Further characterization of Tsol-p27 as a diagnostic antigen in sub-Saharan Africa.

Commercial antigens used to diagnose human neurocysticercosis are obtained from either a soluble parasite extract or a parasite-derived glycoprotein fraction. The aim of the present study was to identify antigenic proteins as potential diagnostic candidates in Mozambique. Soluble proteins from Taenia solium cysticerci were separated by two-dimensional electrophoresis and blotted onto nitrocellulose membranes. Subtracted hybridization was performed with serum samples obtained from patients with neurocysticercosis (NCC) and from a NCC-negative control group. Six antigenic proteins were identified and sequenced by liquid chromatography-mass spectrometry. Among these we found Tsol-p27, which was previously identified as a diagnostic candidate in a study conducted in Nicaragua, Central America. Here, we evaluated Tsol-p27 and the antigen cC1 as potential recombinant diagnostic reagents, and also investigated the localization and partial function of Tsol-p27. Immunoblotting demonstrated that Tsol-p27 was recognized by all 10 serum samples from NCC-positive individuals, whereas cC1 was identified by only five of the 10 positive sera. None of the antigens were recognized by negative control sera. Despite the limited number of serum samples evaluated in this study, the results suggest that Tsol-p27 can be a suitable candidate for diagnosis of human NCC, not only in Central America but also in sub-Saharan Africa.

Evaluation of an immunodot blot technique for the detection of antibodies against Taenia solium larval antigens.

Immunodiagnostic tests represent an important tool for diagnosis of cysticercosis, the disease caused by cysticerci of Taenia solium. Accurate diagnosis of neurocysticercosis (NCC) requires costly neuroimaging techniques (magnetic resonance imaging and computed tomography), which are seldom affordable for people in endemic countries. Hence, new low-cost diagnostic methods offering good sensitivity and specificity are needed. Here, we studied four immunodiagnostic tests immunodot blot Tsol-p27, a commercial ELISA, and Western blot Tsol-p27/TsolHSP36, and compared them with a commercial enzyme-linked immunoelectrotransfer blot (EITB) that we regarded as the gold standard method. The analyzed serum samples were obtained from 160 patients: 94 epileptics suspected of NCC, six individuals confirmed NCC-positive, and 60 with positive (30) or negative (30) serology for Chagas diseases. Of the 100 serum samples from epileptic patients, 13 were positive and 87 negative by EITB. Compared to Western blot Tsol-p27, immunodot blot Tsol-p27 offered similar specificity (97.8% vs. 95.6%) but better sensitivity (86.7% vs. 76.4%). The ELISA was similar to the immunodot blot Tsol-p27 regarding both sensitivity and specificity. Western blot TsolHSP36 provided the lowest sensitivity (61.9%) and specificity (86.1%). None of the antibodies in the serum samples from the Chagas control groups were recognized by immunodot blot Tsol-p27. Our results indicate that the immunodot blot Tsol-p27 provides good sensitivity and specificity. Furthermore, considering the simplicity and low cost of this test, it might be preferable as a diagnostic method in poorly equipped laboratories in endemic countries.

Taenia solium: a two-dimensional Western blotting method combined with the use of an EST-library for the identification of immunogenic proteins recognized by sera from neurocysticercosis patients.

Commercial antigens used to diagnose human neurocysticercosis (NCC) are obtained from either a soluble parasite extract or a parasite-derived glycoprotein fraction. The aim of the present study was to identify antigenic proteins as potential diagnostic candidates in this context. Soluble immunogenic proteins from Taenia solium cysticerci were identified by two-dimensional electrophoresis Western blotting using human sera from Nicaragua confirmed to be positive for NCC by computer tomography. Six antigenic proteins were identified and sequenced by liquid chromatography-mass spectrometry. Among these immunogenic proteins, a novel sequence was found and named Tsol-p27. To determine the antigenicity of Tsol-p27, the previously reported antigen TsolHSP36 and the new Tsol-p27 were expressed as recombinant proteins and evaluated serologically. Immunoblotting demonstrated that Tsol-p27 was recognized by sera from 13 NCC-positive humans, whereas TsolHSP36 was identified by only two of those 13 positive sera. None of the antigens were recognized by negative control sera. Despite the limited number of serum samples evaluated in this study, the results indicate that Tsol-p27 might be a suitable candidate for diagnosis of human NCC.

Analyses of an expressed sequence tag library from Taenia solium, Cysticerca.

BACKGROUND: Neurocysticercosis is a disease caused by the oral ingestion of eggs from the human parasitic worm Taenia solium. Although drugs are available they are controversial because of the side effects and poor efficiency. An expressed sequence tag (EST) library is a method used to describe the gene expression profile and sequence of mRNA from a specific organism and stage. Such information can be used in order to find new targets for the development of drugs and to get a better understanding of the parasite biology. METHODS AND FINDINGS: Here an EST library consisting of 5760 sequences from the pig cysticerca stage has been constructed. In the library 1650 unique sequences were found and of these, 845 sequences (52%) were novel to T. solium and not identified within other EST libraries. Furthermore, 918 sequences (55%) were of unknown function. Amongst the 25 most frequently expressed sequences 6 had no relevant similarity to other sequences found in the Genbank NR DNA database. A prediction of putative signal peptides was also performed and 4 among the 25 were found to be predicted with a signal peptide. Proposed vaccine and diagnostic targets T24, Tsol18/HP6 and Tso31d could also be identified among the 25 most frequently expressed. CONCLUSIONS: An EST library has been produced from pig cysticerca and analyzed. More than half of the different ESTs sequenced contained a sequence with no suggested function and 845 novel EST sequences have been identified. The library increases the knowledge about what genes are expressed and to what level. It can also be used to study different areas of research such as drug and diagnostic development together with parasite fitness via e.g. immune modulation.

Trypanosoma rangeli genotypes association with Rhodnius prolixus and R. pallescens allopatric distribution in Central America.

Previous kDNA polymorphism-based reports have revealed the existence of two Trypanosoma rangeli genotypes (KP1+ and KP1-): SL and SSU rRNA gene polymorphism-based studies have revealed that five genotypes (A-E) are distributed throughout different Latin-American countries. Some evidence has shown that the genotypes' biogeographical distribution is associated with sympatric Rhodnius species. 12 T. rangeli isolates from humans and reservoirs from El Salvador, Guatemala, Honduras, Costa Rica and Panama were characterised by kDNA and mini-exon gene intergene spacer analysis and compared to 12 previously characterised isolates from humans and vectors from Colombia, Guatemala, Honduras and Venezuela. Central American isolates corresponded to genotypes called KP1(+) or lineage A and KP1(-) or lineage C. Such dimorphism was corroborated by randomly amplified polymorphic DNA (RAPD) in 22 selected isolates; a dendrogram was thus produced having two defined branches. One branch grouped KP1(-) or lineage C strains isolated from Rhodnius colombiensis (Colombia), humans (Panama), Procyon lotor and Choloepus hoffmanni (Costa Rica). The other group was formed by KP1(+) or lineage A strains isolated from Rhodnius prolixus (Colombia, Venezuela) and humans (El Salvador, Guatemala, Honduras). These results present evidence that both groups infect different mammals (humans, domestic and silvatic animals) having no association with any particular vertebrate species; however, T. rangeli KP1(+) or (A) strains have been isolated in Central America in areas where R. prolixus circulate (Honduras, El Salvador and Guatemala) and KP1(-) or (C) strains have been isolated in areas where Rhodnius pallescens is the main vector (Panama and Costa Rica) indicating a parasite-vector association. The same lineages circulate in Andean countries (Colombia, Venezuela, Ecuador and Peru), KP1+ being associated with members of the prolixus group (R. prolixus and Rhodnius robustus) and KP1- with members of the pallescens group (R. pallescens, R. colombiensis and Rhodnius ecuadoriensis).