Transactions of the Royal Society of Tropical Medicine and Hygiene (2005) 99, 568—576
Oncospheral peptide-based ELISAs as potential seroepidemiological tools for Taenia solium cysticercosis/neurocysticercosis in Venezuela Elizabeth Ferrer a,i, M. Milagros Cort´ ez a, Zully Cabrera a, Glenda Rojas a, on de Noya c, Hilda A. P´ erez d, Isis Fernandez e, Iris D´ avila b, Belkis Alarc´ Haidee Urdaneta Romero f, Leslie J.S. Harrison g,∗, R. Michael E. Parkhouse h, Teresa G´ arate i a
Centro de Investigaciones Biom´ edicas (BIOMED), Universidad de Carabobo, Maracay, Venezuela Departamento de Parasitolog´ıa, Universidad de Carabobo, Valencia, Venezuela c Instituto de Medicina Tropical, Universidad Central de Venezuela (UCV), Caracas, Venezuela d Instituto Venezolano de Investigaciones Cientificas (IVIC), Centro de Microbiolog´ ıa y Biolog´ıa Celular, Caracas, Venezuela e Departamento de Zoonosis, Ministerio de Sanidad y Asistencia Social, Estado Lara, Venezuela f Instituto de Inmunolog´ ıa Cl´ınica, Universidad de los Andes, M´ erida, Venezuela g Sir Alexander Robertson Centre for Tropical Veterinary Medicine, Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Easter Bush Veterinary Centre, Roslin, Midlothian EH25 9RG, UK h Instituto Gulbenkian de Ciencia, Rua da Quinta Grande 6, P.O. Box 2781, Oeiras, Portugal i Instituto de Salud Carlos III, Centro Nacional de Microbiologia, 28220, Majadahonda, Madrid, Spain b
Received 12 July 2004 ; received in revised form 26 January 2005; accepted 18 February 2005 Available online 23 May 2005 KEYWORDS Cysticercosis; Taenia saginata; Taenia solium; Synthetic peptides; Antibody ELISA; Venezuela
Summary This study evaluates five synthetic peptides derived from four, potentially protective, Taenia saginata oncosphere molecules for the serodiagnosis of T. solium cysticercosis/neurocysticercosis in three distinct Venezuelan endemic regions. The peptides, all of which have been described previously, are designated HP6-3, Ts45W-1, Ts45W-5, Ts45S-10 and TEG-1. In clinically verified and seropositive hospital cases, combining the results of three of the individual peptide-based ELISAs (HP6-3, Ts45W-1 and Ts45W-5) afforded the best balance between sensitivity (85%) and specificity (83.5%), a significant improvement on the 63.6% specificity obtained with the routinely employed T. solium cyst-fluid-based ELISA. Similarly, in the seropositive Venezuelan endemic zone samples, 89.09% of Amerindians, 77.27%
* Corresponding author. Tel.: +44 131 650 6246; fax: +44 131 650 6246.
E-mail address: [email protected]
(L.J.S. Harrison). 0035-9203/$ — see front matter © 2005 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.trstmh.2005.02.003
Peptide-based ELISAs for Taenia solium
of symptomatic rural subjects and 67.83% of non-symptomatic rural subjects were also classed as seropositive by the combined peptide-based ELISAs. The profile of antibody recognition to individual peptides varied between the different groups of samples examined. The relevance of the above findings for the serology and prognosis of T. solium cysticercosis/neurocysticercosis in hospital- and field-based situations is discussed. © 2005 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.
1. Introduction Neurocysticercosis (NCC) is the most common helminth infection of the human central nervous system and is caused by the larval stage of Taenia solium. It is recognized as a serious public health and economic problem in the developing countries of Latin America, Africa and Asia (White, 1997). However, cysticercosis/NCC has also been highlighted as an emerging disease in developed countries, in part due to immigration of infected people from high-prevalence endemic areas to lowprevalence areas (Schantz et al., 1992). Diagnosis of NCC in rural endemic areas is compounded by the fact that neuroimaging techniques such as computed tomography (CT) and magnetic resonance imaging (MRI) are expensive and inaccessible (Del Brutto et al., 1996). However, CT and MRI are still the only techniques available that can be regarded as a ‘gold standard’ for the diagnosis of NCC, as they categorically identify cases with cysts in the brain and can often be used to determine whether cysts are viable (potentially treatable by drugs) or colloidal or calcified. Antibody assays, although potentially valuable diagnostic tools, can be used only to indicate exposure to infection. They do not necessarily indicate current infection, nor do they identify the site of infection, for example in the brain. Currently, routine antibody-based diagnosis of T. solium cysticercosis and NCC still relies on the employment of complex antigens, such as metacestode cyst fluid in ELISA diagnosis and metacestode glycoproteins in the laborious and expensive enzyme-linked immunoelectrotransfer blot (EITB) (Tsang et al., 1989). Although the EITB was recognized as the ‘immunological method of choice for confirmation of the diagnosis of NCC’ by WHO and the Pan American Health Organization, for practical reasons assays such as the T. solium cyst-fluid-based ELISA are still commonly in use in many laboratories in the developing world. A major disadvantage of the above assays and other similar attempts to develop sensitive and specific diagnostic assays (D´ıaz et al., 1992; Larralde et al., 1986; Parkhouse and Harrison, 1987; Rhoads et al.,
1985) is that they demand access to T. soliuminfected pigs as a source of parasite-derived antigen. Clearly, synthetic peptides and recombinant antigens (Felleisen and Gottstein, 1993) potentially provide a reliable, reproducible and cost-effective alternative (Parkhouse and Harrison, 1989), independent of a supply of parasite-derived material. Indeed, several synthetic peptides have already been employed in the immunodiagnosis of various helminth infections (Gonz´ alez-Sapienza et al., 2000; Hern´ andez et al., 2000; Noya et al., 1998). In this study we further assess the diagnostic potential of five peptides with high antigenic indexes (Jameson and Wolf, 1988) predicted from the sequences of four T. saginata oncosphere proteins (see Materials and methods). These proteins were, by definition, immunogenic, as they were antibodyselected from a cDNA expression library of invasive oncospheres. They are therefore logical targets for antibody diagnosis, for example for use as epidemiological tools to determine the degree of exposure to infection, and are clearly distinct from assays based on metacestode-derived antigens. Furthermore, these peptides showed good sensitivity and specificity in the diagnosis of bovine cysticercosis (Ferrer et al., 2003a). As T. saginata is taxonomically very closely related and cross-reactive to T. solium (Harrison and Parkhouse, 1989; Parkhouse and Harrison, 1987), diagnostic assays developed for the T. saginata system may be equally applicable to the T. solium system; indeed, promising results were obtained when these peptides were tested with cerebrospinal fluid (CSF) from NCC patients (Fleury et al., 2003). However, serum samples are much easier and less invasive to obtain, in both the rural and the hospital situation. We have thus proceeded to the next logical step in evaluating these five peptides by using human serum samples from confirmed hospital cases and then from less well-defined rural endemic Venezuelan samples (Ferrer et al., 2002, 2003b). Two factors governed our examination of the results. First, the antibody responses to individual peptides may differ between groups with differing disease history. Second, it is naive to expect that one peptide would be able to function across
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the board as a 100% reliable diagnostic tool in an out-bred population with a spectrum of pathogenesis. Thus, as we observe here, greater and satisfactory sensitivity and specificity was observed by combining the results of three of the five peptide assays.
2. Materials and methods 2.1. Peptides Peptides corresponding to sequences with predicted high antigenic indexes (Jameson and Wolf, 1988) were synthesized (Chiron Technologies, Australia) (Table 1). They consisted of: peptide HP6-3 derived from the sequence of the protective 18 kDa surface/secreted oncospheral antigen identified by McAb-HP6 (Ben´ıtez et al., 1996; Bonay et al., 2002; Harrison et al., 2005); peptides Ts45W-1 and Ts45W-5 derived from the sequence of the T. saginata homologue of the T. ovis 45W protective gene family (Johnson et al., 1989; Waterkeyn et al., 1995); peptide TS45S-10 derived from the T. saginata homologue of the T. ovis 45S protective antigen (Waterkeyn et al., 1995); and peptide TEG1 derived from the sequence of the T. saginata homologue of the main surface protein of Echinococcus (Ben´ıtez et al., 1998; Felleisen and Gottstein, 1993, 1994; Frosch et al., 1991, 1994; Rosas et al., 2002).
2.2. Taenia solium cyst fluid Taenia solium metacestodes were dissected from naturally infected pigs and the cyst fluid was ex-
Amino acid sequencea
Ts45W-5 Ts45S-10 TEG-1
2.3. Human sera This study was conducted in conformation with Venezuelan ethical practices. The ethical review board of Carabobo University, Venezuela approved the study. In addition, before starting the villagebased studies, contact and agreement were first established with the community leaders and permission was obtained from the local health authorities. Serum samples were collected from the following sources. 2.3.1. Clinically and brain scan-verified cases of active and inactive NCC Forty samples were taken from patients with confirmed NCC. Diagnosis was based on: (1) clinical findings, all patients were symptomatic (neurological disorders, seizures, headache etc.); (2) serology, all the samples were positive by an ELISA antibody using T. solium cyst fluid as antigen, determined by the method described by Larralde et al. (1986); and (3) imaging findings, all the patients had cysticerci in their brain, as diagnosed by CT or MRI. On the basis of the neuroimaging findings, patients were categorized into two groups: active NCC, with viable cysts (20 patients); and inactive NCC, with multiple calcifications in their CT/MRI scan (20 patients). The monoclonal-antibody-based HP-10 antigen detection assay, which detects the excretory/secretory products of live metacestodes (Harrison et al., 1989), was used to verify this classification. Aliquots of these serum samples were pooled and used as the positive reference control
Sequences and origin of the five peptides selected from cloned Taenia saginata oncosphere proteins
tracted and stored as previously described (Ferrer et al., 2003a).
Protein description and GenBank numbers
Major secreted ‘functional’ antigen; identified by McAb-HP6 (GenBank no. X95983) AYEQPIERTVVGHQTLRDIFVWGC Taenia ovis 45W gene family homologue (GenBank no. AJ430567) CVTVVTTSGS CGGTEESVVTASRS Taenia ovis 45S homologue (GenBank no. AJ430566) CRDPSKMRDIDRHHEYNVREGND Echinococcus surface protein homologue (GenBank no. X97000)
A cysteine residue (C) was added to each peptide to facilitate their coupling to carrier proteins.
Sequence designation HP6
Peptide-based ELISAs for Taenia solium
serum. All patients were from the central-west of Venezuela. 2.3.2. Sera from three Venezuelan endemic areas All three areas have differing climatic, geographic and socio-economic characteristics. The sera were characterized by (1) the clinical criteria indicated below; (2) reactivity, positive or negative in Ab-ELISA using T. solium cyst fluid as antigen (Larralde et al., 1986); and (3) reactivity in the monoclonal-antibody-based HP-10 antigen detection assay, which detects the excretory/secretory products of live (that is, viable) metacestodes (Harrison et al., 1989). Symptomatic and Ab-positive NCC cases from the Andes: 44 samples from cases with Ab-positive serology and clinical diagnosis conducted according to Del Brutto et al. (1996) and thus based on neurological symptoms (seizures, headache etc.). The patients were all from rural areas of M´ erida State, Venezuela, which is located in the Andes mountain range. Asymptomatic, Ab-positive and Ab-negative samples from Venezuelan Amerindians, (Amazonas State): a total of 55 Ab-positive samples and three Ab-negative samples were selected. Amazonas has a jungle environment and is in the south of Venezuela. Our previous studies indicated that there was strong evidence of very recent infection and/or exposure to T. solium in this community (Ferrer et al., 2002). Asymptomatic, Ab-positive and Ab-negative samples from three rural endemic areas in centralwest Venezuela: all three areas have similar climatic, socio-economic, cultural and geographical characteristics (Ferrer et al., 2003b). There were 108, 23 and 12 Ab-positive samples (total 143) and 18, 15 and 12 Ab-negative samples (total 45) collected from the communities of Canoaba, Sanare and R´ıo Tocuyo, respectively. 2.3.3. Sera from clinically and serologically verified helminthic, protozoal and viral infections These 142 sera covered a range of helminth, protozoal and viral infections found in the areas endemic for T. solium and were obtained from the Instituto de Inmunolog´ıa Cl´ınica, Universidad De Los Andes, M´ erida, Venezuela, Centro de Investigaciones Biom´ edicas, Universidad de Carabobo, Maracay, Venezuela and Instituto de Salud Carlos III, Majadahonda, Madrid, Spain. Helminthic infections (total 78): cystic hydatidosis (20, ultrasonography, serology and clinical findings); Schistosoma mansoni schistosomiasis
(13, coprology exams, serology and clinical findings); fascioliasis (9, serology, clinical findings and epidemiological data); toxocariasis (15, clinical findings, serology and consistent with epidemiological data); onchocerciasis (4, skin snip examination and serology); and finally all determined by coprological examination, Hymenolepis nana hymenolepiasis (2), ancylostomiasis (3), trichuriasis (5) and ascariasis (7). Protozoal infections (total 54): malaria (3, microscopic demonstration of parasite in blood smears); toxoplasmosis (15, ophthalmological exam and serology); Chagas’ disease (8, xenodiagnosis, clinical findings and serology); Leishmania chagasi visceral leishmaniasis (9, microscopic demonstration of parasite in bone marrow aspirates and serology); Entamoeba histolytica amebiasis (11), Blastocystis hominis infection (2), Endolimax nana infection (2) and giardiasis (4) all determined by coprological examination. Viral infections (total 10): hepatitis (5) and cytomegalovirus (1) determined by clinical findings and serology; measles (4, clinical findings only). 2.3.4. Control negative sera Thirty samples from healthy individuals collected from the staff of the laboratory and urban-dwelling university students who had never been to the endemic areas and who had never, to their knowledge, eaten rurally killed or uninspected pork. Aliquots of these 30 sera were also pooled and used as the negative reference control serum. These sera were used to determine cut-off values.
2.4. Peptide and cyst-fluid ELISA for antibody detection (Ab-ELISA) The optimum reagent dilutions were determined by checkerboard titration. In our standardized system, peptides (2 g/ml, 100 l/well) or T. solium cyst fluid (5 g/ml, 100 l/well) were used to sensitize 96 well plates (Dynatech Immunolon 2), and the assay ran as previously described (Ferrer et al., 2003a). After washing and blocking the plates, serum samples (100 l/well at 1/100 dilution in PBS) were added followed by goat anti-human IgG conjugated with alkaline phosphatase (Sigma), (H and L chains) (Sigma), diluted 1/5000. Following addition of the substrate p-nitro-phenyl-phosphate 1 mg/ml (Sigma) in 0.2 mol/l NaHCO3 , 5 mmol/l MgCl2 (pH 9.5), the colour reaction was stopped by adding 50 l of 3N NaOH to each of the wells. The optical density (OD; 405 nm) of the reaction product was read on a Microplate reader (Dynatech MR 700, Helsinki, Finland). ELISA plates were routinely set
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up to include six positive, six negative and six diluent control wells. Each of the samples was run in duplicate. The mean sample ODs minus the mean diluent ODs were corrected for any minor day-today or plate-to-plate variation using a correction factor determined by the formula
formed markedly better than the T. solium cystfluid-based assay, with specificities ranging from 81.0—98.6%. None of the 48 endemic background controls (45 from central-west Venezuela and three from the Amazon region) were positive in any of the peptide assays.
mean PO − mean NO mean Pt − mean Nt
3.2. Sensitivity of the various assays based on serum samples from clinically defined hospital cases (active and inactive NCC)
where P is the positive control, N the negative control, O the reference day and t the test day. ELISA results were rejected if the correction factor for any particular plate varied more than 10% from the reference day. A sample was considered positive if the corrected OD value was greater than the cut-off value. The cut-off value for each individual Ab-ELISA was calculated from the mean ODs of 30 normal serum samples plus 3 standard deviations. Once all the assays were run, the results were verified by repeat assay and once again if considered necessary. The results of the individual assays were examined either singly or in various combinations. In each of these, a sample was classed as seropositive if it was thus classed in any one of the individual assays under consideration, while samples were classed as negative when thus classed in all assays under consideration.
2.5. ELISA for detection of circulating antigens (Ag-ELISA) Ag-ELISA was performed with slight modifications from the original report (Harrison et al., 1989) and as described (Ferrer et al., 2002).
2.6. Data handling and statistical analysis The data was processed using Microsoft Excel and statistical analysis was carried out using either the 2 or Fisher’s exact tests.
3. Results 3.1. Specificity of the various assays based on serum samples from clinically defined cases (various helminth, protozoal and viral infections) As already established, the specificity of the HP10 Ag-ELISA was very high (96.5%), while that of the T. solium cyst-fluid Ab-ELISA was low (63.4%) (Table 2). All the peptide-based Ab-ELISAs per-
Again, as may be anticipated from previous studies, the sensitivity of the HP10-based Ag-ELISA was high (75%) in cases of confirmed active NCC. This is because the assay detects the products of live metacestodes. By contrast, only 10% of cases classed as inactive NCC were positive in this assay (Figure 1). By definition and selection, all (100%) of the serum samples from the cases designated active and inactive NCC were classified as seropositive in the T. solium cyst-fluid-based Ab-ELISA. None of the peptide-based Ab-ELISA assays displayed this level of sensitivity. However, apart from the TEG1-based assay, in which only 10% of active NCC cases were classed as seropositive, all the peptidebased ELISAs displayed similar sensitivity whether the samples were from active or inactive NCC cases (Figure 1).
3.3. Best balance between sensitivity and specificity in the various peptide Ab-ELISAs based on serum samples from clinically defined cases In general, peptide-based assays may be expected to perform better when sera are tested with a selection of peptides rather than simply one. This proved to be the case when we examined the sensitivity and specificity of the individual peptide-based assays in all possible combinations. The indications were that the best combination of sensitivity for clinically defined active NCC (17/20; 85%) and clinically defined inactive NCC (also 17/20; 85%) and specificity (119/142; 83.8%), i.e. samples from other defined infections, occurred when the results from the individual HP6-3, Ts45W-1 and Ts45W-5 peptide assays were considered together (Figure 1).
3.4. Rural symptomatic and rural asymptomatic communities A very high percentage of the asymptomatic Amerindians (67.3%) were positive in the HP10 Ag-
Peptide-based ELISAs for Taenia solium
Table 2 Detection of secreted HP10 antigen from viable Taenia solium metacestodes and of antibody to T. solium metacestode cyst fluid and the five T. saginata-derived oncospheral peptides, in sera from patients without cysticercosis but with other helminthic, protozoal and viral infections Infection
Cystic hydatidosis S. mansoni schistosomiasis Fascioliasis Toxocariasis H. nana hymenolepiasis Onchocerciasis Ancylostomiasis Trichuriasis Ascariasis Malaria Toxoplasmosis Amebiasis B. hominis E. nana Giardiasis Chagas disease Leishmaniasis Hepatitis Measles Cytomegalovirus Total Specificity (%)b
1/20 0/13 0/9 1/15 0/2 0/4 0/3 0/5 2/7 0/3 0/15 0/11 0/2 0/2 0/4 1/8 0/9 0/5 0/4 0/1 5/142 96.5
17/20 5/13 3/9 4/15 1/2 3/4 2/3 2/5 7/7 1/3 0/15 2/11 2/2 0/2 0/4 3/8 0/9 0/5 0/4 0/1 52/142 63.4
6/20 2/13 2/9 0/15 0/2 3/4 0/3 1/5 2/7 1/3 0/15 1/11 0/2 0/2 0/4 1/8 0/9 0/5 0/4 0/1 19/142 86.6
0/20 0/13 0/9 1/15 0/2 0/4 0/3 0/5 0/5 0/3 1/15 0/11 0/2 0/2 0/4 0/8 0/9 0/5 0/4 0/1 2/142 98.6
0/20 0/13 0/9 1/15 0/2 0/4 0/3 0/5 0/5 0/3 0/15 0/11 0/2 0/2 0/4 1/8 0/9 0/5 0/4 0/1 2/142 98.6
3/20 4/13 2/9 0/15 0/2 0/4 0/3 0/5 0/5 1/3 0/15 0/11 0/2 0/2 0/4 0/8 0/9 0/5 0/4 0/1 10/142 93
5/20 2/13 1/9 2/15 1/2 4/4 2/3 4/5 5/7 1/3 0/15 0/11 0/2 0/2 0/4 0/8 0/9 0/5 0/4 0/1 27/142 81
a The ELISAs employed were either the monoclonal antibody HP10-based antigen detection ELISA (Ag-ELISA) or indirect antibody detection ELISAs. b Specificity was calculated as the percentage of correctly designated negative samples.
Figure 1 Percentage of samples classed as positive in the HP10 Ab-ELISA or Ab-ELISAs using the HP6-3, Ts45W-1, Ts45W-5, Ts45S-10 or TEG-1 Taenia saginata oncospheral synthetic peptides. ‘Combined’ denotes the results obtained by combining the results of the HP6-3 + Ts45W-1 + Ts45W-5 peptide Ab-ELISAs; these gave the best overall balance between sensitivity and specificity.
ELISA, indicating infection with viable parasites, and while 43.2% of the symptomatic rural subjects from M´ erida were positive, only 20% of the asymptomatic rural subjects were positive (Figure 1). As the combined results of the HP6-3, Ts45W1 and Ts45W-5 peptide Ab-ELISAs gave the best balance between sensitivity (85%) and specificity (83.8%) for the clinically confirmed hospital cases, the same peptide combination was examined for the three rural Venezuelan communities. The pattern was similar to that for the Ag-ELISA, with higher numbers of the Amerindian subjects (49/55; 89.09%) and rural symptomatic subjects from M´ erida (34/44; 77.27%) positive in comparison with the rural asymptomatic subjects (97/143; 67.83%) (Figure 1). Some variations were apparent between the different peptide-based Ab-ELISA results from the three rural communities. For example, a huge number (around 95%) of the Amerindian samples were positive in the TEG-1 Ab-ELISA, while considerably fewer (around 38%) of the rural symptomatic and asymptomatic samples were positive. Similarly, only 2.27% of the rural symptomatic cases were positive using the HP6-3 Ab-ELISA, in comparison with around 60% of the other two populations (Figure 1).
4. Discussion This work focuses on the serodiagnosis of the important helminth parasite of humans, T. solium. In particular, we examined the diagnostic potential of five T. saginata peptides, which were selected from the sequences of four potentially protective oncospheral proteins. The peptides have proven serodiagnostic utility for T. saginata infections in cattle (Ferrer et al., 2003a) and, as determined in this study, also for T. solium infections in humans. There is clearly an advantage in the development of assays focused on specific recombinant antigens or synthetic peptides as they obviate the requirement for access to parasite material. With the exception of the HP6-3-based Ab-ELISA, none of the individual peptides performed with the necessary high levels of sensitivity and specificity. However, as determined using clinically verified and T. solium cyst-fluid-based Ab-ELISA positive sera, when examined together, peptides HP6-3, Ts45W-1 and Ts45W-5 gave a sensitivity of 85% and a specificity of 83.8%. While the sensitivity was lower than that for the T. solium cyst-fluid-based Ab-ELISA, the specificity improved, i.e. from 63.4% to 83.8%. Indeed, all the individual peptide-based Ab-ELISAs, and all the peptide combinations examined, functioned with greater specificity than the frequently
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employed T. solium cyst-fluid-based Ab-ELISA. In our previous similar study employing CSF (Fleury et al., 2003), peptides HP6-3 and Ts45W-1 detected the majority of the active and inactive NCC patients, but, in this study, the inclusion of peptide Ts45W-5 further improved the sensitivity. It should be noted that the negative control sera consisted of 30 samples from non-endemic areas of Venezuela and an additional 48 samples from the rural communities (endemic background controls), which were all negative in the T. solium cyst-fluidbased Ab-ELISA. We also examined 142 sera from rural Venezuela covering a range of helminthic, protozoal and viral infections (see Materials and methods). When the group of 30 control samples was used to determine the cut-off values, all the second group of 48 endemic background control sera were negative in all the peptide assays. The specificity estimates were based on the third group, comprising helminth, protozoal and viral infections, which provided a more stringent alternative to the endemic background controls. The study did not include sera from identified Taenia spp. carriers (either T. saginata or T. solium). However, the prevalence of taeniasis in an endemic community is widely accepted to be very much lower than that of cysticercosis. That study may be conducted later. Given the proven utility of the peptides with the clinically verified hospital cases of cysticercosis, we tested serum samples from three different rural Venezuelan communities with the same peptidebased Ab-ELISAs and found that the results from these three communities were variable. Markedly few samples were Ab-positive in the HP6-3 assay in the rural symptomatic (M´ erida) samples compared with samples from the central-west and the Amazon. Many more samples from the Amazon region were positive in the TEG-1-based Ab-ELISAs. Interestingly, our previous studies provided strong evidence of very recent infection and/or exposure to T. solium in this community (Ferrer et al., 2002). It may also be significant that a high number of the rural symptomatic cases were Ab-positive in the Ts45W-1 Ab-ELISA, while the TEG-1-based assay seemed to be a poor indicator of active NCC, detecting only 10% of these cases. It is possible that the different patterns of peptide recognition may be genetic or related to the geographical and behavioural differences between the different communities. Another possibility is that the observed variations reflect differences between communities in the balance between recent and longer-term infections. Since there was strong evidence for very recent exposure in the Amerindian community, it is interesting to speculate about an association between recent expo-
Peptide-based ELISAs for Taenia solium
sure and the percentage of positive samples in the TEG-1-based Ab-ELISA. Thus, further studies with these and other peptides might have interesting prognostic value, indicating correlations of immunity/resistance. Clearly, positivity in these peptide-based assays indicates exposure to oncospheral antigens and not necessarily a current infection with metacestodes. Thus, the assays have potential for use in epidemiological surveys designed to measure such exposure in a population. As the peptides were derived from potentially protective antigens, the profile of the antibody responses to them in a population may be not only an indicator of the level of exposure to the parasite but also to the level of immunity. Finally, the assays perform well when serum, instead of CSF, is used as the sample source, a factor that is crucial for the employment of the assays in epidemiological studies and in situations where collection of CSF is difficult or ethically unsupportable. Conflicts of interest statement The authors have no conflicts of interest concerning the work reported in this paper.
Acknowledgements This work was supported by the European Union INCO-DC programme Contract IC18CT950002. Elizabeth Ferrer was supported by a grant from AECI and ISCIII. Special thanks are given to Dra. Nurys Mendoza, Dra. Sonia Santeliz, Dr Cesar Mendez, teacher Nelia Mota and all members of sanitary teams of Sanare and R´ıo Tocuyo for the valuable help and collaboration in the fieldwork for this study. We also thank the Consejo de Desarrollo Cient´ıfico y Humanistico (CDCH) from Carabobo University for transport facilities for the fieldwork. We are indebted to Mr Jose Luis Escalona for his help in planning the fieldwork and Ms Moraima Suarez, the social worker who helped to contact local leaders and patients in Canoabo. The authors would also like to thank Mar´ıa Lares for her help in handling the serum samples, Laura Ben´ıtez (ISCIII, and currently Departamento de Microbiolog´ıa, Facultad de Biolog´ıa UCM, Spain) for her collaboration in the design of the synthetic peptides and Mildred Foster (IAH, Pirbright, UK and currently Sir William Dunn School of Pathology, University of Oxford, Oxford, UK) for her input regarding the synthetic peptide-based ELISAs.
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