RESUMO
Different H-2 congenic strains of mice were immunized with a P. falciparum sporozoite vaccine currently being tested in humans, or with different segments of the vaccine molecule. Specific IgG production or lymph node cell proliferation in response to different antigens was then determined. Only four of seven strains (representing three of eight possible different class II restriction molecules) responded to the vaccine. Of those restriction molecules, only one, I-Ab, was associated with a response to a malaria-encoded T epitope [contained within NP(NANP)3NA], while the other two molecules (E alpha dE beta d and E alpha kE beta s) were associated with a T cell response to a nonmalarial epitope(s) carboxyterminal to the malaria sequence and encoded by a tetracycline resistance gene, read out of frame. If an analogous situation applies in humans, natural boosting by sporozoites will be very restricted. This has serious implications for the effectiveness of the vaccine, since constant high levels of antisporozoite antibodies and possibly antibody-independent T cell effector functions are required for immunity.
Assuntos
Antígenos de Protozoários/imunologia , Genes MHC da Classe II , Plasmodium falciparum/imunologia , Linfócitos T/imunologia , Vacinas/imunologia , Animais , Formação de Anticorpos , Epitopos/imunologia , Tolerância Imunológica , Ativação Linfocitária , Malária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Plasmodium falciparum/crescimento & desenvolvimentoRESUMO
Proteosomes are hydrophobic, membranous, multimolecular preparations of meningococcal outer membrane proteins that are also B cell mitogens. These characteristics suggested that proteosomes may serve as carrier proteins and adjuvants to enhance peptide immunogenicity. Although high titers of malaria circumsporozoite (CS) antibodies protect against malaria, vaccines thus far tested in humans have been insufficiently immunogenic to be clinically useful. Here it is shown that synthetic CS peptides hydrophobically complexed to proteosomes by way of lauroyl-cysteine become highly immunogenic in mice without other adjuvants. The high titers of antibodies produced and the safety of proteosomes in humans suggest that this novel system is widely applicable for the development of peptide vaccines to protect against many diseases.
Assuntos
Antígenos de Superfície/imunologia , Antígenos/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Neisseria meningitidis , Proteínas de Protozoários , Vacinas Sintéticas/imunologia , Adjuvantes Imunológicos , Animais , Especificidade de Anticorpos , Antígenos de Protozoários/imunologia , Apicomplexa/imunologia , Epitopos/imunologia , Imunização , Imunoglobulina G/análise , Imunoglobulina G/biossíntese , Lipossomos/imunologia , Camundongos , Plasmodium falciparum/imunologia , Plasmodium vivax/imunologiaRESUMO
Immunization with radiation-attenuated malaria sporozoites induces potent cellular immune responses, but the target antigens are unknown and have not previously been elicited by subunit vaccines prepared from the circumsporozoite (CS) protein. A method is described here for inducing protective cell-mediated immunity to sporozoites by immunization with attenuated Salmonella typhimurium transformed with the Plasmodium berghei CS gene. These transformants constitutively express CS antigens and, when used to immunize mice orally, colonize the liver, induce antigen-specific cell-mediated immunity, and protect mice against sporozoite challenge in the absence of antisporozoite antibodies. These data indicate that the CS protein contains T cell epitopes capable of inducing protective cell-mediated immunity, and emphasize the importance of proper antigen presentation in generating this response. Analogous, orally administered vaccines against human malaria might be feasible.
Assuntos
Antígenos de Superfície/imunologia , Vacinas Bacterianas/imunologia , Malária/imunologia , Plasmodium berghei/imunologia , Proteínas de Protozoários , Salmonella typhimurium/imunologia , Animais , Feminino , Fígado/microbiologia , Malária/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos , Salmonella typhimurium/genéticaRESUMO
The circumsporozoite (CS) protein of the human malaria parasite Plasmodium falciparum may be the most promising target for the development of a malaria vaccine. In this study, proteins composed of 16, 32, or 48 tandem copies of a tetrapeptide repeating sequence found in the CS protein were efficiently expressed in the bacterium Escherichia coli. When injected into mice, these recombinant products resulted in the production of high titers of antibodies that reacted with the authentic CS protein on live sporozoites and blocked sporozoite invasion of human hepatoma cells in vitro. These CS protein derivatives are therefore candidates for a human malaria vaccine.
Assuntos
Formação de Anticorpos , Antígenos de Superfície/imunologia , Malária/prevenção & controle , Plasmodium falciparum/imunologia , Proteínas de Protozoários , Vacinas/imunologia , Animais , Antígenos de Superfície/genética , Carcinoma Hepatocelular , Linhagem Celular , Clonagem Molecular , Reações Cruzadas , DNA Recombinante , Escherichia coli/genética , Humanos , Neoplasias Hepáticas , Camundongos , Plasmodium/imunologia , Plasmodium falciparum/genética , Plasmodium falciparum/fisiologiaRESUMO
The gene for the circumsporozoite (CS) protein of Plasmodium falciparum has been cloned and its nucleotide sequence determined. The gene encodes a protein of 412 amino acids as deduced from the nucleotide sequence. The protein contains 41 tandem repeats of a tetrapeptide, 37 of which are Asn-Ala-Asn-Pro and four of which are Asn-Val-Asp-Pro. Monoclonal antibodies against the CS protein of Plasmodium falciparum were inhibited from binding to the protein by synthetic peptides of the repeat sequence. The CS protein of Plasmodium falciparum and the CS protein of a simian malaria parasite, Plasmodium knowlesi, have two regions of homology, one of which is present on either side of the repeat. One region contains 12 of 13 identical amino acids. Within the nucleotide sequence of this region, 25 of 27 nucleotides are conserved. The conservation of these regions in parasites widely separated in evolution suggests that they may have a function such as binding to liver cells and may represent an invariant target for immunity.
Assuntos
Antígenos de Superfície/genética , Genes , Malária/imunologia , Plasmodium falciparum/genética , Proteínas de Protozoários , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Antígenos de Superfície/imunologia , Sequência de Bases , Epitopos/genética , Humanos , Fígado/parasitologia , Plasmodium/genética , Plasmodium falciparum/imunologiaRESUMO
A micro enzyme-linked immunosorbent assay utilizing antigen dotted onto nitrocellulose filter discs (Dot-ELISA) was developed for the rapid diagnosis of visceral leishmaniasis. Leishmania donovani promastigotes applied to filter discs in volumes of 1 microliter were placed in 96-well microtiter plates, blocked with bovine serum albumin, then incubated with 4-fold dilutions of patient sera. After incubation with peroxidase-conjugated anti-human antibody, washing and addition of precipitable substrate, positive reactions appeared as blue dots on a white background which were easily read by eye. The procedure is performed at room temperature, takes about 2 h and is economical. At a reciprocal diagnostic titer of greater than or equal to 32, 41 of 42 (98%) leishmaniasis patients were positive, and positive titers ranged from 512 to 524,288. Control sera from healthy individuals showed 1 of 50 (2%) false positive reactions. Sera from patients with African trypanosomiasis, Chagas' disease, and lupus erythematosus were cross-reactive in the Dot-ELISA. No cross-reactivity was noted with sera from patients with amebiasis, coccidioidomycosis, cutaneous leishmaniasis, viral hepatitis, hydatidosis, malaria, schistosomiasis, syphilis, toxoplasmosis or trichinosis. In replicate experiments, 90% of 167 sera tested did not vary in titer. This rapid and inexpensive test should prove to be an important field diagnostic technique for visceral leishmaniasis.
Assuntos
Leishmaniose Visceral/diagnóstico , Complexo Antígeno-Anticorpo , Antígenos/análise , Técnicas de Laboratório Clínico , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina G/análise , Leishmaniose Visceral/imunologia , MicroquímicaRESUMO
Using the cloned circumsporozoite (CS) protein gene of a Brazilian strain of Plasmodium falciparum as probe, we have analyzed the structure of the CS protein gene from 17 other Asian, African, Central and South American parasite strains by nucleic acid hybridization. Each strain appears to have one CS protein gene which hybridizes readily to the Brazilian strain probe. The 5' and 3' thirds of the genes are invariant in size in all 18 strains whereas the central third containing the 12 base pair tandem repeats varies in size over a range of about 100 base pairs. Several differences were found in the locations of Sau3A sites in the genes. The Sau3A sites are significant because each of the minority Asn-Val-Asp-Pro repeats in the cloned gene has a Sau3A site. DNA melting of hybrids revealed a high degree of homology between the sequences of the cloned gene and genes from an Asian strain and an African strain. A 14 base oligodeoxynucleotide with a sequence from the central repeat region hybridized to all strains tested. We conclude that the CS protein gene is highly conserved among strains of P. falciparum and that malaria vaccine development with the CS protein is unlikely to be complicated by strain variation.
Assuntos
Antígenos de Superfície/genética , Genes , Plasmodium falciparum/genética , Proteínas de Protozoários , África , Animais , Ásia , Sequência de Bases , América Central , Mapeamento Cromossômico , Clonagem Molecular , DNA/isolamento & purificação , Eletroforese em Gel de Ágar , Hibridização de Ácido Nucleico , Plasmídeos , América do Sul , Especificidade da EspécieRESUMO
The Dot-ELISA, a rapid, visually read micro enzyme immunoassay for visceral leishmaniasis utilizing minute volumes of antigen "dotted" on nitrocellulose filter discs and precipitable chromogenic substrate, was analyzed under a variety of experimental parameters. Raising assay incubation temperatures from 23 degrees C to 28 degrees C resulted in titer increases in three of five leishmaniasis patient sera; at 37 degrees C, all five patient sera and one of five normal human sera showed titer increases. The amount of antigen used could be reduced 50% by incubating patient serum overnight at 4 degrees C. Antigen discs stored at - 20 degrees C were optimally reactive with leishmaniasis sera over a 270-day period. Antigen discs stored at 4 degrees C and 23 degrees C showed reproducible titer decreases at 90 days. Aging either peroxidase-conjugated antibody or substrate for up to 28 days at 4 degrees C did not adversely affect titers of positive and negative control sera and reagent controls. Activated substrate stored at 23 degrees C was optimally reactive in the assay for at least 24 hours. No changes in titers of positive and negative control sera or nonspecific reactions in reagent controls occurred when using different brands of microtiter plates. The long shelf lives and stabilities of Dot-ELISA antigen and reagents indicate this test should prove useful both in the laboratory and in the field.
Assuntos
Ensaio de Imunoadsorção Enzimática , Técnicas Imunoenzimáticas , Leishmaniose Visceral/diagnóstico , Antígenos/imunologia , Relação Dose-Resposta Imunológica , Humanos , Leishmania/imunologia , Leishmaniose Visceral/imunologia , Preservação Biológica , TemperaturaRESUMO
We report the characterization of Leishmania (L. infantum, L. donovani, and L. major) kinetoplast DNA (kDNA) by the use of restriction endonuclease digestion patterns and Southern hybridizations. Overall, the sizes and fragment patterns of MspI restriction endonuclease-produced DNA fragments vary from species to species. However, kDNA isolates from different species and strains cross-reacted to a great extent in Southern hybridization experiments. Only kDNA isolated from L. infantum and L. major had little homology during hybridization reactions. To prepare DNA probes that would differentiate between species of Leishmania, minicircle kDNA was digested with restriction enzymes and ligated to an E. coli plasmid. Several plasmids were isolated that specifically detect in hybridization experiments as few as 5 X 10(3) L. donovani or L. infantum promastigotes lysed on nitrocellulose filters.
Assuntos
Clonagem Molecular , DNA Circular , DNA Mitocondrial , Leishmania/classificação , Hibridização de Ácido Nucleico , Animais , Enzimas de Restrição do DNA , DNA Circular/genética , DNA de Cinetoplasto , DNA Mitocondrial/genética , Desoxirribonuclease HpaII , Escherichia coli/genética , Leishmania/genética , Leishmania/patogenicidade , Plasmídeos , Especificidade da EspécieRESUMO
Canine sera frequently become anti-complementary when heat-inactivated at 56 degrees C for 30 min, and generally cannot be used in standard complement-fixation (CF) assays. Therefore, a procedure was developed for decomplementing canine sera by absorption with particulate immune complexes consisting of sheep erythrocyte stroma optimally sensitized with anti-sheep erythrocyte antibody (hemolysin). Canine sera incubated for 20 min at 30 degrees C with sensitized stroma consistently showed less than 10% residual complement and were not anti-complementary. This decomplementation procedure was applied in a complement-fixation (CF) test for detection of serum antibodies during canine visceral leishmaniasis. Two groups of German shepherd dogs were injected intravenously with Leishmania donovani or L. donovani chagasi amastigotes, and the course of infections was followed for 12 weeks. Using freeze-thaw sonicate preparations of L. donovani parasites as antigen, reciprocal CF antibody titers above 24 were detectable in sera 7 weeks after infection and gradually increased to a maximum titer of 775 at 12 weeks. Sera from control dogs had mean titers of 24. This improved methodology enhances the potential of the CF test in the serodiagnosis of canine leishmaniasis.
Assuntos
Testes de Fixação de Complemento , Doenças do Cão/diagnóstico , Leishmaniose Visceral/veterinária , Animais , Anticorpos/análise , Complexo Antígeno-Anticorpo , Antígenos/imunologia , Proteínas do Sistema Complemento/imunologia , Cães , Técnicas de Imunoadsorção , Leishmania/imunologia , Leishmaniose Visceral/diagnósticoRESUMO
Of 16 patients with kala-azar treated with sodium stibogluconate (0.1 ml/kg body weight a day), one died on the 12th day of treatment and nine were cured by a 30-day course, although two subsequently relapsed. Extending the course cured a further five patients, and in one patient allopurinol was used in addition before a cure was achieved. Clinical and hematological recovery began within a few days of the start of treatment, but parasites continued to be seen in splenic aspirates for 3 weeks or more.
Assuntos
Gluconato de Antimônio e Sódio/uso terapêutico , Gluconatos/uso terapêutico , Leishmaniose Visceral/tratamento farmacológico , Índices de Eritrócitos , Feminino , Hemoglobinas/análise , Humanos , Quênia , Leishmania/efeitos dos fármacos , Leishmaniose Visceral/sangue , Leishmaniose Visceral/parasitologia , Contagem de Leucócitos , Masculino , Baço/parasitologiaRESUMO
Lymphocyte proliferative responses to the candidate malaria sporozoite vaccine antigen R32tet32 were evaluated in 29 patients with acute Plasmodium falciparum malaria, 20 convalescent patients, 11 nonimmune individuals, and 22 healthy residents of two endemic malarious areas in Thailand. The results indicate that 14 of 20 (70%) convalescent patients and 14 of 22 (64%) residents of endemic areas responded to the R32tet32 antigen. However, only 8 of 29 (28%) patients with acute P. falciparum malaria responded. When 4 of the convalescent patients who remained in a malaria-free area were restudied 5-10 months after the acute infection, they were either not responsive or their responses had greatly diminished. These findings show that sensitization to R32tet32 occurs following a natural P. falciparum infection, but the cellular immune response to sporozoite antigens may be short-lived and may be suppressed during acute P. falciparum malaria.
Assuntos
Antígenos de Protozoários/imunologia , Ativação Linfocitária , Malária/imunologia , Plasmodium falciparum/imunologia , Vacinas/imunologia , Doença Aguda , Animais , Humanos , Imunidade Celular , Vacinas Sintéticas/imunologiaRESUMO
The dot enzyme-linked immunosorbent assay (Dot-ELISA) was compared to the microscopic agglutination test (MA test) for the diagnosis of human leptospirosis. Of 177 sera from 68 soldiers who trained in the Republic of Panama, 102 sera were positive in the MA test and 93 of these sera were positive in the IgM-specific Dot-ELISA. Incidence of infection was 50 of 68 patients with the MA test and 48 of 68 in the IgM Dot-ELISA. Five MA test-positive sera were reactive only in the IgG-specific Dot-ELISA, suggesting previous exposure. All 21 infecting serovars of Leptospira interrogans, as determined by positive reactions in the MA test or culture of blood and urine specimens, were reactive in the Dot-ELISA. Of 75 sera negative in the MA test, 61 were nonreactive in the Dot-ELISA. However, 9 of these 14 Dot-ELISA-positive/MA test-negative sera were acute samples from patients whose later sera were MA test-positive. Positive reactions in the IgM Dot-ELISA occurred in 2 of 30 control, 4 of 10 Lyme disease, 1 of 11 relapsing fever, and 1 of 8 yaws sera; 10 syphilis patient sera were nonreactive. The IgM-specific Dot-ELISA appears to be sensitive and specific for the serodiagnosis of acute leptospirosis. In addition, this rapid test is inexpensive, simple to perform, utilizes minute volumes of killed leptospiral antigen and is easily adaptable to field use.
Assuntos
Ensaio de Imunoadsorção Enzimática , Técnicas Imunoenzimáticas , Doença de Weil/diagnóstico , Testes de Aglutinação , Anticorpos Antibacterianos/análise , Antígenos de Bactérias/imunologia , Humanos , Imunoglobulina G/análise , Imunoglobulina M/análise , Leptospira/imunologia , Leptospira interrogans/imunologia , Fatores de Tempo , Doença de Weil/imunologiaRESUMO
An enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody that recognizes a repetitive epitope on the circumsporozoite protein of Plasmodium falciparum was used in Kenya to assess malaria infections in Anopheles gambiae s.l. and An. funestus. The ELISA confirmed that 88% of 44 sporozoite-positive gland dissections were P. falciparum. The ELISA infection rate of 18.6% (n = 736) for individually tested mosquitoes for both species was significantly higher than the 10.4% (n = 537) salivary gland sporozoite rate determined by dissection. This difference was due to ELISA detection of medium and large sized oocysts on the midguts of infected mosquitoes which did not contain salivary gland sporozoites. From a series of 379 Anopheles that were cut at the thorax, ELISA tests on "head" and "body" portions showed that 29.5% of 95 positive mosquitoes contained circumsporozoite antigen in the body portion in the absence of salivary gland infections. This field evaluation demonstrates that the ELISA can most accurately be used to estimate sporozoite rates by cutting mosquitoes at the thorax and testing anterior portions.
Assuntos
Anopheles/parasitologia , Plasmodium falciparum/isolamento & purificação , Proteínas de Protozoários , Animais , Anticorpos Monoclonais , Antígenos de Protozoários/análise , Antígenos de Protozoários/imunologia , Antígenos de Superfície/análise , Antígenos de Superfície/imunologia , Ensaio de Imunoadsorção Enzimática , Quênia , Plasmodium falciparum/imunologiaRESUMO
Malaria was transmitted to six normal human volunteers by mosquitoes infected from cultured gametocytes of Plasmodium falciparum. This method, which offers advantages over other methods of infecting volunteers, will be useful for evaluating the efficacy of human malaria vaccines.
Assuntos
Malária/transmissão , Adulto , Anopheles/parasitologia , Células Cultivadas , Humanos , Insetos Vetores/parasitologia , Masculino , Plasmodium falciparum/crescimento & desenvolvimentoRESUMO
Leishmania braziliensis panamensis promastigotes, temperature-induced in vitro-cultivated amastigotes, Vero cell-derived amastigotes, and rodent lesion-derived amastigotes were evaluated as antigens in the indirect immunofluorescent antibody (IFA) test for American cutaneous leishmaniasis. Test sensitivity was determined using sera from 34 U.S. soldiers with leishmaniasis diagnosed by demonstrating parasites in their skin lesions. Sera were collected from 3 to 24 months after exposure to Leishmania. Positive IFA reactions among patient sera were 82% with promastigotes or lesion amastigotes, 79% with in vitro amastigotes, and 76% with Vero cell amastigotes (P = N.S.). Positive titers ranged from 1:8 to 1:128 using all antigens. Test specificity was determined with 30 sera from healthy individuals. False positive reactions ranged from 0-5% depending on the antigen and all titers were less than or equal to 1:8. Test cross-reactivity was assessed with 47 sera from patients with other diseases. Depending on the antigen, cross-reactions occurred with sera from patients with Chagas' disease, lupus erythematosus, malaria, toxoplasmosis and amebiasis. None of the antigens cross-reacted with sera from patients with viral hepatitis, coccidioidomycosis, syphilis, schistosomiasis, and trichinosis. In replicate experiments, 99-100% of the sera varied no more than +/- 1 titer dilution. As sensitivity, specificity, cross-reactivity, and reproducibility of the four antigens were statistically similar, promastigotes, which can be easily and economically cultured in large numbers in vitro are recommended for use in the IFA test for American cutaneous leishmaniasis.
Assuntos
Anticorpos/análise , Antígenos/imunologia , Imunofluorescência , Leishmania/imunologia , Leishmaniose/diagnóstico , Adulto , Reações Cruzadas , Estudos de Avaliação como Assunto , Humanos , Leishmania/crescimento & desenvolvimento , Leishmaniose/imunologia , Leishmaniose Mucocutânea/parasitologia , MasculinoRESUMO
The kinetoplast DNA (kDNA) from promastigotes of Leishmania responsible for Old and New World cutaneous and visceral disease was characterized to determine if species and strains causing similar or different diseases could be identified. Restriction enzymes were used to digest kDNA into fragments that were separated into characteristic banding patterns after electrophoresis in agarose or linear gradient polyacrylamide gels. Hybridization was conducted with a 32P-kDNA probe and kDNA fragments transferred from agarose gels to nitrocellulose paper. Leishmania species causing cutaneous diseases in the New and Old Worlds all had different kDNA digest patterns. Visceralizing Leishmania from the New and Old Worlds also had different kDNA restriction fragment patterns although Leishmania donovani parasites with similar fragment patterns were isolated from several humans from central Kenya. Nucleotide sequences were shared among kDNA networks from L. donovani, Leishmania d. chagasi, Leishmania d. infantum, Leishmania tropica, and Leishmania major as determined by hybridization with a 32P-kDNA probe from L. donovani. However, no hybridization was detected between the L. donovani 32P-kDNA probe and kDNA from Leishmania aethiopica or Leishmania braziliensis panamensis. Leishmania characterization results for the same isolates from the published literature were compared and kinetoplast DNA analysis was found to be one of the most sensitive procedures for species and strain identification.
Assuntos
DNA/genética , Leishmania/classificação , Leishmaniose Visceral/parasitologia , Leishmaniose/parasitologia , DNA/análise , Enzimas de Restrição do DNA , Eletroforese em Gel de Poliacrilamida , Humanos , Leishmania/genética , Leishmania/ultraestrutura , Organoides/ultraestruturaRESUMO
The immunogenicity of synthetic peptides representing the repeating portions of circumsporozoite proteins of Plasmodium sporozoites was greatly increased by complexing them to proteosomes via hydrophobic moieties added to their amino termini. Proteosomes have been used safely in people in the development of meningococcal vaccines and therefore proteosome-peptide vaccines are prime candidates for use against malaria.
Assuntos
Adjuvantes Imunológicos , Antígenos de Protozoários/imunologia , Malária/prevenção & controle , Proteínas de Protozoários , Vacinas Sintéticas/imunologia , Vacinas/imunologia , Animais , Anticorpos Antiprotozoários/biossíntese , Proteínas da Membrana Bacteriana Externa/imunologia , Humanos , Substâncias Macromoleculares , Oligopeptídeos/imunologia , Plasmodium falciparum/imunologia , Plasmodium vivax/imunologiaRESUMO
Allopurinol was used in the treatment of 10 patients with kala-azar. Of six patients who had previously failed to respond satisfactorily to Pentostam, "cures" were achieved in four. However, it was necessary to add Pentostam to the allopurinol in one, and another relapsed after apparent "cure" but again responded to allopurinol. The response of four patients who had had no previous treatment for kala-azar was less satisfactory.
Assuntos
Alopurinol/uso terapêutico , Leishmaniose Visceral/tratamento farmacológico , Adolescente , Adulto , Gluconato de Antimônio e Sódio/uso terapêutico , Criança , Pré-Escolar , Feminino , Hemoglobinas/análise , Humanos , Leishmaniose Visceral/sangue , Leishmaniose Visceral/parasitologia , Masculino , Baço/parasitologiaRESUMO
The dot enzyme-linked immunosorbent assay (Dot-ELISA), standard ELISA and the complement fixation (CF) tests were compared in the serodiagnosis of African visceral leishmaniasis (kala-azar). Assay sensitivity was determined using sera from 44 patients with parasitologically confirmed kala-azar. Using the Dot-ELISA, 42 of 44 patients (95%) were positive at a reciprocal titer of greater than or equal to 32 (titer range 512-524 288). In the standard ELISA technique, 43 of 44 patients (98%) were positive (titer range 32-32 768). At a reciprocal titer of greater than or equal to 8 in the CF test, 35 patients (80%) were positive, 1 (2%) was negative and 8 patients (18%) showed anticomplementary (AC) activity (titer range 8-2048). Specificity, determined using 33 sera from healthy individuals not living in endemic areas, was 97% in both the Dot-ELISA and the standard ELISA (32 of 33 sera); in he CF test, all sera were negative except 1 (3%) which showed AC activity. Sera from patients with Chagas' disease cross-reacted in the dot-ELISA up to a titer of 512. In the standard ELISA, cross-reactions occurred mainly using sera from patients with Chagas' disease, malaria and syphilis, and to a lesser extent with sera from amebiasis, schistosomiasis and trichinosis patients. Overall titer agreement in replicate experiments was highest in the Dot-ELISA (89%), followed by the standard ELISA (80%) and the CF test (72%).