Combined human and porcine mass chemotherapy for the control of T. solium.

A combined (human and porcine) mass chemotherapy program was tested in a controlled design in 12 village hamlets in the Peruvian highlands. A single dose of 5 mg of praziquantel was given to eliminate intestinal taeniasis in humans, and two rounds of oxfendazole (30 mg/kg) were administered to all pigs. The total population in the study villages was 5,658 resident individuals, and the porcine population at the beginning of the study was 716 pigs. Human treatment coverage was 75%, ranging from 69% to 80%. There were only a few refusals of owners for porcine treatment of their animals. The effect of the intervention was measured by comparing incidence rates (seroconversion in pigs who were seronegative 4 months before) in treatment versus control villages, before and up to 18 months after treatment. There was a clear effect in decreasing prevalence (odds ratio, 0.51; P < 0.001) and incidence (odds ratio, 0.39; P < 0.013) in the treatment area after the intervention, which did not leave to extinction of the parasite but stabilized in slightly decreased rates persisting along the follow-up period. Mass chemotherapy was effective in decreasing infection pressure in this hyperendemic area. However, the magnitude of the effect was small and did not attain the goal of eliminating transmission.

Epilepsy, cysticercosis, and toxocariasis: a population-based case-control study in rural Bolivia.

OBJECTIVE: To assess the relationship between epilepsy and infection with Taenia solium and Toxocara canis with a case-control study, in the rural area of the Cordillera Province, Bolivia. METHODS: A preliminary two-phase door-to-door prevalence survey determined the prevalence of epilepsy and identified cases and control subjects. At least two control subjects per case were selected, matching on sex, age, and community of residence. Cases and control subjects were assessed serologically for antibodies against T. canis by ELISA and against T. solium by enzyme-linked immunoelectrotransfer blot (EITB). RESULTS: The prevalence survey found 130 confirmed cases of epilepsy, of which 113 were eligible for the case-control study (59 partial seizures and 54 generalized seizures). Two hundred thirty-three control subjects were selected. Multivariable analysis for a matched case-control study was carried out. There was an association between EITB positivity for T. solium and epilepsy with an OR of 1.85 (95% CI 0.99 to 3.4) for all cases. A stronger association was found in those with partial epilepsy with a late onset of disease (15 years and older), where the OR was 3.66 (95% CI 1.10 to 12.10). A positive association was also found with T. canis for all cases with an OR of 2.70 (95% CI 1.41 to 5.19). This increased for those with late-onset partial epilepsy to an OR of 18.22 (95% CI 2.10 to 158.10). CONCLUSION: This finding suggests that both neurocysticercosis and toxocariasis may in part explain the higher prevalence of epilepsy, particularly partial epilepsy, in developing countries.

Proposed diagnostic criteria for neurocysticercosis.

Neurocysticercosis is the most common helminthic infection of the CNS but its diagnosis remains difficult. Clinical manifestations are nonspecific, most neuroimaging findings are not pathognomonic, and some serologic tests have low sensitivity and specificity. The authors provide diagnostic criteria for neurocysticercosis based on objective clinical, imaging, immunologic, and epidemiologic data. These include four categories of criteria stratified on the basis of their diagnostic strength, including the following: 1) absolute--histologic demonstration of the parasite from biopsy of a brain or spinal cord lesion, cystic lesions showing the scolex on CT or MRI, and direct visualization of subretinal parasites by funduscopic examination; 2) major--lesions highly suggestive of neurocysticercosis on neuroimaging studies, positive serum enzyme-linked immunoelectrotransfer blot for the detection of anticysticercal antibodies, resolution of intracranial cystic lesions after therapy with albendazole or praziquantel, and spontaneous resolution of small single enhancing lesions; 3) minor--lesions compatible with neurocysticercosis on neuroimaging studies, clinical manifestations suggestive of neurocysticercosis, positive CSF enzyme-linked immunosorbent assay for detection of anticysticercal antibodies or cysticercal antigens, and cysticercosis outside the CNS; and 4) epidemiologic--evidence of a household contact with Taenia solium infection, individuals coming from or living in an area where cysticercosis is endemic, and history of frequent travel to disease-endemic areas. Interpretation of these criteria permits two degrees of diagnostic certainty: 1) definitive diagnosis, in patients who have one absolute criterion or in those who have two major plus one minor and one epidemiologic criterion; and 2) probable diagnosis, in patients who have one major plus two minor criteria, in those who have one major plus one minor and one epidemiologic criterion, and in those who have three minor plus one epidemiologic criterion.

Optimum dissociating condition for immunoaffinity and preferential isolation of antibodies with high specific activity.

Using an immunoaffinity model consisting of a high performance matrix (Affi-prep-10) with normal human IgG as ligand, and hyperimmune goat anti-human IgG (heavy and light chain active) antibodies, we compared the efficacies of 13 elution reagents. Efficacy was considered in terms of specific activity and total quantitative recovery of the eluted antibody. The optimum, general-purpose dissociation reagent for this immunoaffinity system is 3.0 M MgCl2.6H2O, 0.075 M Hepes/NaOH, with 25% ethylene glycol pH 7.20. The antibodies recovered from diluted (1/2) goat serum with this dissociation reagent have a SpAct of 1.87 times and a total recovery of 6.33 times that of a comparable experiment using the usual eluant of 1.0 M glycine/HCl, pH 2.00. We also demonstrated that the SpAct of antibodies recovered from immunoaffinity procedures performed under antibody excess and antigen limiting conditions is 2.36-8.00 times higher than that produced by antigen excess and antibody limiting configurations.

Development and optimization of the FAST-ELISA for detecting antibodies to Schistosoma mansoni.

The Falcon assay screening test (F.A.S.T.) system was used to develop a rapid, sensitive, and quantitative kinetic-based enzyme-linked immunosorbent assay (k-ELISA) for detecting antibodies against Schistosoma mansoni adult microsomal antigens (MAMAs). The FAST-ELISA uses polystyrene beads on sticks molded to the lid of a microtitration plate. The beads are coated with antigen. Reagents and sera are placed in microtitration plates and the beads exposed to reagents by immersion. The exposure time required for a single dilution of serum or other antibody source, conjugate, and substrate is 5 min each. Excluding preparation time, two plates can easily be assayed in 30 min. The optima for assay conditions, reproducibility, quantitative linearity, and sensitivity are delineated. A battery of sera from patients with both homologous and heterologous infections was tested, and a dilution series of a standard reference serum pool was included with each test. Results were expressed in number of units as calibrated against the standard reference sera pool. Antigen-coated bead storage studies were performed with untreated and three chemically treated antigens. The storage stability of MAMA, ability to perform the assay with minimal equipment, sensitivity, short assay time, and ease of operation make the FAST-ELISA ideal for field studies.

Quantitative capacities of glutaraldehyde and sodium m-periodate coupled peroxidase-anti-human IgG conjugates in enzyme-linked immunoassays.

Covalently linked peroxidase-anti-human IgG conjugates were prepared by either glutaraldehyde or NaIO4 coupling techniques. Sodium dodecyl sulfate polyacrylamide gel electrophoresis shows that the glutaraldehyde coupled conjugate is composed of generally lower molecular weight components than the NaIO4 coupled product. The NaIO4 conjugate, when used to quantitate human immunoglobulin (Ig) in enzyme-linked immunoassays, appears to be highly sensitive in that small amounts of Ig elicited relatively high reactivities. The quantitative range of this type of conjugates, where reactivities are linearly proportional to the amount of human Ig present, is, however, extremely narrow (0.01-0.10 micrograms/ml of human IgG). Conversely, the glutaraldehyde coupled type conjugate is capable of sustaining a much wider range of linearity (0.01-0.6 micrograms/ml), but with a more gradual rise of reactivity which corresponds well to the amount of human Ig present. Conjugates prepared with glutaraldehyde are thus more useful in quantitative assays where wide quantitative ranges are desirable. NaIO4 conjugates on the other hand, are more suited to qualitative assays where sensitivity is more important.

Optimization of binding capacity and specificity of protein G on various solid matrices for immunoglobulins.

Streptococcal protein G is a more versatile and efficient alternative to staphylococcal protein A in purifying immunoglobin G (IgG) isotypes from various animal species. Optimizations are most dramatic with goat IgG, which binds protein G 55 times better than protein A. Using GammaBind G (a recombinant form of protein G (Genex Corp.)), we optimized binding capacity and specificity for IgG. Protein G was covalently coupled to three different matrices (CNBr-Sepharose, Tresyl-Sepharose, and Affigel-10) and compared with protein A-CNBr-Sepharose. Equal volumes of human, mouse, and goat serum samples were equilibrated into Hepes/NaOH buffers with various ionic strengths (i.e., concentrations of NaCl) and pH values and allowed to bind to affinity columns of proteins G and A. Bound ligands were eluted with 8.0 M urea, 0.05-M Tris/HCl, pH 8.00. Bound fractions were assayed for protein concentration and analyzed by sodium dodecyl sulfate polyacrylamide electrophoresis. The optimal conditions for binding IgG to protein G are 1.0 M NaCl and pH 8.0 for human, mouse, and goat.

Enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophoresis.

PIP: The enzyme-linked immunoelectrotransfer blot technique (EITB) combines the high resolving power of gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the high sensitivity of the enzyme-linked immunosorbent assay (ELISA) to produce an extremely powerful qualitative tool for studying antigen-antibody pairs. The EITB is conducted in 3 stages: 1) the antigen mixture is resolved by gel electrophoresis, 2) the resolved gel is then electrophoretically blotted onto nitrocellulose sheets, and 3) the blotted nitrocellulose is then develped by ELISA. This article details all the procedures involved in this methodology and describes the necessary materials. There is the theoretical danger that not all the antigenic sites can retain the native configuration after SDS treatment to allow recognition by appropriate antibodies. However, experiments with IgG, BSA, and other defined antigens tend to negate this argument. The general use of transfer blotting electrophoretically separated biologic molecules began with the DNA-RNA hybridization studies of Southern, with a technique now referred to as the Southern blot.^ieng

Comparison of particulate 3,3',5,5'-tetramethylbenzidine and 3,3'-diaminobenzidine as chromogenic substrates for immunoblot.

In horseradish peroxidase (EC: 1.11.1.7)-dependent immunoblot assays, particulate 3,3',5,5'-tetramethylbenzidine (TMB) is shown to be a more efficient immunoblot substrate than the standard substrate 3,3'-diaminobenzidine (DAB), because TMB is easily prepared, stable, and less carcinogenic than is DAB. Assays of antibody in a serially diluted human immunodeficiency virus (HIV) control serum (CDC reference CAT# VS2151) have the same sensitivity limits with both DAB and TMB (1:312,500). Complete, working substrate solutions of H2O2/TMB/enhancer and of H2O2/DAB were stored at room temperatures and at 48 degrees C respectively. Periodic tests showed the TMB substrate system to be functional after four weeks at 48 degrees C and after eight weeks at room temperature, while the DAB system was functional after one week at 48 degrees C and after four weeks at room temperature. The stability, safety, and convenience of the commercially available TMB kits make this substrate ideal for immunoblot tests.

Sequence variation in the cytochrome oxidase I, internal transcribed spacer 1, and Ts14 diagnostic antigen sequences of Taenia solium isolates from South and Central America, India, and Asia.

We examined the genetic variability in the pig-human tapeworm, Taenia solium, by sequencing the genes for cytochrome oxidase I, internal transcribed spacer 1, and a diagnostic antigen, Ts14, from individual cysts isolated from Peru, Colombia, Mexico, India, China, and the Philippines. For these genes, the rate of nucleotide variation was minimal. Isolates from these countries can be distinguished based on one to eight nucleotide differences in the 396 nucleotide cytochrome oxidase I (COI) sequence. However, all of the 15 isolates from within Peru had identical COI sequences. The Ts14 sequences from India and China were identical and differed from the Peru sequence by three nucleotides in 333. These data indicate that there is minimal genetic variability within the species T. solium. Minimal variability was also seen in the ITS1 sequence, but this variation was observed within the individual. Twenty-two cloned sequences from six isolates sorted into 13 unique sequences. The variability observed within the sequences from individual cysts was as great as the variability between the isolates.

Epilepsy and neurocysticercosis in an Andean community.

BACKGROUND: Taenia solium neurocysticercosis (NCC) has been documented as one of the major causes of epilepsy in developing countries. However, methodological limitations have hindered the evaluation of the epidemiological relationship between cysticercosis and epilepsy at the community level. METHODS: We used the WHO protocol for epidemiological evaluation of neurological disorders to conduct a door-to-door survey among 2723 residents of San Pablo del Lago, an Ecuadorean rural community in which T. solium taeniasis/cysticercosis was known to be endemic. The WHO protocol was complemented by neuroimaging and immunological tests to confirm the diagnosis of this infection. RESULTS: In all 31 people suffering from active epilepsy were detected (prevalence 11.4 per 1000, 95% CI:7.7-15.4); 26 agreed to undergo a computer tomography (CT) examination, and 28 agreed to have blood drawn for serodiagnosis. Fourteen of the 26 (53.8%) had CT changes compatible with NCC and six of the 28 (21.4%) tested positive in the enzyme-linked immunoelectro-transfer blot (EITB) assay. In a seizure-free random sample of this population, 17 of 118 (144 per 1000) subjects examined by CT and 10 out of 96 (104 per 1000) examined by EITB had evidence of this infection. The differences between the epilepsy group and the random sample of the population were statistically significant (OR = 6.93, 95% CI: 2.7-17.5, P < 0.001) for CT diagnosis, but not for EITB results (OR = 2.75, 95% CI: 0.8-7.1, P > 0.12, NS). CONCLUSIONS: These findings confirm that T. solium NCC is a significant cause of epilepsy at the community level in Andean villages of Ecuador. It is important to initiate effective public health interventions to eliminate this infection, which may be responsible for at least half of the cases of reported epilepsy in Ecuador.

Demonstration of species-specific and cross-reactive components of Taenia solium metacestode antigens.

Analysis of human serum reactivities to antigenic components of soluble Taenia solium metacestode proteins showed the predominant presence of determinants shared by T. solium, Echinococcus multilocularis and E. granulosus. Two polypeptides were demonstrated by SDS-PAGE and Western blot or enzyme-linked immunoelectrotransfer blot (EITB) assay to bind serum and CSF antibodies only from T. solium cysticercosis patients. These species-specific antigenic polypeptides focused between pH 4.6 and 3.9 after resolution by isoelectric focusing followed by EITB. The high species-specificity demonstrated by the present techniques offers the opportunity to confirm serologically an infection by T. solium metacestode.

Coagulation factor XIIa (activated Hageman factor) inhibitor from adult Schistosoma mansoni.

An inhibitory activity for the contact phase of the intrinsic coagulation pathway was demonstrated in an extract of adult Schistosoma mansoni. Inhibition is apparently specific for the enzymatic activation of Factor XI (pre-PTA) by Factor XIIa (activated Hageman factor). This phenomenon offers an explanation for the schistosomal evasive mechanism of the host contact hemostatic defense system.

Isotypic analysis of humoral immune responses in rhesus monkeys to an adult microsomal antigen of Schistosoma mansoni: an indicator of successful treatment.

A study was undertaken to examine the potential role of immunodiagnostic methods in determining successful chemotherapy in schistosomiasis. Fifteen rhesus monkeys were infected with 1,500 Schistosoma mansoni (Puerto Rico strain) cercariae, and 10 of the monkeys were then treated with a curative dose of praziquantel 13 weeks after infection. Five monkeys remained untreated. One monkey was not successfully cured, as confirmed by the presence of both male and female worms at the time of perfusion. Serum samples were longitudinally collected and specific Ig isotypes were quantified with an adult microsomal antigen of S. mansoni using the FAST-ELISA. Specific isotypes were detected with monoclonal antibodies specific for each human Ig isotype, followed by a peroxidase-conjugated anti-mouse Ig. Longitudinally, all monkeys showed similar isotype patterns. Isotypes increased for the first nine weeks following infection, and then began to decrease. Ten to 14 days following treatment, all isotypes increased. The Ig isotype responses of all monkeys followed classic patterns of isotype expression. A ratio of pretreatment (week 13) IgG1 absorbance values to post-treatment IgG1 absorbance values was generated for each monkey. All successfully treated monkeys, determined to be worm-free by perfusion, had IgG1 ratios at week 53 greater than 2.4 (range 2.4-181). The untreated monkeys and the single monkey that was a treatment failure had IgG1 ratios less than 2.1 (range 0.09-2.05) for the same time period.

Application of immunodiagnostic assays: detection of antibodies and circulating antigens in human schistosomiasis and correlation with clinical findings.

In an initial cross-sectional survey, serum, urine, and stool samples were collected from 370 participants representing about 10% of the population (n = 4,438) in Behbeet village, 50 km south of Cairo, Egypt, an area well known to be endemic solely for Schistosoma haematobium. Diagnosis was approached in two parallel ways. The first approach, which simulated actual conditions in many endemic areas in Egypt, was based on physical examination and urine and stool microscopic analysis. The second approach was based on two advanced immunodiagnostic assay systems. One system detected antibodies to species-specific microsomal antigens, the other detected circulating schistosomal antigens. Microsomal antigens from S. haematobium and S. mansoni were used to detect antibodies in the Falcon assay screening test (FAST)-ELISA and the enzyme-linked immunoelectrotransfer blot (EITB). Circulating anodic antigen (CAA) and circulating cathodic antigen (CCA) were quantified in serum and urine samples in a sandwich ELISA using monoclonal antibodies. Parasitologically, the prevalence of S. haematobium was 7.01% in females and 25.82% in males, giving an overall prevalence of 15.8%. The combination of urine CCA and serum CAA for detecting circulating antigens and the combination of the S. haematobium adult worm microsomal antigens (HAMA) FAST-ELISA and the HAMA EITB for detecting antibodies significantly improved the sensitivity of detecting S. haematobium circulating antigens and antibodies. Also, including a medical examination as an integral part of field studies and correlating immunodiagnostic results with other clinical and investigational data allowed us to calculate an accurate estimation of S. haematobium prevalence in this area of low endemicity.

Serodiagnosis of Schistosoma mansoni with microsomal adult worm antigen in an enzyme-linked immunosorbent assay using a standard curve developed with a reference serum pool.

A standardized microtest plate enzyme-linked immunosorbent assay was developed using the microsomal fraction of adult worms of Schistosoma mansoni (MAMA) as antigen. The standard reference serum pool was prepared from acutely and chronically infected rhesus monkeys and was shown to be appropriate as a standard for measuring the levels of reactivity of the unknowns. The standard serum pool was arbitrarily designated as having 100 activity units per microliter. The levels of reactivity of the unknowns were expressed as activity units per microliter. Serum specimens were obtained from 190 patients infected with S. mansoni in the Caribbean, South America, and Africa. Serum was obtained from small numbers of patients infected with S. haematobium, S. japonicum, or S. mekongi. Controls were 136 patients with other helminthic infections, 142 patients with protozoal or other diseases with liver involvement, and 81 healthy serum donors. The J index (or predictability) of the assay was calculated to determine the significant level of reactivity. The assay has a predictability of 95% for both patients with S. mansoni infections and those with other infections. The sensitivity of the assay for S. mansoni infections was 96%, and the specificity (in terms of cross-reactions with infections with other parasite genera or with other liver diseases) was 99%. The heterologous Schistosoma species showed a markedly lower level of reactivity, with an overall sensitivity of 55%. This is in accord with the species-specificity previously recognized in MAMA, and emphasizes the need for standard reference pools of human sera prepared from patients infected with single species of each of the Schistosoma. Use of these pools in assays with antigens of the respective schistosome species would allow optimum serologic evaluation.

Factors associated with Taenia solium cysticercosis: analysis of nine hundred forty-six Peruvian neurologic patients. Cysticercosis Working Group in Peru (CWG).

In most developing countries, 10% of acute neurologic cases are patients with neurocysticercosis (NCC). Determining specific factors associated with contracting NCC will facilitate its diagnosis and prevention. We examined multiple socioeconomic, demographic, environmental, medical, and behavioral characteristics of 946 Peruvian neurologic patients for a correlation with NCC, which was diagnosed by the highly specific and sensitive electroimmunotransfer blot (EITB) or immunoblot assay. Eighteen percent (172 of 932) of serum samples and 28% (101 of 362) of cerebrospinal fluid samples were EITB-positive. The proportion of EITB-positive persons was similar for all socioeconomic levels. Significant factors associated with NCC were: 1) being born outside Lima, 2) having raised pigs, 3) more than 20 years of age, 4) a history of seizures, and 5) a history of taeniasis. Of these factors, raising pigs is the only one that is amenable to intervention, via improvements in animal husbandry.

Effective, single-dose treatment or porcine cysticercosis with oxfendazole.

The pig is a vital link in the transmission cycle of Taenia solium, the cestode responsible for human-porcine cysticercosis. Infected pigs also represent an important source of economic loss to farmers in developing countries. Past efforts to find an adequate therapeutic regimen to treat this parasite disease in swine have failed because of low efficacy, high cost, side effects, or the need for multiple doses. In this randomized, no treatment-controlled study, the efficacy and safety of oxfendazole and praziquantel for the treatment of porcine cysticercosis were evaluated in 16 naturally infected pigs. Four groups of four pigs were treated with oxfendazole, praziquantel, oxfendazole plus praziquantel, or untreated. The pigs were humanely killed 12 weeks post-treatment, the number of cyst was counted, and parasite viability was assessed by cyst evagination. No detectable side effects were seen in any of the pigs. Praziquantel treatment alone appeared to reduce the number of cysts, but did not decrease the viability of the remaining parasites. Treatment with oxfendazole alone or oxfendazole plus praziquantel killed all of the parasites, and left only microcalcifications in the meat. Oxfendazole provides, for the first time, a practical, effective, inexpensive, and single-dose therapy for porcine cysticercosis.

Discrepancies between cerebral computed tomography and western blot in the diagnosis of neurocysticercosis. The Cysticercosis Working Group in Peru (Clinical Studies Coordination Board).

Serum samples from sequential patients who underwent cerebral computed axial tomography (CT) scan in a Peruvian radiologic clinic were tested by the highly sensitive and specific enzyme-linked immunoelectrotransfer blot (EITB) test to detect antibodies to Taenia solium. The results of the EITB test were compared with those obtained by CT scan for the diagnosis of neurocysticercosis. Of the 383 patients sampled, 32 (8%) were seropositive. The results of CT and EITB were frequently discrepant. When compared with the EITB assay, the CT scan was 44% sensitive and 95% specific. The sensitivity of CT increased to 63% if less specific images (single calcifications, granulomas, or hydrocephalus) were included. The CT scan for diagnosis of cysticercosis can best be used in conjunction with a reliable serologic test such as the EITB.

Use of sentinel pigs to monitor environmental Taenia solium contamination. The Cysticercosis Working Group in Peru (CWG).

We tested a novel approach to assay Taenia solium prevalence using the enzyme-linked immunoelectrotransfer blot assay in sentinel piglets to determine environmental contamination with T. solium eggs in a disease-endemic zone in Peru. Twelve sentinel piglets from an area where the disease is not present were tested at two months of age, moved to an area where the disease is endemic, and retested at the of age nine months. Sentinel piglets native from this T. solium-endemic area were also tested concurrently at two and nine months of age. Of the non-native pigs, 33% (4 of 12) acquired new infection. Of the 28 native pigs tested, 64% (18 of 28) acquired the infection. In a subset of the native piglets from seronegative sows, 44% (4 of 9) were infected at five months of age. Serodiagnosis of sentinel piglets is a practical method to detect T. solium eggs in the environment. Furthermore, it permits indirect assessment of human risk, which may be useful for monitoring the efficacy of intervention programs.