A modified lateral flow assay, using serum, for the rapid identification of human and bovine cysticercosis in the absence of false positives.

Background: Previously we reported the use of a monoclonal antibody-based (HP10) antigen (Ag) detection lateral flow assay (LFA) for the diagnosis of extraparenchymal neurocysticercosis (EP-NCC). The assay performed well when used with cerebrospinal fluid (CSF) samples but not with their paired serum samples, due to false-positive reactions in some known negative control cases. Methods: Our novel modification involves pretreatment of serum samples using a combination of sodium deoxycholate and dithiothreitol. Results: The modification overcomes the problem of false positives when using negative serum samples from clinically characterized cases of EP-NCC and bovine cysticercosis. In general, there was good agreement between HP10 Ag enzyme-linked immunosorbent assay (ELISA) and the HP10 Ag-LFA, but the HP10 Ag-ELISA was marginally more sensitive than the modified HP10 Ag-LFA. Conclusions: The modified HP10 Ag-LFA provides a field test for the rapid identification of endemic human and bovine cysticercosis.

Sero-prevalence of Taenia spp. cysticercosis in rural and urban smallholder pig production settings in Uganda.

The pork tapeworm, Taenia solium, is prevalent in Uganda although the prevalence has not been determined in all areas of the country. A cross-sectional study, to determine the sero-prevalence of the parasite in pigs kept under rural and urban production settings, was carried out in three Ugandan districts, Masaka, Mukono and Kamuli. Serum samples from 1185 pigs were tested for the presence of T. solium cysticercosis antigen using the HP10 antigen-ELISA (Ag-ELISA) and the ApDia Ag-ELISA assays. Using parallel interpretation of the two tests showed lower levels of observed prevalence of T. solium in rural production settings (10.8%) compared to urban (17.1%). Additionally, Maximum Likelihood Estimation for evaluating assays in the absence of a gold standard, using TAGS on the R platform, estimated the true sero-prevalence to be lower in rural production setting, 0.0% [0.0-3.2%; 95% confidence interval (CI)] than in urban production setting, 12.3% (4.2-77.5% CI). When the sensitivity/specificity (Se/Sp) of the assays were estimated, assuming conditional independence of the tests, HP10 Ag-ELISA was more sensitive and specific [(Se=53.9%; 10.1-100% CI), (Sp=97.0%; 95.9-100% CI)] than the ApDia assay [(Se=20.2%; 1.5-47.7% CI), (Sp=92.2%; 90.5-93.9% CI)]. Subject to parasitological verification, these results indicate there may be a need to implement appropriate control measures for T. solium in the study areas.

A lateral flow assay (LFA) for the rapid detection of extraparenchymal neurocysticercosis using cerebrospinal fluid.

A lateral flow assay (LFA) for the diagnosis and monitoring of extraparenchymal neurocysticercosis, has been developed. The assay is based on the use of the monoclonal antibody HP10, and when applied to cerebrospinal fluid, correctly identified 34 cases of active extraparenchymal neurocysticercosis, but was negative with 26 samples from treated and cured neurocysticercosis patients and with 20 samples from unrelated neurological diseases. There was complete agreement between the HP10 Ag-ELISA results and the HP10-LFA. The HP10-LFA thus has utility for diagnosis and treatment of extraparenchymal neurocysticercosis, frequently a more dangerous form of the infection.

Correction: The Influence of Socio-economic, Behavioural and Environmental Factors on Taenia spp. Transmission in Western Kenya: Evidence from a Cross-Sectional Survey in Humans and Pigs.

[This corrects the article DOI: 10.1371/journal.pntd.0004223.].

The Influence of Socio-economic, Behavioural and Environmental Factors on Taenia spp. Transmission in Western Kenya: Evidence from a Cross-Sectional Survey in Humans and Pigs.

UNLABELLED: Taenia spp. infections, particularly cysticercosis, cause considerable health impacts in endemic countries. Despite previous evidence of spatial clustering in cysticercosis and the role of environmental factors (e.g. temperature and humidity) in the survival of eggs, little research has explored these aspects of Taenia spp. EPIDEMIOLOGY: In addition, there are significant gaps in our understanding of risk factors for infection in humans and pigs. This study aimed to assess the influence of socio-economic, behavioural and environmental variables on human and porcine cysticercosis. A cross-sectional survey for human taeniasis (T. solium and T. saginata), human cysticercosis (T. solium) and pig cysticercosis (T. solium) in 416 households in western Kenya was carried out. These data were linked to questionnaire responses and environmental datasets. Multi-level regression was used to examine the relationships between covariates and human and porcine cysticercosis. The HP10 Ag-ELISA sero-prevalence (suggestive of cysticercosis) was 6.6% for humans (95% CI 5.6%-7.7%), and 17.2% for pigs (95% CI 10.2%-26.4%). Human taeniasis prevalence, based on direct microscopic observation of Taenia spp. eggs (i.e. via microscopy results only) was 0.2% (95% CI 0.05%-0.5%). Presence of Taenia spp. antigen in both humans and pigs was significantly associated with a range of factors, including positive correlations with land cover. The presence of HP10 antigen in humans was correlated (non-linearly) with the proportion of land within a 1 km buffer that was flooding agricultural land and grassland (odds ratio [OR] = 1.09 and 0.998; p = 0.03 and 0.03 for the linear and quadratic terms respectively), gender (OR = 0.58 for males compared to females, p = 0.02), level of education (OR = 0.62 for primary level education versus no formal education, p = 0.09), use of well water for drinking (OR = 2.76 for those who use well water versus those who do not, p = 0.02) and precipitation (OR = 0.998, p = 0.02). Presence of Taenia spp. antigen in pigs was significantly correlated with gender and breeding status of the pig (OR = 10.35 for breeding sows compared to boars, p = 0.01), and the proportion of land within a 1 km buffer that was flooding agricultural land and grassland (OR = 1.04, p = 0.004). These results highlight the role of multiple socio-economic, behavioural and environmental factors in Taenia spp. transmission patterns. Environmental contamination with Taenia spp. eggs is a key issue, with landscape factors influencing presence of Taenia spp. antigens in both pigs and humans.

Neurocysticercosis: HP10 antigen detection is useful for the follow-up of the severe patients.

BACKGROUND: The most severe clinical form of neurocysticercosis (NC) occurs when cysticerci are located in the subarachnoid space at the base of the brain (SaB). The diagnosis, monitoring and treatment of NC-SaB, constitutes a severe clinical challenge. Herein we evaluate the potential of the HP10 antigen detection enzyme-linked immunosorbent assay (HP10 Ag-ELISA) in the long term follow-up of NC-SaB cases. Assay performance was compared with that of Magnetic Resonance Imaging (MRI). In addition, the robustness of the HP10 Ag-ELISA was evaluated independently at two different institutions. METHODOLOGY/PRINCIPAL FINDINGS: A double-blind prospective cohort trial was conducted involving 38 NC-SaB cases and a total of 108 paired serum and cerebrospinal fluid (CSF) samples taken at intervals of 4 to 8 months for up to 43 months. At each medical visit, results of sera and CSF HP10 Ag-ELISA and MRI obtained at last visit were compared and their accuracy was evaluated retrospectively, considering radiological evolution between appointments. In the long-term follow-up study, HP10 Ag-ELISA had a better agreement than MRI with retrospective radiological evaluation. High reproducibility of HP10 Ag-ELISA between laboratories was also demonstrated. CONCLUSIONS: Results reported in this study establish for the first time the usefulness of the comparatively low cost HP10 Ag-ELISA for long term follow-up of NC-SaB patients.

Prevalence of porcine cysticercosis and associated risk factors in Homa Bay District, Kenya.

BACKGROUND: Taenia solium is an important zoonosis in many developing countries. Cysticercosis poses a serious public health risk and leads to economic losses to the pig production industry. Due to scarcity of data on the epidemiology of porcine cysticercosis in Kenya, the present study was conducted to determine the prevalence and risk factors for porcine cysticercosis within Homa Bay district. A cross-sectional survey was carried out in 2010, and a total of 392 pigs were recruited in a household survey, with all being tested by ante-mortem lingual palpation (together with questionnaire data on pig production, occurrence and transmission of porcine cysticercosis, risk factors and awareness of porcine cysticercosis collected from the households from which pigs were sampled). Sufficient serum was collected from 232 of the pigs to be tested for the presence of circulating parasite antigen using a monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (Ag-ELISA). RESULTS: Seventy six pigs were found positive by the Ag-ELISA (32.8%, 95% C.I. 26.8-39.2%), while by tongue inspection cysticerci were detected in 22/ 392 pigs (5.6% 95% C.I. 3.6-8.4%).The most important risk factor for porcine cysticercosis in the Homa Bay area was for pigs to belong to a farm where latrine use by members of the household was not evident (OR = 1.9, 95% CI = 1.13-2.37). CONCLUSION: The present findings indicate that porcine cysticercosis is endemic in Homa Bay District, and that latrine provision, in conjunction with free-range pig keeping contributes significantly to porcine cysticercosis transmission.

Diagnostic epitope variability within Taenia solium 8 kDa antigen family: implications for cysticercosis immunodetection.

To study diagnostic epitopes within the Taenia solium 8 kDa antigen family, six overlapping synthetic peptides from an 8 kDa family member (Ts8B2) were synthesized and evaluated by ELISA and MABA with sera from patients with neurocysticercosis (NCC), from infected pigs and from rabbits immunized with recombinant Ts8B2 protein. The pre-immune rabbit sera and the Ts8B2 recombinant protein served as negative and positive controls, respectively. A similar analysis was done with the already described antigenic peptides from another member of the 8 kDa family, highly similar to Ts8B2, the CyDA antigen. Surprisingly, neither the Ts8B2 peptides nor the CyDA peptides were recognized by infected human and porcine sera. However, the entire Ts8B2 recombinant, as well as amino and carboxy-terminal halves were recognized by the positive serum samples. The observed lack of recognition of linear Ts8B2 peptides suggests that the principal serological response to the Ts8B2 family is focused on conformational epitopes in contrast to the previously observed antigenicity of the CyDA peptides. This differential antigenicity of 8 kDa family peptides could be related with parasite antigenic variability. The fact that rabbits experimentally immunized with Ts8B2 did make anti-peptide antibodies to peptides Ts8B2-6 and CyDA-6, located in the carboxy-terminal region demonstrated that the Ts8B2 peptides are not intrinsically non-immunogenic.

Evidence that active transmission of porcine cysticercosis occurs in Venezuela.

There is a paucity of quantitative data on the status of porcine cysticercosis in Venezuela, information which is essential for understanding the level of disease transmission. This study was, therefore, conducted in a typical small rural community in Yaracuy State, Venezuela, where previous cases of human Taenia solium taeniasis/cysticercosis had been reported and where the free-ranging pig management practices and the lack of rudimentary sanitary facilities indicated an obvious risk for transmission of the disease. Serum samples from 52 village pigs were screened by enzyme-linked immunosorbent assays for anti-cysticercal antibodies (Ab-ELISA), using T. solium cyst fluid as the antigen and the HP10, monoclonal antibody-based, antigen trapping ELISA for parasite antigen (HP10 Ag-ELISA). Significantly, a high proportion of the animals (65.4% for the Ab-ELISA and 42.3% for the HP10 Ag-ELISA) were sero-positive. Five of the pigs, which were selected on that basis of positive tongue palpation, were killed for autopsy, and large numbers of viable cysticerci were found in the carcases. This unequivocal documentation of porcine cysticercosis in Venezuelan pigs presents clear evidence that T. solium is actively transmitted in Venezuela. Further detailed studies and implementation of appropriate control measures are therefore indicated.

Peptide epitopes of the Taenia solium antigen Ts8B2 are immunodominant in human and porcine cysticercosis.

Ts8B2 is a gene which encodes for a member of the Taenia solium metacestode 8kDa antigen family. Since the Ts8B2-GST recombinant protein compares very favourably with other diagnostic antigens, and in order to study the antigenic nature and structure of this molecule, the Ts8B2 was expressed in prokaryotic and eukaryotic systems. The diagnostic potential of the recombinant Ts8B2 proteins was evaluated by enzyme-linked immunosorbent assays (ELISA) using a collection of serum and cerebrospinal fluid (CSF) samples from patients with clinically defined neurocysticercosis (NCC), and also sera from T. solium infected pigs. Despite the predicted glycosylation of the Ts8B2-Bac recombinant protein, there was very little difference in assay sensitivity/specificity when the Ts8B2 reagent was expressed in either prokaryotic or eukaryotic systems, suggesting that peptidic Ts8B2 epitopes are immunodominant in porcine cysticercosis and human neurocysticercosis. Conveniently, production of recombinant Ts8B2 in Escherichia coli is economical and facile, making it a feasible and practical choice as a diagnostic reagent for use in endemic areas. The Ts8B2 ELISA is particularly useful for the diagnosis of active as opposed to inactive cases of NCC and conduct of the assay is also facilitated by the fact that assay sensitivity is significantly greater when serum as opposed to CSF samples are employed.

TSOL18/HP6-Tsol, an immunogenic Taenia solium oncospheral adhesion protein and potential protective antigen.

In this study, we employed Taenia solium mRNA extracted from a tapeworm of Venezuelan origin to clone express and test the recombinant protein of the T. solium homologue of the 18-kDa oncospheral adhesion molecule of Taenia saginata (HP6-Tsag/TSA18). We first confirm the conserved nature of the sequence of the T. solium homologue (TSOL18/HP6-Tsol) and demonstrate that the recombinant protein, which, as with its T. saginata homologue, is characterised by a fibronectin type III homology region, functions as an adhesion molecule. This emphasises the possible importance of TSOL18/HP6-Tsol in tissue invasion, thus providing a rational explanation for its efficacy as a vaccine. As protection against Taenia spp., oncospheres is antibody mediated, logically, therefore, TSOL18/HP6-Tsol may also serve as a diagnostic antigen, as is indeed the case for recombinant HP6-Tsag/TSA18.

The Taenia saginata homologue of the major surface antigen of Echinococcus spp. is immunogenic and 97% identical to its Taenia solium homologue.

The TEG-Tsag gene of Taenia saginata is homologous to the genes expressing the two major surface antigens of Echinococcus spp. (EM10 and EG10). Surface antigens of parasites are logical candidates for vaccines, and in this paper we demonstrate that cattle vaccinated with the recombinant TEG-Tsag protein, either used singly or in conjunction with the recombinant HP6-Tsag protein, the major 18 kDa surface/secreted antigen of T. saginata oncospheres, produce excellent antibody responses to both these recombinant proteins. Thus TEG-Tsag may have utility as a vaccine and also as a diagnostic tool for bovine cysticercosis. In addition, as we now demonstrate a 97% homology between TEG-Tsag and its Taenia solium homologue, TEG-Tsol, this latter molecule may have similar potential in the control of human and porcine cysticercosis. The TEG molecule is characterized by an N-terminal FERM domain and a C-terminal ERM domain which are found in a number of cytoskeletal-associated proteins located at the interface between the plasma membrane and the cytoskeleton and in proteins that interact with lipid membranes. The FERM domain is also postulated to bind to adhesion proteins, in a PIP2-regulated fashion, providing a link between cytoskeletal signals and membrane dynamics. Thus TEG protein may play a role in tegument function and interaction with the host.

Evaluation of recombinant HP6-Tsag, an 18 kDa Taenia saginata oncospheral adhesion protein, for the diagnosis of cysticercosis.

With the objective of providing inexpensive and reproducible assays for the detection of antibodies indicating exposure to Taenia saginata and Taenia solium, we have evaluated the diagnostic utility of the T. saginata oncosphere adhesion protein (HP6-Tsag), expressed in baculovirus (HP6-Bac) and bacteria (HP6-GST [glutathione S-transferase]), employing enzyme-linked immunosorbent assays (ELISAs) and sera from T. saginata infected cattle, T. solium infected pigs and serum and cerebrospinal fluid (CSF) samples from clinically defined T. solium neurocysticercosis (NCC) patients. The two recombinant proteins were antigenic in all three systems, with the signal to background ratio of the HP6-Bac ELISA slightly higher than that for the HP6-GST ELISA. Assay performance in cattle was similar to previously described peptide-based ELISA assays, although NCC sample sensitivity/specificity was marginally better. The sensitivity of the HP6-Bac and HP6-GST ELISAs was close for active human NCC (77.4 and 80.6% for serum and 76.9 and 73.1% for CSF samples, respectively). In inactive human NCC, however, the sensitivity of the HP6-Bac ELISA was almost twice that of the HP6-GST ELISA. Because peptides are relatively expensive and recombinant proteins are simple and economical to produce, the latter may provide useful reagents for antibody detection in countries with endemic cysticercosis/NCC.

Taenia solium: identification and preliminary characterization of a lipid binding protein with homology to the SEC14 catalytic domain.

The objective of this work is to identify proteins of the human and porcine parasite, Taenia solium, which may be exploited for control of the parasite. Through screening a cDNA library of T. solium metacestodes, we have identified a novel Sec-14-like Taenia lipid-binding protein that may play an important role in membrane trafficking. The Sec14-like sequence is a single copy gene, encoding a putative polypeptide of 320 amino acids and 36.1 kDa (sec14Tsol protein). Secondary amino acid structural analysis suggested that the sec14Tsol protein might contain two distinct structural domains, an amino-terminal alpha-helix rich domain and a mixed alpha-helix/beta-stand carboxy-terminal zone, showing homology with the conserved SEC14 domain found in a great number of proteins that bind lipids, as the regulators of membrane trafficking between Golgi membrane bilayers. Significantly, therefore, in a phosphoinositide-binding assay, sec14Tsol purified recombinant protein specifically interacted with important lipid regulators of membrane trafficking, with a preference for PI(3)P(2), PI(3,4)P(2), PI(4,5)P(2) and phosphatidic acid. Moreover, the sec14Tsol protein was localized in the Golgi apparatus of transfected cells and in the spiral canal region of T. solium metacestode tegument. As sec14Tsol protein may play an important role in membrane trafficking, its demonstrated localisation in the intact parasite tegument suggests its involvement in the function of the tegument and thus perhaps interaction with the host.

Molecular cloning and characterisation of Ts8B1, Ts8B2 and Ts8B3, three new members of the Taenia solium metacestode 8 kDa diagnostic antigen family.

Antibody screening of a lambdaZAP-XR Taenia solium metacestode cDNA library yielded a clone (Ts8B1), with an insert of 345 bp, and an open reading frame of 258 bp, that coded for a protein with 85 amino acid residues. Alignment of the predicted amino acid sequence with sequences from SWISSPROT revealed an 88% identity with TcA5.5, a 10 kDa immunodiagnostic antigen of T. crassiceps, 75% identity with CyDA a T. solium metacestode antigen, 40-50% identity with several variants of the 8 kDa subunit of antigen B of Echinococcus spp. and with members of the T. solium metacestode 8 kDa antigen family. Two other Ts8B1 related molecules, Ts8B2 and Ts8B3, were identified in the metacestode cDNA library by PCR, coding for 85 and 66 amino acid polypeptides, respectively. Both Ts8B1 and Ts8B2 were characterized as E/S antigens through their subcellular localisation in the secretory membrane system when expressed in NRK cells. The three cDNA inserts were expressed, purified and probed in enzyme-linked immunosorbent assays (ELISA) with sera and cerebro-spinal fluid from patients with confirmed neurocysticercosis, and with sera from pigs infected with T. solium. The most promising antigen, Ts8B2, performed with a sensitivity of 96.8% and specificity of 93.1% in the detection of active NCC when using serum samples in the assay and performed similarly in the porcine system. The implications of these findings are discussed.

Molecular and functional characterization of a Taenia adhesion gene family (TAF) encoding potential protective antigens of Taenia saginata oncospheres.

Two clones from an activated Taenia saginata oncosphere cDNA library, Ts45W and Ts45S, were isolated and sequenced. Both of these genes belong to the Taenia ovis 45W gene family. The Ts45W and Ts45S cDNAs are 997- and 1,004-bp-long, each corresponding to 255 amino acids and with theoretical molecular masses of 27.8 and 27.7 kDa, respectively. Southern blot profiles obtained with Ts45W cDNA as a probe suggest that these two genes are members of a multigene family with tandem organization. The full genomic sequence was determined for the Ts45W gene and a new family member, the Ts45W/2 gene. The genomic sequences of the T. saginata Ts45W and Ts45W/2 genes were at least 2.2 kb in length with four exons separated by three introns. Exons 1 and 4 coded for hydrophobic domains, while, importantly, exons 2 and 3 coded for fibronectin homologous domains. These domains are presumably responsible for the demonstrated cell adhesion and, perhaps, the protective nature of this family of molecules and the acronym TAF (Taenia adhesion family) is proposed for this group of genes. We hypothesize that these TAF proteins and another T. saginata-protective antigen, HP6, have evolved the dual functions of facilitating tissue invasion and stimulating protective immunity to first ensure primary infection and subsequently to establish a concomitant protective immunity to protect the host from death or debilitation through superinfection by subsequent infections and thus help ensure parasite survival.

Subarachnoidal and intraventricular human neurocysticercosis: application of an antigen detection assay for the diagnosis and follow-up.

BACKGROUND: Neurocysticercosis (NC) is a parasitic disease of the central nervous system caused by the larval stage of Taenia solium. Although imaging studies are recommended for diagnosis and follow-up of patients, their high cost and restricted availability limit their use. Among various immunological tests, the detection of HP10 antigen in cerebral spinal fluid (CSF) has proved to be a useful tool for the diagnosis of NC in the case of viable but not dead parasites. OBJECTIVES: This study was designed to evaluate the usefulness of the detection of HP10 antigen for the diagnosis and follow-up of NC patients. METHODS: The effectiveness of this HP10 assay was analysed for the CSF of 46 confirmed NC cases (21 men, 25 women) who had been clinically and radiologically described. RESULTS: In 21 of 24 NC patients (87.5%) with viable parasites localized in the SA space at the base of the brain or in the ventricles these were detected by means of the HP10 assay, whilst none of the three patients with viable parasites in the parenchyma or sulci had these detected. Used for the follow-up of patients after cysticidal treatment, it was showed that levels of HP10 dropped significantly only among those patients whose cysticerci were clearly damaged. CONCLUSIONS: HP10 antigen assay is recommended as a support for diagnosis and follow-up in NC patients with viable parasites localized in the SA space at the base of the brain or in the ventricles, thereby potentially reducing the number of imaging studies required.

Taenia solium: characterization of a small heat shock protein (Tsol-sHSP35.6) and its possible relevance to the diagnosis and pathogenesis of neurocysticercosis.

A cDNA encoding for a predicted small heat shock protein (sHSP), Tsol-sfISP35.6, has been isolated by antibody screening of a Taenia solium c-DNA library. The clone was a full-length sequence (1172 bp) with an open reading frame of 945 bp and encoded for a 314 amino acid protein with deduced molecular mass of 35.6 kDa, isoelectric point of 5.6 arid the characteristic HSP20/alpha-crystallin domain duplicated. It was highly conserved, with a high sequence similarity with other platyhelminth sHSPs. Western blot analysis, using serum from neurocysticercosis patients (NCC), indicated that the purified Tsol-sHSP35.6 expression product was immunogenic, while in indirect ELISA, using the purified Tsol-sHSP35.6 expression product as antigen and serum samples from pigs and humans, 80% of T. solium infected pigs and 84% of patients with active, or 71% of patients with inactive NCC were sero-positive. The possible relevance of Tsol-sHSP35.6 in the diagnosis and pathogenesis of NCC is discussed.