Extraparenchymal human neurocysticercosis induces autoantibodies against brain tubulin and MOG35-55 in cerebral spinal fluid.

Neurocysticercosis (NC) presents two broad clinical entities: extraparenchymal (EP-NC) and parenchymal (P-NC). Using ELISA methodology, we demonstrate autoantibodies to tubulin and the Major oligodendrocyte glycoprotein (MOG) in the CSF of most, but not all, EP-NC samples. Levels of these autoantibodies were considerably reduced or absent in the P-NC samples. There was a striking correlation between levels of anti-tubulin and anti-MOG, and the significant correlation between the levels of autoantibodies and cellularity in the CSF, suggests that stimulation of the autoantibody response may be a function of cerebral inflammation. A hypothetical model to describe the pathogenesis of EP-NC is presented.

Diagnostic value of glycoprotein band patterns of three serologic enzyme-linked immunoelectrotransfer blot assays for neurocysticercosis.

The enzyme-linked immunoelectrotransfer blot (EITB) assay to detect antibodies in serum is a complementary tool for the diagnosis of neurocysticercosis (NCC). Presence of at least one glycoprotein band corresponding to a Taenia solium (T. solium) antigen indicates a positive result; however, EITB assays have multiple glycoprotein bands, and previous work has suggested that band patterns may have additional diagnostic value. We included 58 participants with a definitive diagnosis of NCC who received care at the Instituto Nacional de Neurología y Neurocirugía in Mexico City. Three different EITB tests were applied to participants' serum samples (LDBio, France; US Centers for Disease Control and Prevention [CDC]; and Instituto de Diagnóstico y Referencia Epidemiológicos [InDRE]). There was substantial variability in specific glycoprotein band patterns among the three assays. However, in age- and sex-adjusted logistic regression models, the number of glycoprotein bands was positively associated with the presence of vesicular extraparenchymal cysts (InDRE adjusted odds ratio [aOR] 1.60 p < 0.001; CDC aOR 6.31 p < 0.001; LDBio aOR 2.45 p < 0.001) and negatively associated with the presence of calcified parenchymal cysts (InDRE aOR 0.63 p < 0.001; CDC aOR 0.25 p < 0.001; LDBio aOR 0.44 p < 0.001). In a sensitivity analysis also adjusting for cyst count, results were similar. In all three EITB serum antibody tests, the number of glycoprotein bands consistently predicted cyst stage and location, although magnitude of effect differed.

Anti-brain protein autoantibodies are detectable in extraparenchymal but not parenchymal neurocysticercosis.

Neurocysticercosis (NC) presents a spectrum of clinical manifestations, with two broad clinical entities based on the central nervous system location of the parasite: extraparenchymal (EP-NC) and parenchymal (P-NC). In this work, using quantitative immunoblot methodology, we demonstrate the presence of autoantibodies to brain proteins in CSF from EP-NC, but not P-NC, patients. There was striking correlation between the level of autoantibodies and the levels of the secreted metacestode glycoprotein HP-10, suggesting that the level of stimulation of the autoantibody response may be a function of the number of viable parasites. Nine corresponding proteins autoantigens were provisionally identified by mass spectroscopy.

Diagnosis of Taeniosis in rural Venezuelan communities: Preliminary characterization of a Taenia solium specific monoclonal (VP-1) Coproantigen ELISA.

The objective of this study was to identify and treat carriers of adult Taenia solium present in two rural Venezuelan communities through examination of faecal samples by coproscopical analysis, and by the application of a polyclonal and a monoclonal (VP-1) coproantigen ELISA. Both the polyclonal and monoclonal ELISA's were negative when tested with soluble extracts of adults of Ascaris lumbricoides, Hymenolepis nana and Trichuris trichura. The polyclonal ELISA was positive for soluble extracts adults of T. solium and T. saginata, whereas the monoclonal ELISA, which recognizes a glycoprotein, was restricted to T. solium, and was also negative with faecal samples from five cases of T. saginata adult infections. In the first community studied, Potrero Largo (Total population: 300), of 248 faecal samples examined, 2 individuals were positive for Taenia spp eggs by coproscopical analysis and the VP-1 ELISA, and yielded T. solium adults upon purging. In contrast, when the polyclonal coproAg ELISA was applied to the same 248 faecal samples, there were a considerable number of positives. Indeed, seven patients highly positive in the polyclonal ELISA did not yield a Taenia spp upon purging and were negative in the VP-1 ELISA. In the second community studied La Yuca (Total population 560), none of the 333 individuals who donated faeces was positive for Taenia spp eggs. Many, however, were infected with a range of intestinal helminth and protozoan parasites. A total of 76 faecal samples with representative intestinal parasite were then tested in the polyclonal and VP-1 assays. Of these, many gave an unacceptable number of significant optical densities in the polyclonal coproAg ELISA. In contrast, all were negative in the VP-1 ELISA, thus providing evidence for the species specificity of the VP-1 ELISA in faecal samples. These results with the VP-1 coproAg ELISA, although preliminary, justify further validation through the testing of more faecal samples from T. solium and T. saginata adult infected individuals.

Immunodiagnosis of human neurocysticercosis: comparative performance of serum diagnostic tests in Mexico.

Immunodiagnosis has a supportive role in the diagnosis of neurocysticercosis (NCC). The aim of this study was to compare the validity of seven immunodiagnostic tests among serum samples from 58 patients with NCC, 26 patients with neurological diseases other than NCC, and 15 healthy controls. One test for viable parasite detection (HP10 antigen assay) and six for antibody detection were evaluated. For the entire sample, sensitivities ranged from 55.2% (NOVALISA) to 81.0% (enzyme-linked immunosorbent assay [ELISA] Taenia solium antibody), with the sensitivity of the latter test significantly higher than that of the in-house ELISA Taenia crassiceps, NOVALISA, enzyme-linked immunoelectrotransfer blot (EITB) CDC, and HP10. Overall, specificities were high, ranging from 85.4% (ELISA Ts) to 97.1% (NOVALISA), with no statistically significant differences. Detection of HP10 antigen was significantly associated with the presence of vesicular parasites. The simple and low-cost ELISA Taenia solium antibody Ab instead of EITB is recommended to support NCC diagnosis in both rural and hospital settings in Mexico.

Short communication: Four cases of Taenia saginata taeniasis in urban Venezuelan communities.

We report four cases of Taenia saginata taeniasis in different urban communities of Aragua state, Venezuela. After subsequent treatment with praziquantel and a saline purge, adult tapeworms were collected from all four patients and demonstrated to be T. saginata by morphological and molecular characterization. The finding of T. saginata in four distinct and separate urban municipalities of the Aragua state indicates the pertinence of rigorous meat inspection, and the importance of establishing parasite prevalence in human and bovine Venezuelan populations.

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.

Comparison of complementary diagnostic tests in cerebrospinal fluid and serum for neurocysticercosis.

The role of immunologic tests in the diagnosis of neurocysticercosis (NC) is controversial and few studies have made comparisons among them. The objective of this study was to compare immunological tests in both serum and cerebrospinal fluid (CSF) for the diagnosis of NC. We conducted a case-control study in Cuenca, Ecuador, enrolling patients with NC (N = 24) and matching them with other neurosurgical patients (N = 18). To detect cysticercal antigen, we used an HP10 antigen assay in serum and CSF ("HP10 Ag -serum -CSF") and a commercial antigen assay in serum (apDia, "ELISA-Ag-serum"), and to detect cysticercal DNA, we used a polymerase chain reaction (PCR) assay in CSF ("PCR-CSF"). Assay sensitivities were: HP10 Ag-serum (41.7%, 95% confidence interval [CI] 22.1-63.4), HP10 Ag-CSF (87.5%, 95% CI: 67.6-97.3), ELISA-Ag-serum (62.5%, 95% CI: 40.6-81.2), and PCR-CSF (79.2%, 95% CI: 57.9-92.9). Sensitivities were higher when limiting to participants with extraparenchymal NC. Specificity was 100% for all assays except ELISA-Ag-serum (72.2%). This preliminary study demonstrated the potential usefulness of the PCR and HP10 Ag assay in CSF, especially for extraparenchymal NC; thus, they could be considered as complementary diagnostic tools when neuroimaging is not conclusive.

Reciprocal contribution of clinical studies and the HP10 antigen ELISA for the diagnosis of extraparenchymal neurocysticercosis.

To evaluate diagnosis of active neurocysticercosis, paired cerebral spinal fluid (CSF) and serum samples from 24 neurocysticercosis (NCC) patients and 17 control neurological patients were assayed in the HP10 Taenia antigen (Ag) ELISA. The CSF samples were also tested with an HP10 Lateral Flow Assay (LFA). The HP10 Ag was detected by ELISA in the CSF of 5/5 patients with Definitive extraparenchymal NCC, and in 4/5 of the corresponding sera. In the Definitive parenchymal group, on the other hand, the HP10 Ag was absent in 2/3 CSF (with a very low value in the one positive sample) and all the corresponding serum samples. Samples of CSF from 4/7 patients in the Probable parenchymal group, were also significantly HP10 Ag positive, suggesting the presence of extraparenchymal cysts not identified by the imaging studies. With the possible exception of one patient, the corresponding serum samples of the Probable parenchymal NCC group, were all HP10 Ag negative. Samples of CSF from 9 NCC patients diagnosed with Mixed parenchymal and extraparenchymal NCC were all significantly HP10 Ag positive, confirming the presence of extraparenchymal cysts, with only 7/9 of the corresponding serum samples being HP10 positive. Thus detection of the HP10 Ag indicates extraparenchymal and not parenchymal cyst localization and is more sensitive with CSF than serum. Three neurological patients clinically diagnosed as subarachnoid cyst, hydrocephalus and tuberculoma, respectively, were clearly positive for HP10 Ag. Of these, two were confirmed as NCC by subsequent imaging; the third died prior to further examination. Thus, a total of 8 patients had their clinical diagnosis questioned. Finally, there was good agreement between the HP10 Ag ELISA and LFA with CSF samples giving an optical density ≥0.4 in the ELISA assay. In conclusion, the HP10 Ag assay should provide a valuable and reciprocal tool in the clinical diagnosis and follow up of extraparenchymal NCC.

The HP10 Taenia monoclonal antibody-based ELISA detects a similar protein in the vesicular fluid of Taenia hydatigena.

Diagnosis of Taenia solium cysticercosis in endemic rural communities depends on serological tests, as typically there is no access to imaging facilities. The HP10 antigen ELISA (HP10 Ag ELISA), which detects a high molecular weight secreted protein of viable metacestodes, has been employed for the diagnosis of both human and porcine cysticercosis in such communities. In this communication, we formally demonstrate that the HP10 Ag ELISA, already known to function for the detection of T. saginata and T. solium cysticercosis, also detects a similar high molecular weight antigen of T. hydatigena. Thus, the HP10 Ag assay, while specific for human cysticercosis, may not be recommended for the diagnosis of porcine cysticercosis where there is co-infection of pigs with T. solium and T. hydatigena.

Approaches and Perspectives for Development of African Swine Fever Virus Vaccines.

African swine fever (ASF) is a complex disease of swine, caused by a large DNA virus belonging to the family Asfarviridae. The disease shows variable clinical signs, with high case fatality rates, up to 100%, in the acute forms. ASF is currently present in Africa and Europe where it circulates in different scenarios causing a high socio-economic impact. In most affected regions, control has not been effective in part due to lack of a vaccine. The availability of an effective and safe ASFV vaccines would support and enforce control-eradication strategies. Therefore, work leading to the rational development of protective ASF vaccines is a high priority. Several factors have hindered vaccine development, including the complexity of the ASF virus particle and the large number of proteins encoded by its genome. Many of these virus proteins inhibit the host's immune system thus facilitating virus replication and persistence. We review previous work aimed at understanding ASFV-host interactions, including mechanisms of protective immunity, and approaches for vaccine development. These include live attenuated vaccines, and "subunit" vaccines, based on DNA, proteins, or virus vectors. In the shorter to medium term, live attenuated vaccines are the most promising and best positioned candidates. Gaps and future research directions are evaluated.

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.

Restriction of HIV-1 replication in primary macrophages by IL-12 and IL-18 through the upregulation of SAMHD1.

Monocyte-derived macrophages (MDM) can polarize into different subsets depending on the environment and the activation signal to which they are submitted. Differentiation into macrophages allows HIV-1 strains to infect cells of the monocytic lineage. In this study, we show that culture of monocytes with a combination of IL-12 and IL-18 led to macrophage differentiation that was resistant to HIV-1 infection. In contrast, M-CSF-derived MDM were readily infected by HIV-1. When monocytes were differentiated in the presence of M-CSF and then further treated with IL-12/IL-18, cells became resistant to infection. The restriction on HIV-1 replication was not dependent on virus entry or coreceptor expression, as vesicular stomatitis virus-pseudotyped HIV-1 replication was also blocked by IL-12/IL-18. The HIV-1 restriction factor sterile α motif and HD domain-containing protein-1 (SAMHD1) was significantly overexpressed in IL-12/IL-18 MDM compared with M-CSF MDM, and degradation of SAMHD1 by RNA interference or viral-like particles carrying the lentiviral protein Vpx restored HIV-1 infectivity of IL-12/IL-18 MDM. SAMHD1 overexpression induced by IL-12/IL-18 was not dependent on IFN-γ. Thus, we conclude that IL-12 and IL-18 may contribute to the response against HIV-1 infection through the induction of restriction factors such as SAMHD1.

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.

Crystal structure of a poxvirus-like zalpha domain from cyprinid herpesvirus 3.

Zalpha domains are a subfamily of the winged helix-turn-helix domains sharing the unique ability to recognize CpG repeats in the left-handed Z-DNA conformation. In vertebrates, domains of this family are found exclusively in proteins that detect foreign nucleic acids and activate components of the antiviral interferon response. Moreover, poxviruses encode the Zalpha domain-containing protein E3L, a well-studied and potent inhibitor of interferon response. Here we describe a herpesvirus Zalpha-domain-containing protein (ORF112) from cyprinid herpesvirus 3. We demonstrate that ORF112 also binds CpG repeats in the left-handed conformation, and moreover, its structure at 1.75 Å reveals the Zalpha fold found in ADAR1, DAI, PKZ, and E3L. Unlike other Zalpha domains, however, ORF112 forms a dimer through a unique domain-swapping mechanism. Thus, ORF112 may be considered a new member of the Z-domain family having DNA binding properties similar to those of the poxvirus E3L inhibitor of interferon response.

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.

Modeling of the Toll-like receptor 3 and a putative Toll-like receptor 3 antagonist encoded by the African swine fever virus.

African swine fever virus (ASFV) is a large double-stranded DNA virus responsible for a lethal pig disease, to which no vaccine has ever been obtained. Its genome encodes a number of proteins involved in virus survival and transmission in its hosts, in particular proteins that inhibit signaling pathways in infected macrophages and, thus, interfere with the host's innate immune response. A recently identified novel ASFV viral protein (pI329L) was found to inhibit the Toll-like receptor 3 (TLR3) signaling pathway, TLR3 being a crucial "danger detector." pI329L has been predicted to be a transmembrane protein containing extracellular putative leucine-rich repeats similar to TLR3, suggesting that pI329L might act as a TLR3 decoy. To explore this idea, we used comparative modeling and other structure prediction protocols to propose (a) a model for the TLR3-Toll-interleukin-1 receptor homodimer and (b) a structural fold for pI329L, detailed at atomistic level for its cytoplasmic domain. As this later domain shares only remote sequence relationships with the available TLR3 templates, a more complex modeling strategy was employed that combines the iterative implementation of (multi)threading/assembly/refinement (I-TASSER) structural prediction with expertise-guided posterior refinement. The final pI329L model presents a plausible fold, good structural quality, is consistent with the available experimental data, and it corroborates our hypothesis of pI329L being a TLR3 antagonist.

Genetic variability of the 18 kDa/HP6 protective antigen in Taenia saginata and Taenia asiatica: implications for vaccine development.

Genomic characterization of the genes encoding the Taenia 18 kDa/HP6 protective antigens was carried out for Taenia saginata and T. asiatica using 42 taeniid isolates comprising 23 samples of T. saginata, 13 samples of T. asiatica and 6 samples of T. solium. The corresponding sequences from all taeniid isolates were PCR-amplified with specific primers and then sequenced. All the genes, and other described taeniid gene homologues, had the same genomic structure. Surprisingly, the T. saginata TSA18 gene showed nucleotide variability within the 23 samples analyzed. This resulted in two distinct genotypes with 96% DNA sequence similarity and deduced amino acid sequences with 21 substitutions, mainly located in the second exon which contains the fibronectin type III domain. In regards to T. asiatica, the 18 kDa gene (TASI18) was very similar to the T. saginata antigen homologues, both at the DNA and deduced amino acid sequence levels, and the TSOL18 gene was conserved among T. solium isolates as previously described. The implications of these findings on the future development of taeniid vaccines are discussed.

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.