Armando Gonzales

Screening for cystic echinococcosis in an endemic region of Peru using portable ultrasonography and the enzyme-linked immunoelectrotransfer blot (EITB) assay

Cystic echinococcosis (CE) caused by the larval form of Echinococcus granulosus is a major public health problem in sheep-raising regions of the World. This study compared portable ultrasound with the enzyme-linked immunoelectrotransfer blot (EITB) assay as screening methods to estimate the prevalence of human CE in a remote village in the Peruvian Andes. Three hundred eighty-nine villagers were examined by portable ultrasound and blood samples were drawn by venipuncture. Sera were collected and tested for antibodies against CE using an EITB assay. Cystic lesions were classified based on their ultrasound morphologic characteristics. The prevalence of human CE using portable ultrasound and the EITB assay were 4.9% and 2.6%, respectively. Fifty-three percent of subjects with CE were EITB positive. Portable ultrasound was well received by the community, augmented CE detection and allowed a faster estimate of human infection than the EITB assay.

DEVELOPMENT OF AN ENZYME-LINKED IMMUNOELECTROTRANSFER BLOT (EITB) ASSAY USING TWO BACULOVIRUS EXPRESSED RECOMBINANT ANTIGENS FOR DIAGNOSIS OF TAENIA SOLIUM TAENIASIS

Taeniasis diagnosis is an important step in the control and elimination of both cysticercosis and taeniasis. We report the development of 2 serological taeniasis diagnostic tests using recombinant antigens rES33 and rES38 expressed by baculovirus in insect cells in an EITB format. In laboratory testing with defined sera from nonendemic areas, rES33 has a sensitivity of 98% (n = 167) and a specificity of 99% (n = 310) (J index: 0.97); rES38 has a sensitivity of 99% (n = 146) and a specificity of 97% (n = 275) (J index: 0.96). Independent field testing in Peru showed 97% (n = 203) of the taeniasis sera were positive with rES33, and 100% of the nontaeniasis sera (n = 272) were negative with rES33; 98% (n = 198) of taeniasis sera were positive with rES38, and 91% (n = 274) of the nontaeniasis sera were negative with rES38. Among the Peruvian sera tested, 17 of 26 Peruvian Taenia saginata sera were false positive with rES38 test. Both tests were also examined with cysticercosis sera, with a positive rate ranging from 21% to 46%. rES33 and rES38 tests offer sensitive and specific diagnosis of taeniasis and easy sample collection through finger sticks that can be used in large-scale studies. They are currently being used in cysticercosis elimination programs in Peru.

Taenia solium oncosphere antigens induce immunity in pigs against experimental cysticercosis

Immunity to Taenia solium infection was investigated using an experimental intramuscular oncosphere infection assay (IMOA) model in pigs. Three naturally infected pigs with cysticercosis were treated with oxfendazole (OFZ), a drug demonstrated to kill cysts in porcine muscle. These animals were then challenged with oncospheres but did not develop any cysts while three uninfected pigs that were similarly challenged, did develop intramuscular cysts. In another study, two groups of three pigs each were immunized with crude T. solium oncosphere and metacestode antigens, respectively, and tested with the IMOA. Immunization with crude oncosphere antigens (OAs) induced 100% protection, while metacestode antigens provided only partial protection. Immunoblots showed that pigs with complete immune protection to oncosphere intramuscular challenge had antibodies to two OAs at 31.3 and 22.5 kDa, respectively. Antibody to these two antigens was absent in pigs immunized with metacestodes or in uninfected control pigs. This study demonstrated the presence of two antigens that are unique to the oncosphere. Although, antibody to these two antigens is consistently present in pigs that are protected from an oncosphere intramuscular challenge their role in preventing infection by T. solium larval cysts is still hypothetical.

Neurocysticercosis: A natural human model of epileptogenesis

To develop a better understanding of mechanisms of seizures and long‐term epileptogenesis using neurocysticercosis.

Taenia solium infection in a rural community in the Peruvian Andes.

Abstract An epidemiological study was conducted in a highland, rural community in Peru, to determine the seroprevalences of human and porcine infection with Taenia solium and the risk factors associated with human infection. The seroprevalences, determined using an assay based on enzyme-linked-immuno-electrotransfer blots (EITB), were 21% (66/316) in the humans and 65% (32/49) in the pigs. The human subjects aged <30 years were more likely to be positive for anti-T. solium antibodies than the older subjects (P < 0.001). The risk factors associated with human seropositivity were lack of education beyond the elementary level [odds ratio (OR)=2.69; 95% confidence interval (CI)=1.09-6.65] and pig-raising (OR=1.68; CI=0.96-2.92). Curiously, sheep-raising was inversely associated with human T. solium infection (OR=0.50; CI=0.28-0.90). The study site appears to be a new endemic focus for T. solium in the central Peruvian Andes. Although, in earlier studies, the seroprevalence of T. solium infection has generally been found to increase with age, the opposite trend was observed in the present study. The results of follow-up studies should help determine if the relatively high seroprevalence in the young subjects of the present study is the result of a transient antibody response.

Immunoinformatics prediction of linear epitopes from Taenia solium TSOL18

Cysticercosis is a public health problem in several developing countries. The oncosphere protein TSOL18 is the most immunogenic and protective antigen ever reported against porcine cysticercosis, although no specific epitope has been identified to account for these properties. Recent evidence suggests that protection might be associated with conformational epitopes. Linear epitopes from TSOL18 were computationally predicted and evaluated for immunogenicity and protection against porcine cysticercosis. A synthetic peptide was designed based on predicted linear B cell and T cell epitopes that are exposed on the surface of the theoretically modeled structure of TSOL18. Three surface epitopes from TSOL18 were predicted as immunogenic. A peptide comprising a linear arrangement of these epitopes was chemically synthesized. The capacity of the synthetic peptide to protect pigs against an oral challenge with Taenia solium proglottids was tested in a vaccine trial. The synthetic peptide was able to produce IgG antibodies in pigs and was associated to a reduction of the number of cysts, although was not able to provide complete protection, defined as the complete absence of cysts in necropsy. This study demonstrated that B cell and T cell predicted epitopes from TSOL18 were not able to completely protect pigs against an oral challenge with Taenia solium proglottids. Therefore, other linear epitopes or eventually conformational epitopes may be responsible for the protection conferred by TSOL18.

In response: Multifactorial basis of epilepsy in patients with neurocysticercosis

Dear Editors, Carpio and Romo have emphasized the limitations in our understanding of symptomatic seizures and the epileptogenic process that follows in some, but not all patients with neurocysticercosis. Their letter also highlights a critical need for careful, systematic studies of clinical and molecular biomarkers in these patients. Fundamental knowledge gained from these patients could be extrapolated to many other causes of epilepsy. We are glad that in these aspects the authors agree with us on the potential usefulness of neurocysticercosis to unravel the pathophysiology of seizures and epilepsy in neurocysticercosis as well as in other seizure disorders. Our report is not a general review of neurocysticercosis and accordingly does not discuss other issues raised by the Carpio and Romo. The disease is relatively unstudied and many points controversial, so there is room for differences of opinion. For a number of reasons, this is not the forum to discuss these differences. However, our group of epilepsy and neurocysticercosis experts believe most evidence suggests that a small proportion, rather than a “few people with NC” develop epilepsy and that the evidence implicating neurocysticercois as a cause of epilepsy, we believe, is quite strong. Supporting evidence originates from number of kinds of information including numerous epidemiology reports, follow up of treated patients, and studies implicating specific calcifications as seizure foci in patients with epilepsy. In the later specific calcified lesions in patients with epilepsy have been implicated by their semiology and EEG findings implicating specific calcifications in about 50%-60% of patients studied. Similarly, whether repeated seizures over an extended period of time arising from an inflammatory focus is epilepsy or is a series of acute symptomatic seizures is important from a pathophysiology point of view, but applying outdated definitions of epilepsy to recurrent seizures in neurocysticercosis only begs for an improved classification scheme.

A novel enolase from Taenia solium metacestodes and its evaluation as an immunodiagnostic antigen for porcine cysticercosis

Cysticercosis is a worldwide parasitic disease of humans and pigs principally caused by infection with the larvae of the pork tapeworm Taenia solium. Through the use of the recently-made-available T. solium genome, we identified a gene within a novel 1448 bp ORF that theoretically encodes for a 433 amino acid-long protein and predicted to be an α-enolase closely related to enolases of other flatworms. Additional bioinformatic analyses revealed a putative plasminogen-binding region on this protein, suggesting a potential role for this protein in pathogenesis. On this basis, we isolated the mRNA encoding for this presumptive enolase from T. solium metacestodes and reverse-transcribed it into cDNA before subsequently cloning and expressing it in both E. coli (rEnoTs) and insect cells (rEnoTsBac), in a 6xHis tagged manner. The molecular weights of these two recombinant proteins were ∼48 and ∼50 kDa, respectively, with the differences likely attributable to differential glycosylation. We used spectrophotometric assays to confirm the enolase nature of rEnoTs as well as to measure its enzymatic activity. The resulting estimates of specific activity (60.000 U/mg) and Km (0.091 mM) are quite similar to the catalytic characteristics of enolases of other flatworms. rEnoTs also exhibited high immunogenicity, eliciting a strong polyclonal antibody response in immunized rabbits. We subsequently employed rEnoTsBac for use in an ELISA aimed at discriminating between healthy pigs and those infected with T. solium. This diagnostic assay exhibited a sensitivity of 88.4% (95% CI, 74.92%-96.11%) and a specificity of 83.7% (95% CI: 69.29%-93.19%). In conclusión, this study reports on and enzymatically characterizes a novel enolase from T. solium metacestode, and shows a potential use as an immunodiagnostic for porcine cysticercosis.