RESUMEN
The success of the Guinea Worm (GW) Eradication Program over the past three decades has been tempered by the persistence of GW disease in a few African nations and the potential for a future resurgence in cases. Domestic dogs are now a major concern as a disease reservoir as large numbers of cases of canine GW disease are now reported each year, mainly along the Chari River in Chad. As a first step toward the development of a serologic assay for dogs, archived human plasma samples from dracunculiasis-positive donors from Togo were used to select adult female GW antigens for peptide sequencing and cloning. Eight protein sequences of interest were expressed as recombinant glutathione-S-transferase (GST) fusion proteins, and the most promising proteins were coupled to carboxylated microspheres for use in multiplex assays. A thioredoxin-like protein (TRXL1) and a domain of unknown function (DUF148) were assessed for total IgG and IgG4 reactivities using a panel of specimens from GW cases, uninfected donors, and individuals infected with various nematode worms, including Onchocerca volvulus. Both the DUF148-GST and the TRXL1-GST assays cross-reacted with O. volvulus sera, but the latter assay was always the more specific. The IgG4 and total IgG TRXL1-GST assays both had sensitivities > 87% and specificities > 90%. Maximum specificity (> 96%) was obtained with the total IgG assay when reactivity to both antigens was used to define a positive case. Given the good performance of the human assay, we are now working to modify the assay for dog assessments.
Asunto(s)
Anticuerpos Antihelmínticos/inmunología , Antígenos Helmínticos/inmunología , Dracunculiasis/diagnóstico , Dracunculiasis/veterinaria , Inmunoglobulina G/inmunología , Animales , Antígenos Helmínticos/genética , Western Blotting , Reacciones Cruzadas , Erradicación de la Enfermedad , Reservorios de Enfermedades/parasitología , Enfermedades de los Perros/diagnóstico , Enfermedades de los Perros/inmunología , Perros , Dracunculiasis/inmunología , Dracunculiasis/prevención & control , Dracunculus , Electroforesis en Gel de Poliacrilamida , Glutatión Transferasa/genética , Humanos , Onchocerca volvulus , Oncocercosis/diagnóstico , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Sensibilidad y Especificidad , Pruebas Serológicas , Tiorredoxinas/genéticaRESUMEN
Global eradication of the guinea worm (Dracunculus medinensis) is near, although perhaps delayed a little by the discovery of a transmission cycle in dogs. It is therefore an appropriate time to reflect on the severe impact of this infection on the life of the communities where it was endemic prior to the start of the global eradication programme in 1981. From 1971 to 1974, we conducted a series of unpublished studies on guinea worm in a group of villages in Katsina State, northern Nigeria, where the infection was highly endemic. These studies demonstrated the high rate of infection in affected communities, the frequent recurrence of the infection in some subjects and the long-standing disability that remained in some infected individuals. Immunological studies showed a high level of immediate hypersensitivity to adult worm and larval antigens but a downregulation of Th1-type T-cell responses to worm antigens. Freeing communities such as those described in this article from the scourge of guinea worm infection for good will be an important public health triumph.
Asunto(s)
Dracunculiasis/epidemiología , Dracunculus , Enfermedades Endémicas , Animales , Antígenos , Costo de Enfermedad , Personas con Discapacidad , Perros , Regulación hacia Abajo , Dracunculiasis/inmunología , Dracunculiasis/transmisión , Humanos , Hipersensibilidad/epidemiología , Nigeria/epidemiología , Recurrencia , Células TH1RESUMEN
Dracunculiasis is a promising candidate for eradication, but transmission of Dracunculus medinensis and recrudescence of the disease have been observed repeatedly. In the present investigation, the D. medinensis-specific cellular cytokine response profiles and the parasite-specific antibody subclass reactivity were evaluated in dracunculiasis patients at distinct states of infection. Analysis of the cellular cytokine response in dracunculiasis patients disclosed a D. medinensis antigen-specific depression of IFN-gamma production with patent D. medinensis infection, while the T helper type 2 cytokine IL-10 was similar in patent, post-patent and control individuals, and IL-5 production was always the highest in controls. In parallel, diminished IFN-gamma and IL-12 responses to antigens from Ascaris lumbricoides, Entamoeba histolytica and mycobacteria were observed in patent and post-patent dracunculiasis cases. The parasite-specific IgG(1) and IgG(4) subclass reactivity profiles corresponded with the D. medinensis infection state, and a clear distinction between patent and post-patent patients and controls was found. Overall a depressed cytokine release was observed with patent D. medinensis, which extended beyond the parasite-specific immune responsiveness. The detection of D. medinensis-specific IgG(1) and IgG(4) isotypes may help to distinguish newly exposed, patent and post-patent D. medinensis infections.
Asunto(s)
Anticuerpos Antihelmínticos/análisis , Citocinas/inmunología , Dracunculiasis/inmunología , Dracunculus/inmunología , Inmunoglobulina G/inmunología , Adolescente , Adulto , Anciano , Animales , Anticuerpos Antihelmínticos/sangre , Niño , Dracunculiasis/epidemiología , Femenino , Humanos , Inmunidad Celular , Masculino , Persona de Mediana Edad , Estadística como Asunto , Togo/epidemiologíaRESUMEN
Sera from individuals living in a dracunculiasis endemic area of northern Ghana were examined for circulating Dracunculus medinensis antigens by applying protocols previously developed for detection of circulating antigens in other helminth infections. Antisera from rabbits immunised with homogenized first stage D. medinensis larvae were used for antigen capture and detection in three different forms, namely non-treated, biotinylated and horseradish peroxidase (HRP)-labelled. Three different preparations of human sera were examined, namely non-treated, pre-treated with polyethylene glycol/ethylenediaminetetra-acetic acid (PEG/EDTA) for analysis of precipitated immune complexes, and pre-treated with trichloroacetic acid (TCA) for analysis of isolated glycoproteins. In both SDS-PAGE/Western blotting and ELISA, significant reactivity was observed between non-treated and treated rabbit-antisera on the one hand and non-treated and treated human sera on the other. However, no significant response differences were observed between sera obtained from individuals with dracunculiasis and non-endemic controls. The reasons are analysed and possible explanations presented. The study provided no evidence that D. medinensis-specific circulating antigens, detectable by relatively simple means, occur in infected individuals.
Asunto(s)
Antígenos Helmínticos/sangre , Dracunculiasis/diagnóstico , Dracunculus/inmunología , Adolescente , Adulto , Animales , Anticuerpos Antihelmínticos/biosíntesis , Anticuerpos Antihelmínticos/inmunología , Western Blotting , Cáusticos/química , Quelantes/química , Niño , Dracunculiasis/inmunología , Ácido Edético/química , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Femenino , Ghana/epidemiología , Humanos , Sueros Inmunes/inmunología , Masculino , Persona de Mediana Edad , Excipientes Farmacéuticos/química , Polietilenglicoles/química , Prevalencia , Conejos , Ácido Tricloroacético/químicaRESUMEN
To explore the possibility for development of an immunodiagnostic test for Dracunculus medinensis infections, the antibody responses (IgG1, IgG2, IgG3, IgG4 and IgE) to antigen preparations made from adult female worms (ADGW) and first stage larvae (LVGW) of D. medinensis were analysed. By sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western blotting, sera from individuals with patent D. medinensis infection reacted extensively and similarly with ADGW and LVGW over a broad molecular weight range (5-200 kDa). Sera from individuals infected with Onchocerca volvulus (the major cross-reacting infection) had a more pronounced reaction with ADGW than LVGW, and only with antigens of molecular weights above 23 kDa. These findings were used to design an enzyme-linked immunosorbent assay (ELISA) with high sensitivity and specificity for D. medinensis infection. The most promising results were obtained when detecting for specific IgG4. Thus, when using LVGW, the assay had a sensitivity of 83% and a specificity of 97%. Refining the antigen into a low molecular weight filtrate improved the sensitivity to 92%, and the sensitivity was further improved (to 96%) when combining the results from two or more antibody types measured simultaneously, without affecting the specificity. The results were encouraging for the prospects for developing an applicable immunodiagnostic test for patent D. medinensis infections based on detection of specific serum antibodies.
Asunto(s)
Anticuerpos Antihelmínticos/sangre , Antígenos Helmínticos/inmunología , Dracunculiasis/diagnóstico , Dracunculus/inmunología , Ensayo de Inmunoadsorción Enzimática , Adolescente , Adulto , Animales , Niño , Reacciones Cruzadas , Dracunculiasis/inmunología , Femenino , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Masculino , Persona de Mediana Edad , Peso Molecular , Oncocercosis/inmunología , Sensibilidad y EspecificidadRESUMEN
The specific serum IgG1, IgG4, and IgE responses to Dracunculus medinensis and the level of total IgE of individuals living in a highly endemic area of northern Ghana were measured by ELISA. Sera were obtained in the high transmission season from individuals with prepatent, patent, or postpatent infection as well as from individuals from the same endemic area who claimed to have never had a patent infection (i.e., endemic normal individuals). Individuals with prepatent or postpatent infections responded with a significantly lower mean level of specific IgG1 and IgG4 compared with individuals with a patent infection, and with a significantly higher mean level of specific IgG1 and IgG4 compared with endemic normal individuals. For specific IgE, no differences were found in the mean antibody level between the infection status categories. Individuals with a patent infection had a significantly lower mean serum level of total IgE compared with prepatent, postpatent, and endemic normal individuals. Endemic normal individuals had the highest mean level of total IgE. Furthermore, in all clinical categories, high responders for specific IgG1 and IgG4 generally had low levels of total IgE, whereas low responders for specific IgG1 and IgG4 generally had high levels of total IgE. A similar dichotomy, although less distinct, was observed between specific IgG1 and IgG4 on the one hand and specific IgE on the other. Thus, similar to what has been suggested for schistosomiasis and lymphatic filariasis, the relationship between the IgG subclasses and IgE appears to play a role in, or at least to reflect, a mechanism for protective immunity in dracunculiasis.
Asunto(s)
Anticuerpos Antihelmínticos/sangre , Dracunculiasis/inmunología , Dracunculus/inmunología , Adolescente , Adulto , Anciano , Animales , Niño , Dracunculiasis/epidemiología , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Inmunoglobulina G/clasificación , Masculino , Persona de Mediana EdadRESUMEN
The serum antibody responses (specific IgG1, IgG4, and IgE, and total IgE) to Dracunculus medinensis infection in humans from a highly endemic area of northern Ghana were examined regularly by ELISA over a period of one year in cohorts of individuals who developed a patent D. medinensis infection during the study period (actively infected category), or who claimed to have never had a patent infection (endemic normal category). The results were analyzed in relation to seasonality and time of patency of infection. For individuals in the actively infected category, a clear seasonal variation in the mean levels of specific IgG1 and IgG4 was found, with the highest levels late in the dry season and early in the rainy season, when transmission is high, and the lowest levels late in the rainy season and early in the dry season. Endemic normal individuals responded with low and fluctuating levels of specific IgG1 and with low and nonfluctuating levels of specific IgG4. For specific and total IgE, no seasonal variation was observed in any of the two infection status categories. In relation to time of patency of infection (only involving the category of actively infected individuals), the mean levels of specific IgG1 and IgG4 increased from two months before patency of infection, peaked during patency, and then gradually decreased for four months until a constant level was reached. No significant fluctuations in the levels of specific and total IgE were observed in relation to time of patency. The present study thus showed extensive variation in levels of D. medinensis-specific IgG1 and IgG4 (but not IgE) over time. Seasonal variations in antibody responses may also occur in other helminth infections, especially those with seasonal transmission, and these should be taken into consideration when interpreting the results of immunologic studies.
Asunto(s)
Anticuerpos Antihelmínticos/sangre , Dracunculiasis/inmunología , Dracunculus/inmunología , Adulto , Animales , Dracunculiasis/transmisión , Femenino , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Inmunoglobulina G/clasificación , Masculino , Persona de Mediana Edad , Estaciones del AñoRESUMEN
The serum antibody response (total, and isotypes IgG1, IgG4, IgM, IgA and IgE) to Guinea worm infection was examined in humans from a highly endemic area of northern Ghana by ELISA and SDS-PAGE/Western blot techniques using an adult D. medinensis antigen. Sera were obtained early and late in the peak transmission period, from persons with patent and postpatent infections, as well as from persons from the same endemic area who claimed never to have had Guinea worm infection. To observe for potential cross-reactions in the tests, sera were also obtained from areas with no transmission of Guinea worm from patients with hookworm, O. volvulus and W. bancrofti infections, and from non-infected controls. Sera from persons living in the Guinea worm endemic area reacted extensively with Guinea worm antigen in both tests, and large numbers of bands were produced in the Western blots (up to 35 identified for some sera). For most antibody isotypes, the ELISA absorbance values obtained with sera from the same individuals varied between the two transmission seasons, with the highest titres present towards the end of the peak transmission period. The mean antibody titres for persons in the patent and postpatent infection categories were not significantly different when sera were obtained at the same season of the year. Persons from the endemic area, who claimed never to experience patent infections, also had antibodies to Guinea worm, although at significantly lower mean levels than for the patent and postpatent categories. The highest specificity in the ELISA and the most homogenous Western blots were obtained when detecting for antibodies of the IgG4 isotype.
Asunto(s)
Anticuerpos Antihelmínticos/sangre , Dracunculiasis/epidemiología , Dracunculiasis/inmunología , Adolescente , Adulto , Anciano , Animales , Formación de Anticuerpos , Antígenos Helmínticos , Western Blotting , Niño , Dracunculus/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Ghana/epidemiología , Humanos , Isotipos de Inmunoglobulinas/sangre , Masculino , Persona de Mediana Edad , Población RuralRESUMEN
The Falcon assay screening test-enzyme-linked immunosorbent assay (FAST-ELISA) and the enzyme-linked immunoelectrotransfer blot (EITB) technique were used to test human sera with Dracunculus medinensis adult worm antigen in order to assess their potential value in the immunodiagnosis of dracunculiasis. The human sera used were from patients with prepatent and patent D. medinensis infections or from patients infected with other nematodes (Onchocerca volvulus and Loa loa) or trematodes (Schistosoma mansoni and S. haematobium), as well as uninfected Nigerian and Puerto Rican normal controls. In the FAST-ELISA, the sera from prepatent and patent dracunculiasis patients gave the highest absorbance values relative to normal human sera. The highest cross-reactivity was observed with onchocerciasis sera; no cross-reactivity was seen with sera from individuals with loiasis or schistosomiasis mansoni or haematobia. By the EITB, sera from dracunculiasis patients specifically recognized a 16 kDa protein (Dm 16) and antibodies to Dm 16 disappeared 2 months after worm extraction. Recognition of Dm 16 occurred from the late prepatent stage. A 17 kDa protein (Dm 17) was also recognized by dracunculiasis sera, but antibodies to Dm 17 disappeared more slowly and were present 1 year after recovery. The 16 kDa and 17 kDa antigens of D. medinensis may be useful in the immunodiagnosis of dracunculiasis.
Asunto(s)
Antígenos Helmínticos/inmunología , Dracunculiasis/diagnóstico , Dracunculus/inmunología , Ensayo de Inmunoadsorción Enzimática , Immunoblotting , Animales , Anticuerpos Antihelmínticos/sangre , Reacciones Cruzadas , Dracunculiasis/inmunología , Humanos , Loa/inmunología , Onchocerca/inmunología , Schistosoma haematobium/inmunología , Schistosoma mansoni/inmunologíaRESUMEN
Immunoelectroblotting and enzyme-linked immunosorbent assay were used to identify non-cross-reacting antigenic components of Dracunculus medinensis and the filarial worms Onchocerca volvulus, Loa loa, Wuchereria bancrofti, Brugia malayi, and Mansonella ozzardi. Parasite specific serodiagnostic ELISA systems for onchocerciasis and dracunculiasis were devised based on these findings. Phosphate buffered saline extracts of adult worms were passed through a column of monoclonal antibodies to phosphorylcholine (PC). Crude and PC-depleted extracts were reacted on ELISA plates with individual sera from subjects infected with a range of nematodes. Binding of total antibody (Ig) or IgG class antibody and IgG4 subclass antibody was revealed using goat antihuman-Ig-phosphatase conjugate, or appropriate mouse monoclonal antihuman-Ig-type-specific reagents, followed by goat antimouse-Ig-phosphatase conjugate. Specificity of ELISA was improved by restricting reaction to the host's IgG4 antibody subclass, and/or by removing PC determinants from crude antigens. In parallel immunoelectroblots, crude and PC-depleted extracts probed with pooled sera showed potentially useful diagnostic antigens, including a 12 kDa protein from D. medinensis and 14, 18, and 27 kDa proteins from O. volvulus. Two Onchocerca specific ELISA systems non-reactive with antibodies to D. medinensis were devised.