The Future of Fungal Serology

The incidence of invasive fungal infections continues to grow. Early and rapid diagnosis is essential to prevent morbidity and mortality. The number of assays available for the detection of fungal antigens in human body fluids are increasing in number and becoming part of the basic diagnostic workup for many fungal infections. Detection of specific antibody has been an important component in the diagnosis of fungal infections. Complement fixation and immunodiffusion continue to be the gold standard for antibody detection but are complex to perform, require extensive expertise, and are mostly performed in reference labs. Newer assays are being developed to reduce turn-around time, but have not been fully evaluated. A challenge for improving serologic assays is to move from crude antigens and polyclonal antibodies to purified and/or recombinant antigens and monoclonal antibodies, while retaining good sensitivity and specificity. Recent developments using lateral flow methodology have provided novel point-of-care antigen assays requiring little technical expertise. Such innovative techniques will help to keep the future of fungal serology bright.     

A Recombinant Matrix Metalloproteinase Protein from Gnathostoma spinigerum for Serodiagnosis of Neurognathostomiasis

Neurognathostomiasis is a severe form of human gnathostomiasis which can lead to disease and death. Diagnosis of neurognathostomiasis is made presumptively by using clinical manifestations. Immunoblotting, which recognizes antigenic components of molecular mass 21 kDa and 24 kDa in larval extracts of Gnathostoma spinigerum (Gs 21/24), has high sensitivity and specificity for diagnosis of neurognathostomiasis. However, only very small amounts of the Gs 21/24 antigens can be prepared from parasites harvested from natural or experimental animals. To overcome this problem, we recently produced a recombinant matrix metalloproteinase (rMMP) protein from G. spinigerum. In this study, we evaluated this rMMP alongside the Gs 21/24 antigens for serodiagnosis of human neurognathostomiasis. We studied sera from 40 patients from Srinagarind Hospital, Khon Kaen University, Thailand, with clinical criteria consistent with those of neurognathostomiasis, and sera from 30 healthy control adults from Thailand. All sera were tested for specific IgG antibodies against both G. spinigerum crude larval extract and rMMP protein using immunoblot analysis. The sensitivity and specificity for both antigenic preparations were all 100%. These results show that G. spinigerum rMMP protein can be used as an alternative diagnostic antigen, in place of larval extract, for serodiagnosis of neurognathostomiasis.     

Advances and limitations in the early diagnosis of invasive yeast infections

In the last years, the main advances in the serological diagnosis of mycoses caused by yeasts have occurred in the area of antibody and (1-3)-beta-D-glucan detection. Commercialization of the Candida albicans IFA IgG test and detection of antibodies against recombinant antigens Hwp1 and enolase are the most important contributions to the first area. Detection of (1-3)-beta-D-glucan confirms its usefulness as a good marker for the diagnosis of invasive candidiasis. The most recent studies suggest that combination of two tests to detect antígen, antibodies, (1-3)-beta-D-glucan and DNA will be needed to optimize the diagnosis of systemic yeast infections.     

Antibody responses of experimentally infected lambs to antigens collected during in vitro maintenance of the adult, metacestode or oncosphere stages of Taenia hydatigena and Taenia ovis with further observations on anti-oncospheral antibodies

The antigenicity and specificity of crude antigens collected during the in vitro maintenance of Taenia hydatigena and T. ovis, excretory/secretory (ES) antigens, were assessed in a peroxidase microenzyme-linked immunosorbent assay (ELISA), using sera from lambs given experimental monospecific infections with T. hydatigena, T. ovis, Echinococcus granulosus or Fasciola hepatica. ES antigens of larval cysts of T. ovis and T. hydatigena were less reactive than those of adult or oncosphere stages. Strong interspecific cros-reactions occurred between all antigen preparations, and these antigens offered no better specificity than crude somatic extracts. IgG1 was the major immunoglobulin detected in sera from lambs experimentally infected with T. ovis or T. hydatigena using antigens prepared from sonicated oncospheres. Discrete peaks of anti-oncospheral antibodies were detected following initial and challenge infections with eggs (whether the homologous or heterologous species), when sera were assayed with a PBS sonicate or an ES antigen from oncospheres. However, when oncospheres solubilised with sodium deoxycholate were used, the antibody response was prolonged and resembled that reported previously when somatic extracts of adult and metacestode stages were used as antigen. The results showed that oncospheres share antigens in common with other life-cycle stages, but also support the notion that they may possess some unique stage-specific antigenic determinants.     

Demonstration of antibodies during experimental toxocarosis using antigenic extract prepared from the adult parasit]

The detection of antibodies in subjects with visceral larva migrans syndrome caused by Toxocara canis is usually carried out with antigenic larvae, whose preparation presents some difficulties. Results obtained using extracts prepared from adult parasites show that such antigens are quite suitable for the detection of antibodies present in visceral larva migrans syndrome. This phenomenon is easily understood when one considers that there are numerous antigenic points in common between the different stages of the evolution of this parasite.     

Attempt to isolate from Ascaris suum an antigenic fraction for use in the immunologic diagnosis of onchocerciasis

For the purpose of immunological diagnosis of onchocerciasis, the authors attempted to isolate an antigenic fraction from Ascaris suum which would only detect anti-Onchocerca volvulus antibodies. Chromatofocusing was done on a crude antigenic preparation from the ascaris. Immunoelectrophoresis showed that the fraction eluted between pH 5.6 and pH 4.9 contained antigens which reveal anti-Onchocerca antibodies; anti-Loa loa antibodies are revealed by the fraction eluted at pH less than 4. From the fraction which reveal anti-Onchocerca antibodies, affinity chromatography isolated a system in which three components were detected by electrofocusing at a Pi of about pH 5.5. The specificity and sensitivity of this system are being studied.     

Preparation and immunochemical properties of monoclonal antibodies to potato ring rot pathogen Corynebacterium sepedonicum

Five stable hybridoma lines producing monoclonal antibodies to Corynebacterium sepedonicum were obtained. The specificity of monoclonal antibodies obtained was characterized. Interactions of the antibodies with native cells and antigenic preparations from bacterial cell extracts were studied. The epitope specificity of these antibodies to their recognized antigens and the use of the antibodies in advanced immunodiagnostic assays are discussed.     

Production of Polyclonal Antibodies to the Recombinant Coat Protein of Citrus tristeza virus and their Effectiveness for Virus Detection

Citrus tristeza virus (CTV) is distributed worldwide and causes the most economically important virus diseases of citrus. Enzyme‐linked immunosorbent assay (ELISA) and/or immunoprinting have become an indispensable tools for large‐scale diagnosis of CTV worldwide. Several CTV detection kits are commercially available, based on either polyclonal or monoclonal antibodies developed against purified virus preparations. We have developed polyclonal antibodies to recombinant p25 CTV coat proteins (rCP) and determined their effectiveness for both trapping and as the intermediate antibody in double‐antibody sandwich indirect (DASI) ELISA. The p25 coat protein gene of three CTV isolates was amplified by RT‐PCR and further cloned and expressed in Escherichia coli cells. The rCP was injected into rabbits and goats for antibody production. Western blotting assays with the rCP CTV‐specific antibodies reacted positively with the homologous and heterologous rCP of the three CTV isolates and with the corresponding native coat protein present in crude sap extracts of CTV‐infected citrus tissue, but not with extracts from healthy tissue. The rCP antibodies from goat and rabbit reacted as both plate trapping and intermediate antibodies in DASI‐ELISA, discriminating healthy and CTV‐infected citrus, with optical density (OD₄₀₅) values in the range of 0.151–2.415 for CTV‐infected samples and less than 0.100 for healthy tissue. Commercially available anti‐CTV antibodies were used as a reference. Previous reports indicate that antibodies developed to recombinant antigens, including those of CTV, may not be functional for trapping the target antigens under non‐denaturing conditions. Our results showed the feasibility of CTV antibodies developed to the rCP for use as both trapping and intermediate antibodies in DASI‐ELISA, when the recombinant antigen was fractioned with polyacrylamide electrophoresis gel and further extensively dialysed against phosphate buffer saline prior to its use as immunogen.     

Immunohistochemical observation on the antigens inducing IgG and IgM antibodies against sparganum]

Localization and characterization of the antigenic components of sparganum which induced IgG and IgM antibodies in the host were studied by immunohistochemical techniques and SDS-PAGE and Western blotting. The antigen recognized by IgG antibody of rats or mice which were immunized by infection or injection of crude extracts of metacestodes of Spirometra erinacei, was located in the parenchyma of sparganum, especially at the cortex and around the calcareous corpuscles. The immunoreaction was demonstrated not only in the encysted fibrous wall of host but around the arterioles or venules in the connective tissue of host. The antigen recognized by IgM antibody of rats or mice was also observed in the parenchyma of sparganum and in the connective tissue of host. By 5-20% gradient SDS-PAGE and EIBT, we detected antigenic components by IgG and IgM antibodies of the rat or mouse immunized by infection or injection of crude extract of spargana. Twenty-three antigenic bands from crude extracts of spargana were recognized by IgG antibody and 15 components by IgM antibody of immunized rats. Out of the bands recognized by IgG and IgM antibodies, 15 were cross-reacted each other. Twenty components of excretory-secretory proteins from spargana were recognized by IgG, and 5 components by IgM antibody of immunized rats. By IgG and IgM antibodies of immunized mice, 16 components of crude extracts were recognized by IgG antibody and 9 components by IgM antibody. Twenty components of excretory-secretory preparation were recognized by IgG antibody and 5 components by IgM antibody. Thirteen components of crude extracts were cross-reacted by IgG antibody of rats and mice.     

New approaches for allergen-specific immunotherapy

Rates of allergic diseases such as asthma and rhinitis are on the rise as important health problems in every country of the world. Allergen specific immunotherapy with natural allergenic extracts is a treatment directed to changing the natural course of these diseases, and is a treatment that has reported beneficial effects in a majority of allergic patients. However, this treatment is difficult because of the complex composition of the extracts. The composition is difficult to standardize and, consequently, the risk of anaphylactic shock is increased; furthermore, sensitization can occur to other antigens present in the extract. Therefore, new allergen specific immunotherapy approaches are needed. Chemically defined and standardized antigens are more easily managed and provide a safer and more efficient treatment. Vaccines for immunotherapy have already been designed, based on recombinant allergens, variants (or peptides derived from them), that can be administrated alone or in combination with adjutants. Some of these preparations are indicated for facilitating the uptake and antigenic presentation by dendritic cells, or by targeting the mast cells and basophiles. Studies in vitro, in animal models and clinical trials in allergic patients, indicate that these preparations may provide protection against the allergen exposure and improve the symptoms by inducing the production of blocking antibodies of the IgE mediated response, production of regulator T cells and cytokines of Th1 profile.     

Antibodies isolated by using cloned surface antigens recognize antigenically related components of Legionella pneumophila and other Legionella species

We studied the antigenic cross-reactivity of surface proteins among various strains of Legionella pneumophila and other Legionella species by using a novel method of antibody purification. Anti-bacterial antibodies in hyperimmune sera were adsorbed to and eluted from the surface of recombinant E. coli cells that express individual L. pneumophila antigens on their surface. These affinity-purified antibodies were then used to probe protein immunoblots prepared from the test strains to detect cross-reactive domains. We found that antigenic proteins are generally conserved in all L. pneumophila serogroups. Although some of these antigenic domains are shared with members of other Legionella species, they are associated with proteins of different molecular mass. Our approach to the study of antigenic cross-reactivity has potential advantages over similar studies that use either monoclonal antibodies or monospecific antibodies prepared by immunization with purified antigens.     

Is there a role for antibody testing in the diagnosis of invasive candidiasis?

During the last decades, the use of antibody tests for the diagnosis of invasive mycoses has declined as a consequence of the general belief that they are insensitive and non-specific. However, there is a clear evidence that antibodies can be detected in highly immunodeficient patients (such as bone marrow transplant recipients), and that those antibodies are useful for the diagnosis. Antibody tests are currently in use as diagnostic tools for some primary mycoses, such as the endemic mycoses, aspergilloma, allergic bronchopulmonary aspergilosis and sporothrichosis. For invasive candidiasis, diagnostic methods must differentiate Candida colonization of mucous membranes or superficial infection from tissue invasion by this microorganism. Substantial progress has been made in diagnosis of invasive candidiasis with the development of a variety of methods for the detection of antibodies and antigens. However, no single test has found widespread clinical use and there is a consensus that diagnosis based on a single specimen lacks sensitivity. It is necessary to test sequential samples taken while the patient is at greatest risk for developing invasive candidiasis to optimize the diagnosis. Results obtained from a panel of diagnostic tests in association with clinical aspects will likely be the most useful strategy for early diagnosis and therapy.     

Diagnosis of Midwestern Endemic Mycoses

Histoplasmosis and blastomycosis are the two most common midwestern endemic mycoses. A history of exposure to the geographic areas in which these organisms occur is central to raising suspicion for an endemic fungal infection. For infection with these organisms, the diagnosis is definitively established by recovery of the organism in tissue or body fluids, which may take weeks. A rapid diagnosis can be made by finding the distinctive yeasts in tissues or body fluids. Antigen testing allows a presumptive diagnosis of these endemic fungal infections while awaiting culture results, but cross-reactivity between assays for Histoplasma and Blastomyces is routinely seen. Antibody tests provide supportive evidence for infection with either of these endemic mycoses but are less useful in immunosuppressed patients.     

Production and immunochemical characterization of monoclonal antibodies toCorynebacterium sepedonicum, the causative agent of potato ring rot

Five stable hybridoma lines producing monoclonal antibodies toCorynebacterium sepedonicum were obtained. The specificity of monoclonal antibodies obtained was characterized. Interactions of the antibodies with native cells and antigenic preparations from bacterial cell extracts were studied. The epitope specificity of these antibodies to their recognized antigens and the use of the antibodies in advanced immunodiagnostic assays are discussed.     

Serological potential for fungal identification

POLONELLI, L. & MORACE, G., 1989. Serological potential for fungal identification. Specific antigens are valuable for the identification of fungal cultures. Early attempts to immunoidentify fungi were hampered by heterogeneity of antigens, antibody preparations and use of improper serological procedures. In recent years, the double diffusion exoantigcn technique has proved to be the most effective method for immunological identification of mycelial fungus cultures. Additional advances in perfecting methods occurred with the adoption of improved reference antisera obtained either through absorption or by immunizing animals with selected immunoelectrophoretic arcs or precipitin bands (reference antigens). Preliminary studies have shown that serodiagnostically important antigens may be used for accurately and rapidly identifying hyaline as well as dematiaceous fungi. Agglutination techniques consisting oflatex particles sensitized with rabbit anti- Cryptococcus neoformans globulin or Candida monospecific antisera permit the detection of specific yeast antigens in a few minutes. In spite of the great success obtained with the antigen test methods, some limitations in these procedures are apparent. The major problem derives from the occurrence of extensive cross reactions among congeneric species.
Hybridoma technology permits the production of uniform and standardized antisera (monoclonal antibodies) reacting with species-specific or strain-specific antigenic determinants (Western blotting technique) and the availability of functional pure epitopes (affinity chromatography). The current value and limitations as well as further avenues for the advance of the different procedures are reported.     

Comparative immunochemical characteristics and serological activity of the antigens of Cysticercus tenuicollis and Taenia hydatigena

Data are given on immunochemical analysis and serological activity of different antigens from larval and imaginal forms of Taenia hydatigena. Considerable heterogeneity and close antigenic affinity of the parasite's extracts under study both between each other and with the host's proteins, excluding the antigens from T. hydatigena, which has no common components with the latter, are established. In the homologous system in each extract under study there were recognised no less than 5 to 9 antigenic components. It is shown, however, by the method of adsorption of heterologous antibodies that the number of specific antigens in each of them does not exceed 1 or 2. All antigens happened to be serologically active, but the highest diagnostic efficiency was shown by extracts from scolices of C. tenuicollis and T. hydatigena. Antiparasitic antibodies were followed by these antigens in the sera of experimentally infected sucking pigs from the 10th day of the infection. They reached their maximum level on the 24th day and then was observed a gradual fall of the titre of specific antibodies, the level of which by the 115th day did not actually differ from initial values. The highest sensitivity and specificity in the immunoenzyme reaction under experimental conditions was displayed by the extracts from scolices of C. tenuicollis.     

医学真菌菌株保藏方法概述

近年来,随着真菌感染的患者急剧增加,真菌病已经成为一个重要的公共卫生问题。对医学真菌流行病学、致病机理、耐药机理以及防治策略的研究尤为重要,而所有研究的基础都建立在对医学真菌的进行有效的保藏。医学真菌的保藏方法很多,包括传代法、蒸馏水法、冷冻干燥保藏法等,保藏方法的选择因实验室条件、菌种和研究要求不同而不同。本文对目前常用的几种医学真菌菌种保藏方法的优缺点及其应用等做了综述。     

Sero-diagnosis of invasive aspergillosis: Attempts to determine antigen and antibody relevance to infection

Attempt was made to define antigens and antisera which might prove useful in diagnosis of invasive aspergillosis in man. A convalescent antiserum (serum from rabbits after live infection withAspergillus fumigatus conidia) which might be more representative of immunological reaction to fungal growthin vivo, did not react in enzyme-linked immunosorbent assay with commercial antigens which are used at present in attempts to detect antibody response in systemic infections in man. However, this convalescent antiserum reacted with antigens from a range of fungal extracts. Antigens from young culture filtrates, in particular the 24h culture filtrate are advocated as the standard antigens for antibody detection using conventional immunoprecipitation techniques. For the detection of circulating antigens, the use of convalescent antiserum in enzyme-linked immunosorbent assay might be promising in the early diagnosis of invasive aspergillosis.     

Shared and non-shared antigens from three different extracts of the metacestode of Echinococcus granulosus

Hydatid cyst fluid (HCF), somatic antigens (S-Ag) and excretory-secretory products (ES-Ag) of Echinococcus granulosus protoscoleces are used as the main antigenic sources for immunodiagnosis of human and dog echinococcosis. In order to determine their non-shared as well as their shared antigenic components, these extracts were studied by ELISA-inhibition and immunoblot-inhibition. Assays were carried out using homologous rabbit polyclonal antisera, human sera from individuals with surgically confirmed hydatidosis, and sera from dogs naturally infected with E. granulosus. High levels of cross-reactivity were observed for all antigenic extracts, but especially for ES-Ag and S-Ag. Canine antibodies evidenced lesser avidity for their specific antigens than antibodies from human origin. The major antigenic components shared by HCF, S-Ag, and ES-Ag have apparent molecular masses of 4-6, 20-24, 52, 80, and 100-104 kDa, including doublets of 41/45, 54/57, and 65/68 kDa. Non-shared polypeptides of each antigenic extract of E. granulosus were identified, having apparent masses of 108 and 78 kDa for HCF, of 124, 94, 83, and 75 kDa for S-Ag, and of 89, 66, 42, 39, 37, and 35 kDa for ES-Ag.     

Immunochemical studies of Aspergillus fumigatus mycelial antigens by polyacrylamide gel electrophoresis and western blotting techniques

Differences were detectable among strains of the opportunist fungal pathogen Aspergillus fumigatus when water-soluble (WS) preparations were analysed by combined SDS-PAGE and Western blotting procedures. A wide range of molecules of apparent molecular masses from approximately 20 to greater than 100 kDa showed specific binding to antibodies raised in rabbits to A. fumigatus wall and cytoplasmic components. The ability to bind antibody was markedly reduced by treatment of these antigens with sodium periodate or with specific proteases or glucanases. Pretreatment of blotted antigens with either concanavalin A (ConA) or wheat germ agglutinin (WGA) did not, however, inhibit subsequent antibody binding. The antigens of subfractions prepared from a single strain of A. fumigatus WS material were also susceptible to periodate oxidation and enzymic hydrolysis. Slight cross-reactivity was apparent when crude preparations of cellular or culture filtrate antigens, used in this laboratory to detect antibodies to Candida albicans, Coccidioides immitis and Cryptococcus neoformans, were probed with hyperimmune rabbit antisera to A. fumigatus. Efforts were made to characterize the WS preparations of A. fumigatus, used as diagnostic antigens in many laboratories. The electrophoretically separated antigenic moieties were shown to be predominantly glycoproteins. Binding of cytoplasmic antigens to antibodies raised to wall material showed the presence of many common components in both wall and cytosol. Antiserum to wall components revealed most differentiation among A. fumigatus strains.