Potential for diagnosis of intestinal nematode infections through antibody detection in saliva.

The present study was the first to investigate the potential of saliva in community diagnosis of the major human intestinal nematode infections, Trichuris trichiura, Ascaris lumbricoides, and the hookworms. Immunoglobulin G (IgG) antibodies specific to parasite antigens were quantified in saliva samples of 187 individuals (all ages) from a St Lucian community, and 120 school-aged children from Tanga region, Tanzania, and relationships with current infection status (determined by numbers of parasite eggs in stool) were examined. For T. trichiura infection, the age relationships of parasite-specific salivary IgG antibodies mirrored those of infection intensity at the community level. Within both areas, children with current T. trichiura infection exhibited significantly higher anti-T. trichiura salivary IgG responses than uninfected children. Similar trends were apparent for A. lumbricoides and hookworm infections, though not to a level of statistical significance. Comparison of mean T. trichiura infection levels and antibody responses in age-matched children from St Lucia and Tanzania suggested that measurement of parasite-specific salivary IgG may have potential as a marker of transmission intensity at the community level.

Identification and characterization of excreted/secreted products of Trichuris trichiura.

This study provides the first description of the range and immunogenicity of proteins excreted and/or secreted by living T. trichiura adult worms following their recovery from the human large intestine. Metabolic labelling of T. trichiura excretory/secretory (ES) products with [35S]-methionine revealed a range of proteins with prominent components at 52-54 kDa, 35-45 kDa & 17 kDa. In contrast, the major component of unlabelled T. trichiura ES, somatic whole worm and isolated stichosome extracts, and of [35S]-methionine labelled somatic extracts, was present at approximately 47 kDa. Similarly, the major 43 kDa protein present in unlabelled T. muris ES, somatic worm extract and [35S]-methionine labelled somatic worm extract, was only weakly detected in labelled T. muris ES. Pulse chase experiments demonstrated that after 20 h, the 43 kDa was a prominent component of T. muris ES. These data suggest that the 43/47 kDa protein of Trichuris adult worms is not a major constituent of newly synthesized ES but is either synthesized at a slower rate than other proteins, or sequestered or stored, most likely in the stichocytes, before release. Immunoprecipitations using a range of sera from T. trichiura-infected individuals demonstrated that many of the ES components are immunogenic. Antibody responses were vigorous in children with intense infections and negligible in parasitologically negative children. There was marked heterogeneity in responses to a 17 kDa antigen, with the age profile of anti-17 kDa antibody levels reflecting age-dependent infection intensities at the population level.

Comparison of age-dependent antigen recognition in two communities with high and low Trichuris trichiura transmission.

A previous ELISA-based study using whole worm extract, compared age-antibody profiles in two communities with high and low levels of Trichuris trichiura transmission (Needham et al., 1992). This showed that specific IgG1 levels mirrored infection intensity at the population level, while IgA levels exhibited a weak trend to remain elevated in the adult age classes in the area of highest transmission. This was interpreted as preliminary evidence for IgA-mediated resistance in the population with greatest prior experience of infection. The present study extends this work to compare IgG1 and IgA isotype recognition of separated antigens by Western blot between the two communities. Comparison of age-dependent antigen recognition in the two communities shows that both qualitative and quantitative recognition by IgG1 antibodies is related to the current intensity of infection (as assessed by eggs per gram of faeces, epg). The magnitude of the IgA response to separated antigens of 16-17 kDa and 90 kDa exhibits a stronger trend to remain elevated in adults and to reflect the past experience of infection: IgA antibodies are present at significantly higher levels in adults from the high transmission area compared with those from the community with low levels of T. trichiura endemicity, despite infection levels in these age groups being of similar magnitude. This comparative study therefore, provides further evidence to support a role for IgA in acquired immunity to T. trichiura in areas of intense transmission.

Temporal changes in Trichuris trichiura infection intensity and serum isotype responses in children.

The present study describes Trichuris trichiura infections in a cohort of children over a period of approximately 5 years, and examines the relationships between changes in infection intensity and changes in parasite-specific isotype responses (measured by ELISA). The decrease in mean infection intensity with time was mirrored by time-dependent changes in IgG subclass responses, with IgA and IgE levels remaining relatively constant in the cohort. At the individual level, changes in infection intensity between the initial and final time-points correlated positively and significantly with changes in all isotype levels with the exception of IgA.

Immunoepidemiology of intestinal helminthic infections. 2. Immunological correlates with patterns of Trichuris infection.

The significance of parasite-specific serum and secretory immunoglobulin (Ig) isotype responses as determinants of Trichuris trichiura infection intensity in endemic communities is discussed. Comparison of age-dependent isotype responses and the age profiles of infection intensity in 2 endemic communities with markedly different levels of T. trichiura transmission suggest that serum IgA responses may reflect the accumulated past experience of infection and thus may be relevant in acquired immunity to T. trichiura and contribute to the age-convexity of infection intensity in areas of intense transmission. Preliminary analysis of data from a second community-based study shows that parasite-specific secretory IgA in saliva increases with age and correlates negatively with infection intensity, suggesting that secretory IgA may also be implicated in acquired immunity to this gut-dwelling nematode.

Age-dependency of serum isotype responses and antigen recognition in human whipworm (Trichuris trichiura) infection.

The present study examines the age-dependency of parasite-specific isotype responses and antigen recognition profiles of individuals within a Trichuris trichiura endemic community, in order to evaluate the significance of serum antibodies as determinants of observed age-related patterns of infection intensity. A high degree of individual heterogeneity is observed in isotype responses to separated T. trichiura antigens by Western blot. Recognition by IgG1 antibodies exhibits marked age-dependency. The age-profiles of IgG1 responses to selected antigens of 16-17 kDa and 90 kDa molecular weight reflect the age-related changes in current infection intensity at the population level. Similarly, mean age patterns of IgG2 responses to a 90 kDa antigen, and mean IgG4 responses to a 16-17 kDa antigen reflect mean infection levels. IgG3 responses are negligible, and for methodological reasons, both IgE and IgM specificities are not presented. IgA responses to separated antigens of 16-17 kDa and 90 kDa, exhibit age-profiles which may suggest the development of an IgA-mediated acquired resistance to T. trichiura with age. IgA levels remain elevated throughout early adulthood, when infection intensity levels markedly decrease, supporting the hypothesis that IgA antibodies may be significant in generating the convex nature of the age-infection profile of T. trichiura.

Immunodiagnosis of Strongyloides stercoralis infection: a method for increasing the specificity of the indirect ELISA.

Indirect enzyme-linked immunosorbent assay (ELISA) allows sensitive detection of serum immunoglobulin (Ig) G against a soluble extract of Strongyloides stercoralis infective larvae. In this study, 40/40 (100%) human strongyloidiasis sera had high levels of anti-S. stercoralis IgG, but 30/40 (75%) filariasis sera, and 12/40 (30%) necatoriasis sera also had higher levels than control sera from UK residents. In attempts to increase the assay specificity by absorption of cross-reactive IgG, the effectiveness of pre-incubation of sera with extracts of different parasitic nematodes was investigated. One hour of incubation with 20 micrograms/ml aqueous extract of Onchocerca gutturosa absorbed cross-reactive IgG in most filariasis and necatoriasis sera, reducing the proportion with IgG levels above the positivity threshold by more than one-half. Preliminary results suggest that absorption with extracts of other filarial nematodes is equally effective, and that some of the cross-reactive IgG is directed against phosphorylcholine. Cross-reactive IgG in most necatoriasis sera was effectively absorbed with 20 micrograms/ml extract of Necator americanus. Cross-reactive IgG was not effectively absorbed with an extract of Ascaris lumbricoides. Absorption of cross-reactive IgG is an effective means of increasing the specificity of the indirect ELISA, for use in the immunodiagnosis and immuno-epidemiology of S. stercoralis infection.

The relationship between Trichuris trichiura transmission intensity and the age-profiles of parasite-specific antibody isotypes in two endemic communities.

The present study compares parasite-specific antibody responses in two Caribbean communities with high and low levels of Trichuris trichiura transmission. The age-dependency of antibody levels suggest that IgG1 and IgG2 levels relate to the current intensity of infection (as assessed by density of eggs in stool (e.p.g.) and reflect the age-intensity profile at the population level. IgG4, IgE and IgA levels persist into early adulthood and the subsequent decline is gradual. In the low transmission area, lower infection levels are reflected in lower parasite-specific antibody levels (of all isotypes) in the community as a whole. Despite a significantly greater past experience of infection in the high transmission area, antibody levels are not maintained at significantly higher levels throughout adulthood. The production of IgA appears to require a threshold for triggering, and a vigorous IgA response is maintained into early adulthood only in the high transmission village where peak intensity is greatest and the age-convexity of intensity is most marked. Experimental and theoretical studies focusing on the dynamic nature of host-helminth interactions in hosts exposed to high and low infection levels, and the putative role of acquired immunity, are discussed in relation to the data presented.

Age-dependency of infection status and serum antibody levels in human whipworm (Trichuris trichiura) infection.

This study examines the age-dependency of the relationships between human infection with whipworm (Trichuris trichiura) and parasite-specific antibody level measured by ELISA against an extract of adult worms after preincubation of the sera with Ascaris lumbricoides adult worm extract. The convex age-profile of parasite infection intensity is shown to be mirrored by an age-dependent change in age-class mean levels of IgG (all subclasses except IgG3), IgA, IgM and IgE. Mean antibody levels rise with increasing acquisition of infection in childhood and decline as the intensity of infection falls in adulthood. Immunoblot analysis of selected sera from different age-classes indicates that antigen recognition is similarly dependent on infection intensity. In individual children, antibody levels correlate positively with acquisition of infection, consistent with a simple model of antigen dosage specifying the magnitude of the humoral immune response. In adults, IgG4 correlates positively and IgA negatively with intensity of infection, suggesting involvement of these isotypes in functional roles of immune blockade or effector mechanisms, respectively.

Humoral immune responses in human infection with the whipworm Trichuris trichiura.

The humoral immune response to infection with Trichuris trichirua was investigated by ELISA and immunoblotting using human sera from the Caribbean island of St Lucia. Immunoblot analysis of the degree of cross-reactivity with the related trichuroid Trichinella spiralis and with the other commonly co-existent nematodes, Ascaris lumbricoides and Toxocara canis, was carried out using selected sera. The IgM, IgA, IgE, and IgG subclass antibody levels were measured in ELISA using a detergent solubilized extract of adult T. trichiura. The IgG and IgE responses were highly Trichuris specific. Anti-T. trichiura IgM responses were totally cross-reactive with A. lumbricoides and were completely ablated by pre-incubation of sera with Ascaris antigen. The IgG response was predominantly of the IgG1 subclass with a minimal IgG3 response. Only 1 person out of 130 tested had a detectable IgG3 response. The IgG2 response appeared to be directed primarily against carbohydrate or polysaccharide antigens as pre-treatment of the ELISA plates with poly-L-lysine was necessary before a response could be detected. These data are the first demonstration of human isotypic responses to infection with T. trichiura.

Dissociation of antibody responses during human schistosomiasis and evidence for enhancement of granuloma size by anti-carbohydrate IgM.

Immunoglobulin (Ig) G and IgM antibody levels to soluble egg antigens (SEA), adult worm glycoproteins (AWGP), carbohydrate antigens (CHO) and cationic exchange fraction 6 (CEF6) were measured in serum specimens taken from Brazilian patients with acute, intestinal, hepato-intestinal and hepatosplenic schistosomiasis mansoni. The antibody levels varied among the groups, with the highest anti-egg antigen responses in the acute patients and the highest anti-adult worm responses in patients with chronic disease. The responses to the component parts of the egg antigens were dissociated, with anti-carbohydrate IgG and IgM responses being highest in the acute infection group and anti-CEF6 IgG responses being uniform among the clinical groups. The possibility of a direct role for anti-CHO antibody responses in egg-induced pathology was investigated using the mouse lung model. The anti-carbohydrate monoclonal antibody NIMP/M45 significantly enhanced granuloma formation. Mice given NIMP/M45 produced granulomas larger than those of naive mice or mice given an unrelated monoclonal antibody, and as large as those produced by mice which had been presensitized to egg antigens. The independent regulation of responses to egg antigens may indicate that such responses are minimized to reduce the pathological consequences of infection whilst allowing the development of protective anti-worm responses.

Human cysticercosis and intestinal parasitism amongst the Ekari people of Irian Jaya.

A random sample of 242 people showed that 42 had palpable cysts of Taenia solium. Faecal examination recovered eggs of T. solium in seven (3%), while Trichuris (83%), Ascaris (83%), hookworms (76%), Strongyloides stercoralis (10%) and Strongyloides sp. (29%), Entamoeba histolytica (14%), Entamoeba coli (22%), Entamoeba hartmanni (7%), Entamoeba polecki (7%), Balantidium coli (9%) and Dientamoeba fragilis (21%) were the most common other intestinal parasites encountered. ELISA tests, using antigens prepared from adults and eggs of T. solium and from cysticerci of T. saginata were not very sensitive, the last diagnosing less than half of known positives while still retaining good specificity.

The epidemiology of schistosomiasis in the later stages of a control programme based on chemotherapy: the Basrah study. 2. The serological profile and the validity of the ELISA in seroepidemiological studies.

An epidemiological study of Schistosoma haematobium infection in Al-maadan locality, considered to be the main remaining endemic focus in Basrah, southern Iraq was carried out. The association between the serological profile of the population, measured by the enzyme linked immunosorbent assay (ELISA), and various factors including current infection by urine examination, water contact pattern, past history of, and treatment for, schistosomiasis, and cercarial dermatitis was investigated. Further study of the serological data by the relative operating characteristic (ROC) analysis to assess the validity of the ELISA in detecting schistosomal infection, showed that current infection, past history of infection, age, cercarial dermatitis and category of household were significantly associated with the serological profile of the population. The analysis allowed quantification of the effects of past history of infection and cercarial dermatitis on the validity of the ELISA in detecting schistosomiasis.

The recognition of Schistosoma mansoni surface antigens by antibodies from patients infected with S. mansoni and S. haematobium.

Polypeptide surface antigens of Schistosoma mansoni recognized by schistosomiasis patients have been identified and their strain and species specificity investigated. Antibodies from individuals infected with S. mansoni were used in immunoprecipitation assays of 125I-labelled schistosomulum surface antigens. All individuals surveyed from St. Lucia strongly precipitated antigens of approximately Mr 38,000 to 32,000 and 20,000. These antigens were shown by two-dimensional gel electrophoresis to be the same as those recognized by experimentally immunized mice. Although individuals showed a highly heterogeneous response against total polypeptide antigens synthesized in vitro by cell-free translation of adult S. mansoni mRNA, all individuals recognized the same surface antigens. Immunoprecipitation with sera from patients infected with S. mansoni in many different parts of Africa resulted in generally the same antigens being precipitated, although a very high molecular weight antigen(s), not strongly recognized by the St. Lucian sera was also precipitated by most of the African patient sera. One serum from Ghana precipitated the high molecular weight antigen but not the low molecular weight antigens, raising the possibility of the existence of S. mansoni strain(s) exhibiting some diversity in surface antigens. The surface of S. mansoni schistosomula was found to bind strongly antibodies from individuals infected with S. haematobium, demonstrating that most surface antigens are cross-reactive. Immunoprecipitation demonstrated, however, that of the polypeptide surface antigens only the very high molecular weight antigen was recognized by anti-S. haematobium antibodies and that the 38,000 to 32,000 and 20,000 Mr antigens were species-specific. Immunoprecipitation of the polypeptide antigens derived from purified adult surface membranes demonstrated recognition of the same 32,000, 25,000 and 20,000 Mr antigens recognized by chronically infected mice. Again these antigens were found to be species-specific.

Specificity of surface molecules of adult Brugia parasites: cross-reactivity with antibody from Wuchereria, Onchocerca and other human filarial infections.

The extent of structural and immunological similarity between surface antigens of three species of Brugia filarial parasites was studied by lodogen-mediated surface labelling of adult worms of B. malayi, B. pahangi and B. timori. The close homology and cross-reactivity between these antigens reported in previous surface labelling studies with Bolton-Hunter reagent, was verified in this system. The surface antigens of adult B. pahangi are also recognised by antibody from patients with Wuchereria bancrofti, the major human lymphatic filariae, and by antibody to Loa loa and Mansonella perstans. Further experiments have begun to establish the boundaries of these cross-reactions: antibodies to nonfilarial nematodes such as Trichinella, Necator and Strongyloides does not recognise the adult surface antigens; however, although most anti-Onchocerca sera show little or no reaction to the major (29 kDa) surface antigen, there is consistent reactivity to the secondary 20 kDa antigen, and extensive recognition of a minor antigen of 15 kDa.

Hepatosplenic schistosomiasis in Kenya: an assessment of the enzyme-linked immunosorbent assay (ELISA).

Sera from 124 adult Kenyan patients with chronic splenomegaly and from 93 geographically matched controls without splenomegaly were tested for evidence of Schistosoma mansoni infection by enzyme-linked immunosorbent assay (ELISA). Ova of S. mansoni were detected on stool or rectal snip examination in 23.4% of all patients, whereas 57.3% had a positive ELISA. All patients with parasitological or histological evidence of schistosomal infection had a positive ELISA, and a negative test reliably excluded schistosomiasis. On the basis of liver histology, 23 patients (18.5%) were considered to have hepatosplenic schistosomiasis, of whom 17 (73.9%) had a positive stool or snip. The ELISA was positive in 47.5% of cases of non-schistosomal splenomegaly, and in 52.7% of apparently normal controls. This high seropositive rate in the latter two groups emphasizes that schistosomal infection does not signify disease, and limits the diagnostic value of the test in individual cases of splenomegaly. Marked tribal and, therefore geographical, differences were noted in the prevalence of infection.

Serodiagnosis of mansonian schistosomiasis with CEF6, a cationic antigen fraction of Schistosoma mansoni eggs.

Studies of the host-parasite relationship of Schistosoma mansoni in mice revealed that the parasite's eggs were toxic to liver cells of immunosuppressed hosts. Attempts to identify the toxic egg constituent led to the isolation of a fraction CEF6 from crude egg homogenate by cation exchange chromatography. CEF6 contains two cationic antigens, omega 1 and alpha 1, the former being recognized as the putative hepatotoxin. In an initial assessment of its serodiagnostic potential in enzyme immunoassay (ELISA), CEF6 yielded an enhanced degree of sensitivity and specificity when compared with crude egg homogenate, and a subsequent 'blind' inter-laboratory collaborative study confirmed that in certain circumstances the use of a purified fraction such as CEF6 may be advantageous. We here review the experimental work leading up to the isolation of CEF6, and summarize the results so far obtained from its use as a diagnostic aid in human schistosomiasis.

The stage-, strain- and species-specificity of a Schistosoma mansoni egg antigen fraction (CEF6) with serodiagnostic potential.

The immunological properties of CEF6, a cationic fraction of Schistosoma mansoni egg homogenate (SEA) containing two antigens, omega 1 and alpha 1, have been further investigated in two assays, immunoelectrophoresis (IEP) and enzyme immunoassay (ELISA). Although precipitating antibodies to antigen omega 1 were amongst the first to appear in mice with patient infections, IgG reactivity against CEF6 in ELISA trailed somewhat behind that of IgG activity against unfractionated SEA. Specificity of the two antigens in CEF6 to the egg stage of the parasite life-cycle was demonstrated by the failure of immunizations with cercarial or worm antigens to induce antibodies which reacted against either alpha 1 or omega 1 in IEP, or against CEF6 in ELISA. Mice infected with S. mansoni strains derived from geographically distinct areas, including the Caribbean, South America and Africa, produced antibodies which were reactive against CEF6 prepared from eggs of a Puerto Rican S. mansoni strain that had been maintained in the laboratory for many years. Of the different precipitating anti-SEA antibody species induced by the various S. mansoni strains in mice, those reactive against antigen omega 1 appeared to be present in highest titre. Sera from mice chronically infected with S. japonicum and S. haematobium failed to precipitate CEF6 in IEP and were less reactive with CEF6 than with S. mansoni SEA in ELISA. However, similar degrees of ELISA reactivity against S. mansoni CEF6 and SEA were given by sera from mice infected with S. bovis. The results support the notion that the antigens in CEF6 may be useful in the serodiagnosis of schistosomiasis mansoni infections.