Cecilia Thors

Impaired allergy diagnostics among parasite-infected patients caused by IgE antibodies to the carbohydrate epitope galactose-α1,3-galactose

BACKGROUND The carbohydrate epitope galactose-α 1,3-galactose (α-Gal) is abundantly expressed on nonprimate mammalian proteins. We have recently shown that α-Gal is responsible for the IgE binding to cat IgA, a newly identified cat allergen (Fel d 5). OBJECTIVE We sought to investigate the diagnostic relevance of IgE antibodies to Fel d 5 and α-Gal among parasite-infected patients from central Africa without cat allergy compared with patients with cat allergy from the same region. METHODS Sera from 47 parasite-infected patients and 31 patients with cat allergy were analyzed for total IgE and IgE antibodies against cat dander extract (CDE) by using the ImmunoCAP system. Inhibition assay was performed with α-Gal on solid phase-bound CDE. The presence of IgE specific for the major cat allergen Fel d 1, Fel d 5, and α-Gal was analyzed by means of ELISA. RESULTS Among the 47 parasite-infected patients, 85% had IgE antibodies against α-Gal (OD; median, 0.175; range, 0.102-1.466) and 66% against Fel d 5 (OD; median, 0.13; range, 0.103-1.285). Twenty-four of the parasite-infected patients were sensitized to CDE, and 21 of them had IgE antibodies to Fel d 5 and α-Gal. There was no correlation between IgE levels to CDE and rFel d 1 among the parasite-infected patients but a strong correlation between CDE and Fel d 5 and α-Gal (P < .001). Among the group with cat allergy, only 5 patients had IgE to α-Gal, and nearly 75% (n = 23) had IgE to rFel d 1 (median, 7.07 kU(A)/L; range, 0.51-148.5 kU(A)/L). In contrast, among the patients with cat allergy, there was a correlation between IgE levels to CDE and rFel d 1 (P < .05) but no correlation between CDE and Fel d 5 and α-Gal. CONCLUSION IgE to α-Gal causes impaired allergy diagnostics in parasite-infected patients. Screening for IgE to rFel d 1 and other allergens without carbohydrates might identify patients with true cat sensitization/allergy in parasite-infested areas.

Web-Based Virtual Microscopy for Parasitology: A Novel Tool for Education and Quality Assurance

Background The basis for correctly assessing the burden of parasitic infections and the effects of interventions relies on a somewhat shaky foundation as long as we do not know how reliable the reported laboratory findings are. Thus virtual microscopy, successfully introduced as a histopathology tool, has been adapted for medical parasitology. Methodology/Principal Findings Specimens containing parasites in tissues, stools, and blood have been digitized and made accessible as a “webmicroscope for parasitology” (WMP) on the Internet (http://www.webmicroscope.net/parasitology).These digitized specimens can be viewed (“navigated” both in the x-axis and the y-axis) at the desired magnification by an unrestricted number of individuals simultaneously. For virtual microscopy of specimens containing stool parasites, it was necessary to develop the technique further in order to enable navigation in the z plane (i.e., “focusing”). Specimens were therefore scanned and photographed in two or more focal planes. The resulting digitized specimens consist of stacks of laterally “stiched” individual images covering the entire area of the sample photographed at high magnification. The digitized image information (∼10 GB uncompressed data per specimen) is accessible at data transfer speeds from 2 to 10 Mb/s via a network of five image servers located in different parts of Europe. Image streaming and rapid data transfer to an ordinary personal computer makes web-based virtual microscopy similar to conventional microscopy. Conclusion/Significance The potential of this novel technique in the field of medical parasitology to share identical parasitological specimens means that we can provide a “gold standard”, which can overcome several problems encountered in quality control of diagnostic parasitology. Thus, the WMP may have an impact on the reliability of data, which constitute the basis for our understanding of the vast problem of neglected tropical diseases. The WMP can be used also in the absence of a fast Internet communication. An ordinary PC, or even a laptop, may function as a local image server, e.g., in health centers in tropical endemic areas.

Cross reacting antibodies against keyhole limpet haemocyanin may interfere with the diagnostics of acute schistosomiasis

The number of individuals catching schistosomiasis has increased with the popularity of ‘primitive tourism’ in Africa. Highly immunogenic material originating from the intestine of intravascular adult schistosomes gives rise to an antibody response making possible early identification of infected individuals using serology. Antibodies against gut associated antigens (anti‐GAA), detected by indirect immunofluorescence microscopy employing sections of adult worms as antigen may occur before the onset of egg production. In the present study we show that this well known schistosomiasis‐specific anti‐GAA staining reaction can be confused with a similar staining reaction with ducts of both male and female worms. Antibodies with duct reactivity were seen in sera both from schistosomiasis‐patients and patients with some other invasive worm infections. Cross reactive anti‐duct antibodies appear to have different specificity. One cross reactive antibody reacted with antigenic epitopes present in keyhole limpet haemocyanin (KLH). Anti‐duct reactivity could be inhibited by absorption with KLH. This was most obvious in the trichinellosis patient sera.

Thomsen-Friedenreich oncofetal antigen in Schistosoma mansoni : localization and immunogenicity in experimental mouse infection.

Our preliminary observation, that sera from schistosomiasis patients react with carcinomas, raised the possibility of antigenic cross-reactivity. We here extend this observation to show that mice experimentally infected with Schistosoma mansoni react with human urothelial and transitional bladder carcinomas and also with a gastric carcinoma cell line, AGS. To identify cross-reacting epitopes, we looked for the expression of carcinoma markers in schistosome worms and eggs using monoclonal antibodies against tumour antigens MUC1, Tn and TF (also known as the oncofetal Thomsen-Friedenreich antigen or T antigen). Immunohistochemical staining showed that the TF-epitope is present in adult intravascular S. mansoni worms and eggs deposited in tissues of infected animals. The localization of TF-immuno-reactive material in schistosomes was seen at the parasite surface between male and female worms and around trapped eggs in the liver. This localization is consistent with the antigen being secreted. Mice experimentally infected with S. mansoni, developed circulating antibodies against the TF-epitope (identified as Gal(beta1-3) GalNAc-O-R) as seen in ELISA using TF-expressing asialoglycophorin (AGP) as antigen. The observed anti-TF response in S. mansoni-infected mice reflects the complexity of host-parasite interactions in this infection.

Schistosomiasis in Swedish travellers to sub-Saharan Africa: can we rely on serology?

In the absence of egg excretion, laboratory diagnosis of recently acquired schistosomiasis is dependent on serology. 42 of 83 Swedish adventure tourists to sub-Saharan Africa had serum anti-schistosome antibodies indicating recent infection. There is little doubt regarding the specificity and sensitivity of serology for the demonstration of infection, but there is a need for alternative serological methods which could be more widely used than the standard immunofluorescence assay (IFA) for antibodies against gut-derived antigens (anti-GAA). We present results suggesting that 40/42 anti-GAA positive cases also react with keyhole limpet haemocyanin (KLH), a readily available commercial antigen. High anti-GAA titres were seen for more than 2 y despite treatment with praziquantel. Thus we are faced with several questions. How likely is it that positive serology means treatment failure? What is the risk involved in chronic infection? What is the prospect for monitoring treatment outcome by serology? We conclude that there is a need for better information on the risk of becoming infected, for improved methods for testing and for monitoring the therapeutic effects in adventure tourists.

Localization and Identification of Schistosoma mansoni /KLH-crossreactive Components in Infected Mice

KLH (Keyhole limpet hemocyanin) is highly immunogenic, and crossreactive epitopes occur widely in nature. In schistosomiasis, infected hosts generate antibodies reactive with KLH. This is of diagnostic importance but we lack detailed information on the immunogen-carrying molecules and their distribution in the worm. We used anti-KLH antibodies to localize cross-reacting epitopes in the various developmental stages of the parasite in experimental Schistosoma mansoni infection. The staining results show KLH crossreactivity in the life stages of the parasite. By immunoblotting we show that KLH-crossreactive antigenic epitopes in the parasite eggs are carbohydrates, also recognized by antibodies against soluble schistosome egg antigens. The localizations in the larval stages and in adult worms suggest that crossreacting antigenic epitopes are secretory products.

Schistosome antigen gp50 is responsible for serological cross-reactivity with Trichinella spiralis.

The 50-kDa component (gp50) present in Schistosoma mansoni eggs and secretions of the various life stages of the parasite was recognized by experimentally infected mice and by humans with S. mansoni, Schistosoma haematobium, and Schistosoma japonicum infection. All sera reacting with crude S. mansoni-soluble egg antigens (SEA) also reacted strongly with gp50 in enzyme-linked immunosorbent assay. No reactivity against gp50 was seen with sera from individuals without schistosomiasis, with the exception of sera from patients with Trichinella spiralis infection. All of 10 sera from patients with trichinellosis also reacted with schistosomes by immunofluorescence essentially recognizing testes, ovaries, ootype epithelium and ducts of the reproductive system. Cross-reacting antigens were seen in T. spiralis hypodermis, stichocytes and possibly germinal primordia using anti-gp50 monoclonal antibodies and anti-gp50-positive schistosomiasis patient sera. The results suggest that the anti-gp50 antibody response constitutes a significant part of the anti-SEA antibody response in infected individuals and is a major reason for the previously recognized serological cross-reactivity between T. spiralis and schistosome species.

ISOLATION OF AN SBA LECTIN-REACTIVE GLYCOPROTEIN (GP50) AND ITS IDENTIFICATION IN SCHISTOSOMA MANSONI LARVAL AND ADULT WORM SECRETIONS

A major 50-kDa soybean agglutinin (SBA)-reactive component present in extracts of Schistosoma mansoni eggs was isolated by SBA lectin affinity chromatography. In polyacrylamide gel electrophoresis (PAGE), the SBA-reactive component was seen as a 100-kDa polypeptide band that after reduction and alcylation was substituted by a 50-kDa component. This suggests that it occurs in native form as a dimer. Monoclonal antibodies produced against gp50 reacted with miracidial and cercarial secretions and with adult worm components including tegumental structures suggestive of a secretory function.

Expression of low molecular mass cytokeratins in oocytes of Schistosoma mansoni.

We studied the distribution of acidic 45 kDa keratin 18 and 40/42 kDa keratin 19 in Schistosoma mansoni, a trematode of medical importance in many tropical regions. The monoclonal antibodies which were produced against the cytoskeleton of mammary carcinoma cell line BT-20 recognized cytokeratins preferentially in parasite oocytes. As has been described in mammalian oocytes, the acidic cytokeratins were present in a nonfibrillar form. The two monoclonal antibodies also recognized testicular cells. No keratin immunoreactivity could be demonstrated by immunofluorescence microscopy at the larval stage, the miracidium. In immunoblotting, the molecular mass as determined by SDS-polyacrylamide gel electrophoresis of schistosome cytokeratins was about 15 kDa higher than that of the corresponding cytokeratins recognized by the monoclonal antibodies in BT-20 cells. The results suggest that acidic low molecular mass cytokeratins in trematodes have a phylogenetically conserved major function in oocytes which is unrelated to the documented cytoskeletal role in differentiated mammalian epithelial cells.

Evaluation of Serological Assays for Diagnosis of Onchocercosis

Microscopic analysis of skin snips is the most widely used diagnostic technique for onchocercosis in endemic countries. The invasive nature and low sensitivity of that procedure has called for alternative diagnostic methods for this disease. Presently, serological assays detecting filariosis in general are available for routine analysis. However, serological assays specific for onchocercosis are still not universally available. We have evaluated the performance of a dot blot assay (DBA) as a potential method for specific detection of onchocercosis. The DBA, which detects IgG, as compared with 2 IgG4-immunoblot assays, all employing Onchocerca volvulus antigen. Furthermore, an enzyme-linked immunosorbent assay (ELISA) and an immunofluorescence assay (IFA) based on antigens from Acanthocheilonema viteae and Brugia malayi, respectively, were included in the comparison. Samples from microfilariae-positive patients and negative controls from the onchocercosis-endemic country Ghana were analysed. The DBA was significantly more sensitive and specific than the IgG4-assays and the ELISA, respectively. Furthermore, the anti-filarial Ig was increased in patients 1 month post-ivermectin treatment. Sera from patients with suspected filariosis from different parts of the world were analysed using DBA, ELISA and IFA. Patients responding positively in the DBA (12%) had clinical symptoms compatible with onchocercosis whereas those positive in ELISA and IFA (53% and 48%, respectively) had various clinical symptoms. These results indicate that the DBA is more specific than and as sensitive as the ELISA and the IFA presently used for the diagnosis of onchocercosis.