexes showed diagnostic values of 80% sensitivity and 90% specificity. In conclusion, specific IgY have been shown to be a potential immunodiagnostic tool with promising future applications in human ascariasis.

In this study, we have produced a monoclonal antibody (MoAb) against 16 kDa antigen of Paramphistomum gracile (16 kDaAgPg), and developed an accurate sandwich enzyme-linked immunosorbent assay (sandwich ELISA) for the detection of circulating 16 kDaAg in the serum and fecal samples from cattle naturally infected with P. gracile. MoAb 1D10 was immobilized on a microtitre plate, and the antigen in the samples was captured and detected with biotinylated rabbit anti-16 kDaAgPg antibody. The lower detection limit of sandwich ELISA was 3·5 pg mL−1, and no cross-reaction with other parasite antigens was evaluated. The reliability of the assay was examined using the serum and fecal samples from cattle naturally infected with P. gracile, Fasciola gigantica, Moniezia benedeni, Trichuris sp., Strongyloides sp., strongylids and non-infected animals. The sandwich ELISA showed the sensitivity, specificity and accuracy at 98·33, 100 and 99·55% (serum samples), and 96·67, 100 and 99·09% (fecal samples). Therefore, this detection method is a rapid and excellent potential assay for the accurate diagnosis of paramphistomosis.

There is an increasing interest in improving neurocysticercosis (NCC) diagnosis through the search of new and alternative antigenic sources, as those obtained from heterologous antigens. The aim of this study was to obtain potential biomarkers for NCC diagnosis after gel filtration chromatography [gel filtration fraction (GFF)] from the total saline extract (SE) from Taenia saginata metacestodes, followed by protein identification and application in immunodiagnostic. SE and GFF proteic profiles were characterized in gel electrophoresis, and diagnostic performance was verified by testing 160 serum samples through enzyme-linked immunosorbent assay and immunoblotting. Sensitivity (Se), specificity (Sp) and other diagnostic parameters were calculated. Polypeptides of interest in the diagnosis of human NCC present at GFF were analysed by mass spectrometry (MS) and B-cell epitopes were predicted. GFF had the best diagnostic parameters: Se 93·3%; Sp 93%; AUC 0·990; LR+ = 13·42 and LR− = 0·07, and proved to be useful reacting with serum samples in immunoblotting. Proteic profile ranged from 64 to 68 kDa and enolase and calcium binding protein calreticulin precursor were identified after MS. The enolase and calcium-binding protein calreticulin precursor showed 18 and 10 predicted B-cell epitopes, respectively. In conclusion we identified important markers in the GFF with high efficiency to diagnose NCC.

Up to now, parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Hence, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden. In the present study, a monoclonal antibody (MoAb) against recombinant Fasciola gigantica fatty acid binding protein (rFgFABP) has been produced. As well, a reliable sandwich enzyme-linked immunosorbent assay (sandwich ELISA) has been developed for the detection of circulating FABP in the sera of mice experimentally and cattle naturally infected with F. gigantica. MoAb 3A3 and biotinylated rabbit anti-recombinant FABP antibody were selected due to their high reactivities and specificities. The lower detection limit of sandwich ELISA was 5 pg mL−1, and no cross-reaction with other parasite antigens was observed. This assay could detect F. gigantica infection from day 1 post infection. In experimental mice, the sensitivity, specificity and accuracy of this assay were 93·3, 100 and 98·2%, while in natural cattle they were 96·7, 100 and 99·1%. Hence, this sandwich ELISA method showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica.

In regions where Chagas disease is endemic, canine Trypanosoma cruzi infection is highly correlated with the risk of transmission of the parasite to humans. Herein we evaluated the novel TcTASV protein family (subfamilies A, B, C), differentially expressed in bloodstream trypomastigotes, for the detection of naturally infected dogs. A gene of each TcTASV subfamily was cloned and expressed. Indirect enzyme-linked immunosorbent assays (ELISA) were developed using recombinant antigens individually or mixed together. Our results showed that dogs with active T. cruzi infection differentially reacted against the TcTASV-C subfamily. The use of both TcTASV-C plus TcTASV-A proteins (Mix A+C-ELISA) enhanced the reactivity of sera from dogs with active infection, detecting 94% of the evaluated samples. These findings agree with our previous observations, where the infected animals exhibited a quick anti-TcTASV-C antibody response, coincident with the beginning of parasitaemia, in a murine model of the disease. Results obtained in the present work prove that the Mix A+C-ELISA is a specific, simple and cheap technique to be applied in endemic areas in screening studies. The Mix A+C-ELISA could help to differentially detect canine hosts with active infection and therefore with high impact in the risk of transmission to humans.

The objective of this paper is to critically review and summarize available scientific and lay literature, and ongoing studies on human and porcine cysticercosis in Mozambique to identify knowledge gaps and direct immediate and long-term research efforts. Data on the spatial distribution and prevalence of the disease in human and swine populations are scarce and fragmented. Human serological studies have shown that 15–21% of apparently healthy adults were positive for cysticercosis antibodies or antigen, while in neuropsychiatric patients seroprevalence was as high as 51%. Slaughterhouse records indicate a countrywide occurrence of porcine cysticercosis, while studies have shown that 10–35% of pigs tested were seropositive for cysticercosis antibodies or antigen. Current research in Mozambique includes studies on the epidemiology, molecular biology, diagnosis and control of the disease. Future research efforts should be directed at better understanding the epidemiology of the disease in Mozambique, particularly risk factors for its occurrence and spread in human and swine populations, documenting the socio-economic impact of the disease, identifying critical control points and evaluating the feasibility and epidemiological impact of control measures and development of local level diagnostic tools for use in humans and swine.

The surface antigens of the bovine filarial parasite Setaria digitata were isolated by EDTA extraction and purified by affinity chromatography using sepharose bound human filarial (Wuchereria bancrofti) antibodies obtained from chronic human filarial sera. The purified and crude antigens were used in enzyme-linked immunosorbent assay (ELISA) for the detection of serum antibodies in bancroftian filariasis. The purified antigen showed sensitive and specific reactions in ELISA for the detection of antibodies in filarial sera and showed least cross reactivity with other parasitic infections. The crude and purified antigens showed about 18 and 6 peptide bands respectively in SDS-PAGE and about 11 and 6 antigenic bands respectively in enzyme-linked immunoelectrotransfer blot (EITB). The purified antigen was observed to be glycoprotein in nature. It was possible to identify the stage-specific infection in human filariasis by using the crude and purified antigens in EITB.

Specific immunoreactive components present in crude somatic extract and in excretory-secretory (ES) products of Gnathostoma spinigerum advanced third-stage larvae (L3) were identified by Western blotting and their diagnostic potential evaluated by indirect ELISA. Although both crude antigen preparations were highly complex, the ES antigen gave a more satisfactory diagnostic result. Most G. spinigerum specific components present in the somatic and ES preparations had molecular weights below 29 kD and were not glycosylated, judging from the concanavalin A staining pattern. Specific diagnostic antigens were prepared by subjecting the crude preparations to SDS-PAGE. Low molecular weight components were identified and electroeluted from the gel. Excess SDS was removed by the use of an ion retardation resin. The antigens obtained by this relatively simple procedure were found to be highly specific for G. spinigerum. Sensitivity, specificity, and positive and negative predictive values for the assay using the fractionated somatic antigen were 100%.

Infective first stage larvae of Trichinella spiralis were recovered from muscles of laboratory infected mice by digesting the muscles with 1% HC1–1% pepsin and collecting the larvae by modified Baerman's method. The larvae were cultivated in a serum-free medium for 18 h. The ES antigen obtained from the culture medium was used in an enzyme-linked immunosorbent assay (ELISA) for detecting IgG antibodies to T. spiralis in serum samples collected from three groups of individuals. The individuals of the first group were parasitologically confirmed trichinellosis patients, while those of group 2 were patients with other helminthiasis and group 3 were healthy, parasite-free individuals. The specificity of the assay was 100%. The sensitivity of the test was also 100% when performed on sera of group I collected at days 57 and 120 after infection. Sera collected earlier (day 23) and those collected 700 days after infection had negligible reactivity. Thus IgG-ELISA using ES antigen of the L1 was useful not only for diagnosis but also in evaluation of cure. Western blot analysis revealed that specific antigens of T. spiralis were 94, 67, 63, and 39 kilodalton components.

The present paper describes two repetitive proteins representing common antigens of African trypanosomes which are non-variant and which are recognized early in infection by the host immune system. These antigens were identified by their ability to immunoreact with bovine serum taken during the early phase of a cyclic trypanosomal infection. Screening of a cDNA library from T. b. gambiense with such early infection serum identified a protein which contains a repetitive motif consisting of 68 amino acid repeat units (GM6). Immunofluorescence and immunogold electron microscopy revealed that GM6 is located on fibres which connect the microtubules of the membrane skeleton with the flagellum. A second repetitive antigen detected by this serum is MARP1 (microtubule-associated repetitive protein 1), a protein previously characterized in this laboratory as a high-molecular weight component of the membrane skeleton, which consists of more than 50 tandemly repeated, near-identical 38 amino acid repeat units. Beta-galactosidase fusion products of both proteins demonstrated a strong immunoreactivity with sera from T. b. brucei and T. congolense-infected cattle. The result from this preliminary immunological evaluation indicates a high immunodiagnostic sensititivy (90%) of the two recombinant antigens which make them interesting candidates for immunodiagnosis of trypanosomiasis in cattle.

The recombinant Echinococcus multilocularis antigen ll/3–10 is one of the most promising tools for immunodiagnosis of alveolar echinococcosis in human patients. Its nucleic acid sequence represents a part of the E. multilocularis gene encoding the metacestode antigen II/3, the former being basically present and expressed in both E. multilocularis and E. granulosus. Most (94%) patients with alveolar echinococcosis respond to infection with a marked anti-II/3–10 IgG synthesis; in contrast, most of the cystic echinococcosis patients do not, for some reason, recognize the recombinant antigen. We tackled this problem by generating cDNA derived from both E. granulosus and E. multilocularis full length II/3 genes, performed by reverse transcription and PCR amplification. Sequence analysis revealed a very high degree of conservation of the primary sequence of the antigen II/3 in both Echinococcus species. cDNA fragments were subcloned and expressed in E. coli as fusion proteins with Schistosoma japonicum glutathione S-transferase. Recombinant proteins were affinity purified and comparatively assessed by ELISA with respect to antibody-binding characteristics. Sera from patients suffering from cystic echinococcosis showed no significant differences in reactivity with the antigens derived from either E. multilocularis or E. granulosus. Therefore, parameters other than some minor differences in the primary sequence seem to be responsible for the lack of antigen II/3 recognition in cystic echinococcosis.

Note

Nucleotide sequence data reported in this paper have been submitted to the GenBank® data base with the accession numbers U05573 and U05574.

Three ELISA assays, based on hyperimmune rabbit serum raised against adult cestode somatic antigen, were applied in this study for the detection of Taenia- and Echinococcus-specific antigens in host faeces. The first assay, using an antiserum against Taenia pisiformis antigen extract, was used in a time-course of T. pisiformis experimental infection in dogs. The assay was shown to be considerably more sensitive than microscopical detection of eggs in faeces. Antigen was present in faeces before patency and antigen levels were independent of T. pisiformis egg output. The second assay, involving a test for human taeniasis based on antibodies against T. solium, was applied in two field studies carried out in China and Guatemala. The test was highly specific, no false positive reactions occurred with human faecal samples and the test was capable of diagnosing individuals who would not have been detected by coproscopy or treatment to recover the tapeworm. A third assay was designed for E. granulosus and demonstrated 87·5% sensitivity and 96·5% specificity with samples from naturally and experimentally infected dogs with Echinococcus or Taenia infections. In both the human Taenia and canine Echinococcus studies antigen could be detected in faecal samples from infected hosts stored in 5% formalin for 6 months. Further refinements to these tests for field application are discussed.

A recombinant Echinococcus multilocularis antigen (II/3–10), which had previously been shown to exhibit immunodiagnostic characteristics highly specific for human alveolar echinococcosis, and the corresponding native parasite antigen, were further characterized with immunochemical and molecular biological methods. Immunoblot analysis using a polyclonal antiserum raised in rabbits against the recombinant protein, and subsequent Northern hybridization analysis, revealed that the native antigen was expressed by E. multilocularis at the adult as well as at the metacestode stage. In metacestodes, the antigen was shown by using indirect immunofluorescence and the same antiserum to be localized within the germinal layer and membrane structures of developing protoscolices. Electrophoretic analyses revealed remarkable differences in the apparent molecular weight of the antigen under reducing and non-reducing conditions. In further immunoblot analyses, anti-II/3–10 antibodies identified the corresponding epitopes on bands with identical Mr in all E. multilocularis isolates investigated (European, Asian and North American). By Southern hybridization analyses of the respective gene, phylogenetically related sequences were shown to be present in other helminth species such as E. granulosus and several Taenia spp. In the same respect, immunoblotting revealed that anti-II/3–10 antibodies reacted with antigens of different Mr from various E. granulosus isolates and some other cestode species, indicating the presence of shared and thus potentially cross-reacting epitopes. The relevance of these findings for the immunodiagnostic performance of the recombinant antigen is discussed.

A dipstick dot ELISA for detection of Taenia-specific coproantigens was developed. The test was based on a sandwich ELISA using antibodies raised against adult Taenia solium. Antibodies were adsorbed to nitrocellulose paper previously adhered to acetate plastic to form dipsticks. Once blocked with 5% skimmed milk and dried the antibody-coated dipsticks were stable for several weeks at room temperature. Both micro and dot ELISA formats were genus specific although the dot ELISA was less sensitive than the micro ELISA based on the same antiserum. During field studies, in which the majority of samples were tested in rural villages soon after collection, 3728 samples were tested. All samples were also examined by microscopy using formol ether concentration and individuals questioned to determine whether they were aware of being infected. After the initial diagnostic work individuals were treated with taeniacidal drugs for worm recovery. Use of the coproantigen test significantly increased the number of cases diagnosed. Of the 41 cases diagnosed by the three diagnostic techniques combined 31 were detected by the dipstick assay making it the most sensitive technique employed. The specificity of the dipstick assay was 99·9% with a positive predictive value of 88·%. The combined diagnostic approach did not, however, diagnose all cases. The coproantigen test was fast and easy to use. Further improvements may make the dipstick test suitable for wide-scale use in field studies and diagnostic laboratories.

This study describes, for the first time, the characterization of excretory–secretory antigens (ES-Ag) from Echinococcus granulosus protoscoleces, evaluating their usefulness in the immunodiagnosis of human cystic echinococcosis. ES-Ag were obtained from the first 50 h maintenance of protoscoleces in vitro. This preparation contained over 20 major protein components which could be distinguished by 1-dimensional SDS–PAGE with apparent masses between 9 and 300 kDa. The culture of of protoscoleces from liver produced a greater variety of excretory–secretory protein components than those from lung. Determination of enzymatic activities of secreted proteins revealed the presence of phosphatases, lipases and glucosidases, but no proteases. These findings were compared to those obtained from somatic extracts of protoscoleces and hydatid cyst fluid products. Immunochemical characterization was performed by immunoblotting with sera from individuals infected by cystic echinococcosis (n=15), non-hydatidic parasitoses (n=19), various liver diseases (n=24), lung neoplasia (n=16), and healthy donors (n=18). Antigens with apparent masses of 89, 74, 47/50, 32, and 20 kDa showed specificity for immunodiagnosis of human hydatidosis. The 89 and 74 kDa components corresponded to antigens not yet described in E. granulosus, whereas proteins of 41–43 kDa and 91–95 kDa were recognized by the majority of the non-hydatid sera studied.

Infection with Entamoeba histolytica, the protozoan parasite that causes amoebic colitis and liver abscess, results in 34 million to 50 million symptomatic cases of amoebiasis (all illnesses caused by E. histolytica, including amoebic dysentery) worldwide each year, causing 40 thousand to 100 thousand deaths annually. As a result of accruing biochemical, genetic and immunological data, E. histolytica was re-defined in 1993 to recognise the existence of two morphologically identical but genetically distinct human parasites: E. histolytica, the aetiological agent of invasive intestinal and extraintestinal amoebiasis, and Entamoeba dispar, a non-pathogenic intestinal parasite. Because microscopy is unable to distinguish between these two organisms, it should no longer be relied upon to diagnose amoebiasis. Sensitive and specific molecular techniques that are able to distinguish E. histolytica from E. dispar have been developed recently; they include (1) the detection of an E. histolytica antigen using an enzyme-linked immunosorbent assay (ELISA), (2) the use of the polymerase chain reaction (PCR) to amplify amoebic DNA, and (3) the culture of stool samples followed by isoenzyme analysis. Of these three test methods, only antigen detection using ELISA can be performed rapidly and easily, making it the diagnostic test method of choice for clinical use in the developing world, where the morbidity and mortality caused by E. histolytica are greatest. However, the PCR method is a powerful tool for the genetic typing of different amoebic strains. Together these two methods should result in both improved clinical diagnosis and treatment of amoebiasis, and a greater understanding of the epidemiology of E. histolytica. Such knowledge will not only assist public health efforts to control amoebiasis, but also facilitate the careful testing of the anti-amoebic vaccines that are currently being developed.

Parasitic bronchitis is a serious disease of cattle and is caused by the nematode, Dictyocaulus viviparus. For over 30 years, a radiation-attenuated larval vaccine has been used for prevention of this disease. This vaccine has been used with considerable success in the UK and parts of Western Europe, however, it has several disadvantages. It has a short shelf- life and the vaccine has to be produced annually necessitating the use of donor calves. Following vaccination, calves must receive further boosting from natural challenge to maintain protective immunity. Sales of the irradiated larval vaccine have decreased dramatically since the 1970s. This is thought to be due to increased reliance of farmers on anthelmintic programmes to control lungworm infection. It is possible that, under certain circumstances, these programmes do not allow sufficient parasite exposure to stimulate protective immunity to further Dictyocaulus challenge. This is borne out by the recent documented increase in the number of outbreaks of parasitic bronchitis in the UK. A stable vaccine against D. viviparus that is capable of stimulating a more prolonged immunity would be beneficial. Recent research has been directed at identification and isolation of components thought to be involved in parasite survival in the host and examination of their potential as vaccine candidates. One of these components is acetylcholinesterase (AChE), an enzyme secreted by adult worms. This review describes the development of the secreted AChE as a vaccine candidate, as well as documenting recent developments in the immunodiagnosis of D. viviparus.

Schistosomiasis remains a serious world-wide public health problem with a still unfulfilled need for routine cost-effective methods of diagnosis. Such methods are required not only for people in endemic areas, but increasingly for tourists who may have become infected during visits to such places. This article reviews the wide range of immunoassays and antigenic preparations that have been shown to have potential for diagnosis of schistosomiasis by the indirect method of antibody detection. Antigens in native form derived from cercariae, adult worms and eggs are considered, as well as schistosome antigens produced by recombinant DNA technology and the schistosome cross-reactive antigen, keyhole limpet haemocyanin (KLH). Respective advantages and disadvantages of antibody detection, circulating antigen detection and parasitological methods of diagnosis are analysed. It is suggested that due to the relative insensitivity of both parasitology and antigen detection, antibody detection methods could find increasing use in situations of low infection intensity.