Serodiagnosis of parasitic diseases.

In this review on serodiagnosis of parasitic diseases, antibody detection, antigen detection, use of monoclonal antibodies in parasitic serodiagnosis, molecular biological technology, and skin tests are discussed. The focus at the Centers for Disease Control on developing improved antigens, a truly quantitative FAST-enzyme-linked immunosorbent assay, and the very specific immunoblot assays for antibody detection is highlighted. The last two assays are suitable for field studies. Identification of patient response in terms of immunoglobulin class or immunoglobulin G subclass isotypes or both is discussed. Immunoglobulin isotypes may asist in defining the stage of some diseases. In other instances, use of a particular anti-isotype conjugate may increase the specificity of the assay. Monoclonal antibodies have played important roles in antigen purification and identification, in competitive antibody assays with increased sensitivity and specificity, and in assays for antigen detection in serum, body fluids, or excreta. Molecular biological technology has allowed significant advances in the production of defined parasitic serodiagnostic antigens.     

Presence of high concentrations of circulating Toxoplasma antigens during acute Toxoplasma infection in athymic nude mice.

In mice infected with an avirulent strain of Toxoplasma gondii, circulating Toxoplasma antigens were detectable in the sera during weeks 1 to 3 of infection by a simple agglutination test that uses latex particles coated with anti-Toxoplasma antibodies. An infection in athymic nude mice resulted in high agglutination titers in the anti-Toxoplasma antibody-coated latex particle test and the absence of anti-Toxoplasma antibodies in sera during the acute phase, suggesting that the detection of circulating Toxoplasma antigens is a good tool for the diagnosis of acute toxoplasmosis, especially in severely immunocompromised hosts.     

Use of three immunological techniques for the detection of Toxoplasma spIgA antibodies in acute toxoplasmosis.

AIMS--To assess the performance and efficacy of three immunological techniques for the detection of Toxoplasma specific IgA antibodies in acute toxoplasmosis. METHODS--The following techniques were used to examine 128 serum samples (51 cases of acute toxoplasmosis, 50 cases of heterologous infections, and 27 healthy controls): direct enzyme linked immunosorbent assay (ELISA), antibody capture ELISA, and antibody capture agglutination. RESULTS--Direct ELISA had a sensitivity of 98% and a specificity of 97%, antibody capture ELISA of 100% and 99%, respectively, and antibody capture agglutination had sensitivity and specificity of 100%. CONCLUSIONS--All three immunological techniques performed well with similar efficacy. Detection of Toxoplasma specific IgA antibodies is a useful diagnostic marker for acute toxoplasmosis.     

Pathogen-specific leptospiral proteins in urine of patients with febrile illness aids in differential diagnosis of leptospirosis from dengue

Leptospirosis and dengue are two commonly seen infectious diseases of the tropics. Differential diagnosis of leptospirosis from dengue fever is often difficult due to overlapping clinical symptoms and lack of economically viable and easy-to-perform laboratory tests. The gold standard for diagnosis is the microscopic agglutination test (MAT). In this study, the diagnostic potential of screening for pathogen-specific leptospiral antigens in urine samples is presented as a non-invasive method of disease diagnosis. In a study group of 40 patients, the serum was tested for anti-leptospiral antibodies by MAT and enzyme-linked immunosorbent assay (ELISA). Urine of these patients was screened for leptospiral antigens by ELISA using specific antibodies against LipL32, LipL41, Fla1, HbpA and sphingomyelinase. Group I patients (n = 23) were classified as leptospirosis-positive based on MAT and high titres of circulating IgM-specific anti-leptospiral antibodies. All of these patients excreted all five leptospiral antigens in the urine. The 17 MAT-negative cases included six patients with pyrexia of unknown origin (PUO; Group II) and 11 confirmed dengue patients (Group III). The latter tested negative for both serum anti-leptospiral antibodies and urinary leptospiral antigens. A salient outcome of this study was highlighting the usefulness of screening for urinary leptospiral antigens in disease diagnosis, as their presence confirmed leptospiral aetiology in two PUO patients. Immunoblots of urinary antigens identified well-defined bands corresponding to LipL32, HbpA and sphingomyelinase; the significance of the 42- and 58-kDa sphingomyelinase bands is discussed.     

Granulocyte antibody detection – the role of MPHA

Human neutrophil antigens (HNA) are involved in the pathogenesis of a variety of clinical conditions, such as neonatal immune neutropenia (NIN), refractoriness to granulocyte transfusions, alloimune neutropenia after bone marrow transplantation, febrile transfusion reactions, and transfusion-related acute lung injury (TRALI). The accurate detection of granulocyte antibodies is essential for the diagnosis and prevention of the clinical conditions in which these antibodies are involved, especially TRALI. The international granulocyte immunology workshop recommends the use of granulocyte agglutination test (GAT) and granulocyte immunofluorescence test (GIFT) for the screening of granulocyte antibody, and the use of monoclonal antibody-specific immobilization of granulocyte antigen (MAIGA) assay for the determination of the antibody specificity. Presently, however, no single technique available is enough to detect all clinically relevant granulocyte antibodies. Here, we describe the advantages and disadvantages of the mixed-passive hemagglutination (MPHA) assay in the general context of granulocyte serology.     

Clinical laboratory applications of monoclonal antibodies.

Monoclonal antibody (MAb) technology is well recognized as a significant development for producing specific serologic reagents to a wide variety of antigens in unlimited amounts. These reagents have provided the means for developing a number of highly specific and reproducible immunological assays for rapid and accurate diagnosis of an extensive list of diseases, including infectious diseases. The impact that MAbs have had in characterizing infectious disease pathogens, as well as their current and future applications for use in clinical microbiology laboratories, is reviewed. In addition, the advantages (and disadvantages) of the use of MAbs in a number of immunoassays, such as particle agglutination, radioimmunoassays, enzyme-linked immunosorbent assays, immunofluorescent-antibody assays, and immunohistology, are explored, including the use of these reagents in novel test system assays. Also, nucleic acid probe technology is compared with the use of MAbs from the perspective of their respective applications in the diagnosis of infectious disease agents. There is no question that hybridoma technology has the potential to alter significantly the methods currently used in most clinical microbiology laboratories.     

Enzyme-linked immunosorbent assays and developments in techniques using latex beads

Not surprisingly, most of the publications on enzyme immunoassays or latex agglutination tests in 1990 have been applications for specific antigens or antibodies. Nevertheless, a large number of studies of considerable interest to all users have been reported. In this short review, we have chosen to highlight investigations into: factors affecting assay performance; enzyme immunoassay design and statistical analysis; novel approaches in enzyme immunoassays and related techniques; immunohistochemistry; and latex agglutination tests.     

Techniques applicable to broncho-alveolar lavage fluid in hospital laboratory for the diagnosis of parasitic and fungal pneumopathies in immunosuppressed patients

Parasitic and fungal organisms which are likely to cause pulmonary infections in immunosuppressed patients can be detected in broncho-alveolar fluid (BAL fluid). Single and standard methods, such as direct examination of the pellet, eosine-methylene blue fast (RAL 555), cultures in usual mediums of mycology must be systematically applied to this sample and may help detect these organisms without further exploration. If the results are negative, more recent techniques can be used if they present a real asset: an easier reading and mostly an improved sensitivity. Such is the case of immuno-fluorescence assay with monoclonal antibodies for detection of Pneumocystis carinii, and inoculation of MRC5 fibroblast cell line in tissue culture for isolation of Toxoplasma. Fungal pulmonary infections diagnosis has not yet succeeded in benefiting from modern findings: latex tests proposed for the detection of circulating antigens are nor sensitive nor specific, except the "Crypto LA test". Considering the relatively frequent association with other infectious agents, the detection of a parasitic or fungal organism in the BAL fluid should not interrupt investigation of this sample; neither should it lead to hasty conclusions regarding the responsibility of this agent in acute pneumopathy. This role will have to be evaluated according to criteria which are different for each isolated organism.     

Diagnosis of neuroparacoccidioidomycosis by detection of circulating antigen and antibody in cerebrospinal fluid

Neuroparacoccidioidomycosis (neuroPCM) is the central nervous system infection by the fungus Paracoccidioides brasiliensis. Its diagnosis is a difficult task that depends on neuroimaging techniques such as computed tomography and magnetic resonance imaging. However, the detection of circulating P. brasiliensis antigens in body fluids by inhibition enzyme-linked immunosorbent assay (inh-ELISA) has provided encouraging results. In this study, 14 cerebrospinal fluid (CSF) and 11 serum samples of patients with neuroPCM were analyzed by inh-ELISA for detection of circulating glycoprotein antigens of 43 kDa (gp43) and 70 kDa (gp70). Circulating gp43 and gp70 antigens were detected in all CSF samples from patients with neuroPCM at mean concentrations of 19.3 and 6.8 mug/ml, respectively. In addition, both gp43 and gp70 antigens were detected in 10 of 11 serum samples analyzed at mean concentrations of 4.6 and 4.0 mug/ml, respectively. By immunodiffusion test, CSF samples were determined to be negative in 13 of 14 samples. The detection of anti-gp43 and anti-gp70 antibodies by conventional ELISA showed positive results for all CSF samples, with titers ranging from 1:50 to 1:51,200. Therefore, the high sensitivity of the inh-ELISA technique in detecting gp43 and gp70 antigens in the CSF of neuroPCM patients strongly indicates that this assay can be considered as a powerful diagnostic tool. In addition, the finding of anti-gp43 and anti-gp70 antibodies in CSF samples by conventional ELISA also seems to be a promising diagnostic method for this special modality of PCM.     

Characterization of sperm antigens reacting with human antisperm antibodies

Antibodies reacting with human spermatozoa have been detected by various immunological techniques in the sera of subfertile men. Different patterns of sperm agglutination are observed with different sera, either head-to-head, tail-to-tail, or tail-tip-to-tail-tip. Differences have been detected between the clinically relevant antibodies in spontaneously infertile males and the less important antibodies in males who have undergone reversal of vasectomy. It has been suggested that the variations in agglutination patterns are due either to different classes of antibody or to binding of antibody to different antigens. In the present study immunoblotting techniques were used to characterize the reactivity of solubilized sperm proteins with serum samples exhibiting different modes of sperm agglutination. This involved the electrophoretic transfer of proteins from SDS gels to nitrocellulose sheets followed by overlay with serum antibody. Using these techniques we have attempted to characterize the antigens of spermatozoa which react with sera from both spontaneously infertile and vasovasostomized men. The results showed that although antisperm antibodies bind to discrete and sperm-associated antigens, there is no substantial difference between the antigenic patterns observed with antibodies producing different types of sperm agglutination. Neither the antigens detected, nor the intensity of reaction showed significant differences although there was a tendency for head-to-head agglutinating antibodies to react more strongly with the higher molecular weight antigens. Moreover, although with sequential serum samples the patterns of agglutination may change, the antigenic pattern remains unchanged.     

Detection of Antibodies to Brucella Cytoplasmic Proteins in the Cerebrospinal Fluid of Patients with Neurobrucellosis

The diagnosis of human neurobrucellosis usually relies on the detection of antibodies to Brucella lipopolysaccharide (LPS) in cerebrospinal fluid (CSF) by agglutination tests or enzyme-linked immunosorbent assay (ELISA). Here we describe the detection of immunoglobulin G (IgG) to cytoplasmic proteins (CP) of Brucella spp. by ELISA and Western blotting in seven CSF samples from five patients with neurobrucellosis. While IgG to CP (titers of 200 to 12,800) and IgG to LPS (800 to 6,400) were found in the CSF of these patients, these antibodies were not detected in CSF samples from two patients who had systemic brucellosis without neurological involvement. The latter, however, had serum IgG and IgM to both LPS and CP. No reactivity to these antigens was found in CSF samples from 14 and 20 patients suffering from nonbrucellar meningitis and noninfectious diseases, respectively. These findings suggest that, in addition to its usefulness in the serological diagnosis of human systemic brucellosis, the ELISA with CP antigen can be used for the specific diagnosis of human neurobrucellosis.     

Urinary hydatid antigen detection by coagglutination, a cost-effective and rapid test for diagnosis of cystic echinococcosis in a rural or field setting

We describe here coagglutination (Co-A), a rapid slide agglutination test for the detection of hydatid antigen in the urine for the diagnosis of cystic echinococcosis (CE). Paired urine and serum samples were collected from 16 patients with surgically confirmed CE, 10 patients with ultrasound-proven CE, 14 patients with clinically diagnosed CE, 24 patients with various parasitic diseases other than CE, and 25 healthy control subjects. Co-A detected excreted hydatid antigen in the concentrated urine of 7 of 16 (43.75%) surgically confirmed cases, 6 of 10 (60%) ultrasound-proven cases, and 8 of 14 (57.14%) clinically diagnosed cases of CE. A false-positive reaction was observed with 12.50% of control urine specimens from patients with parasitic diseases other than CE and 12% of urine samples from healthy controls. The circulating antigen was detected in the serum in 13 of 16 (81.25%) surgically confirmed cases, 6 of 10 (60%) ultrasound-proven cases, and 13 of 14 (92.86%) clinically diagnosed cases of CE. False-positive reactions were observed with three sera (12.5%) from controls with other parasitic diseases. The low sensitivity of Co-A for detection of antigen in the urine of a patient whose serum was positive for the antigen is possibly due to low levels of antigen in the urine. Unlike the collection of blood for serum, which is an invasive procedure and also requires technical expertise and disposable syringes, urine can be collected easily and frequently without causing any inconvenience to the patient. Urine as a clinical specimen alternative to serum would be immensely useful in the diagnosis of CE, particularly in a rural or field setting. In such situations as well as in poorly equipped laboratories, Co-A has the potential to be used as a simple, rapid, and economical slide agglutination test for detection of urinary hydatid antigen in the diagnosis of CE.     

Longitudinal study of brucellosis in mice by immunoassay of lipopolysaccharide-related antigens in blood and urine

Immunoassays based on latex agglutination or enzyme labelling (ELISA) were devised for the detection of lipopolysaccharide (LPS) of Brucella abortus, or its degradation products, in biological fluids of infected mice. The agglutination of latex was measured by counting of the remaining non-agglutinated particles in an automated immunoassay analyser. LPS was assayed by agglutination with antibody-coated latex and by competitive inhibition of agglutination of LPS-coated latex by anti-LPS antiserum. The inhibition system was more sensitive for the detection of degradation products of LPS. Correlation between ELISA and agglutination inhibition immunoassay was excellent (r = 0.96). Degradation of LPS occurred during storage, particularly when the samples contained specific antibodies. It could be prevented by removing cells immediately after collecting blood samples and by heating or alkaline denaturation of plasma. CBA/H mice were infected with various doses [65-(65 x 10(6) cfu] of B. abortus biovar 3 cells and the course of infection followed by immunoassay of LPS-related antigens in serum and urine, and by titration of specific antibodies and non-specific circulating immune complexes. The concentration of LPS degradation products, assayed by the agglutination inhibition assay, was related to the severity of the infection, which was assessed by viable counts of B. abortus in the spleen. A close correlation was observed between the values of antigenaemia, the number of cfu (r = 0.97), and the inoculum size (r = 0.99 at day 28).     

The serodiagnosis of infection with Salmonella typhi

BACKGROUND/AIMS: The serodiagnosis of infection with Salmonella typhi, using the Widal agglutination assay, relies on patients' antibodies to the O = 9,12 lipopolysaccharide (LPS) antigens, H = d flagellar antigens, and the Vi capsular antigens. A Vi agglutination titre of > 1/40 has traditionally been regarded as indicative of recent infection with S typhi. In this study, 91 sera were used to assess the reliability of the Widal agglutination assay based on antibodies to the Vi antigens. METHODS: The Widal agglutination assay was carried out using protocols established by the Central Public Health Laboratory, Colindale. Antibodies to the Vi capsular antigen were detected using a standard preparation of S typhi, ViI Bhatnagar variant strain (S typhi, ViI). Sera used in the study comprised 73 from patients who were culture positive for S typhi, 10 from patients who were culture positive for other species of Salmonella not expressing a Vi antigen (namely, S javiana, S enteritidis, S typhimurium, S stanley, S saint paul, S bareilly, or S mbandaka), and eight from healthy blood donors. RESULTS: Agglutination titres of > or = 1/40 were detected to S typhi ViI in 69 of 73 sera from patients with typhoid, although 27 of these also agglutinated an unrelated control antigen. The Widal assay also detected significant amounts of agglutinating antibodies to S. typhi ViI in all eight control sera and seven sera from patients infected with S bareilly, S enteritidis, S javiana, S mbandaka, S saint paul, and S stanley. CONCLUSIONS: Agglutinating antibodies to the Vi antigen can be detected by the Widal assay, but even with the appropriate control antigens the results were unreliable. The serodiagnosis of infections with S typhi should be based on the detection of antibodies to both the O = 9,12 LPS antigen and the H = d flagellar antigen by immunoblotting, and should not use the Vi antigen-based Widal assay. Conclusions should be made in the light of patients' clinical details and any knowledge of previous immunisation for typhoid.     

Immunological agglutination kinetics of latex particles with physically adsorbed antigens

The influence of the properties of antigens and particles on the immunological agglutination kinetics of the antigen-coated latex particles was studied. Horse cytochrome c, hen egg-white lysozyme (HEL), bovine serum albumin (BSA), and Aspergillus sp. glucose oxidase were physically adsorbed onto the surfactant free latices of styrene-methacrylic acid (MAA) copolymer (P (S/MAA)) and polystyrene (PS). The initial rates of the immunological agglutination of these protein-coated particles initiated by the addition of antibodies were quantified by the absorbance change at a wavelength of 680 nm. The initial agglutination rates of the particles covered with smaller antigens were lower. This effect of the molecular size of antigens was larger in P(S/MAA), because small antigens are probably buried in the hydrous polymethacrylic acid layer on the surface of particles. Thus, both the molecular size of antigens and the surface properties of particles affect the sensitivity of the immunological agglutination. On the other hand, the dependence of the initial rate of the immunological agglutination on the ionic strength and pH was similar irrespective of antigen-particle systems. The initial agglutination rates were largest at an ionic strength of approximately 0.05 at pH 7.0 and decreased with increasing pH. This dependence of the sensitivity on the pH and ionic strength is attributed to the electrostatic interactions of particle-particle and antibody-particle.     

Diagnostic use of autoantibodies in myasthenia gravis

The diagnostic use of antibodies is dependent on sensitivity and specificity of the methods of antibody detection, which have been developed and improved over the years. Here, we review the different methods for the detection of acetylcholine receptor and muscle-specific kinase antibodies, which are, so far, the only antibodies recognised as pathogenic in myasthenia gravis (MG). Seronegative MG patients will benefit from more sensitive methods of antibody detection. The most recent developments in antibody detection assays, particularly those based on cells expressing target antigens, allow rapid and reliable identification of autoantibodies, improving the diagnosis and treatment of MG patients. The same approaches to antibody detection are now being applied to a wide range of other autoantigens and other autoimmune diseases.     

Development and Comparative Evaluation of a Plate Enzyme-Linked Immunosorbent Assay Based on Recombinant Outer Membrane Antigens Omp28 and Omp31 for Diagnosis of Human Brucellosis

Brucellosis is an important zoonotic infectious disease of humans and livestock with worldwide distribution and is caused by bacteria of the genus Brucella. The diagnosis of brucellosis always requires laboratory confirmation by either isolation of pathogens or detection of specific antibodies. The conventional serological tests available for the diagnosis of brucellosis are less specific and show cross-reactivity with other closely related organisms. These tests also necessitate the handling of Brucella species for antigen preparation. Therefore, there is a need to develop reliable, rapid, and user-friendly systems for disease diagnosis and alternatives to vaccine approaches. Keeping in mind the importance of brucellosis as an emerging infection and the prevalence in India, we carried out the present study to compare the recombinant antigens with the native antigens (cell envelope and sonicated antigen) of Brucella for diagnosis of human brucellosis by an indirect plate enzyme-linked immunosorbent assay (ELISA). Recombinant outer membrane protein 28 (rOmp28) and rOmp31 antigens were cloned, expressed, and purified in the bacterial expression system, and the purified proteins were used as antigens. Indirect plate ELISAs were then performed and standardized for comparison of the reactivities of recombinant and native antigens against the 433 clinical samples submitted for brucellosis testing, 15 culture-positive samples, and 20 healthy donor samples. The samples were separated into four groups based on their positivity to rose bengal plate agglutination tests (RBPTs), standard tube agglutination tests (STATs), and 2-mercaptoethanol (2ME) tests. The sensitivities and specificities of all the antigens were calculated, and the rOmp28 antigen was found to be more suitable for the clinical diagnosis of brucellosis than the rOmp31 antigen and native antigens. The rOmp28-based ELISA showed a very high degree of agreement with the conventional agglutination tests and promising results for further use in clinical screening and serodiagnosis of human brucellosis.     

Autoimmunity in paraneoplastic neurological syndromes

In patients with Paraneoplastic Neurological Disorders, the researcher detected several autoantibodies reacting with neuronal antigens and tumors; their characteristics supported the hypothesis that autoimmunity plays a part in these diseases and gave impetus to the study of these neurological disorders. The relationship between detection of anti-neuronal antibodies, clinical syndromes, and certain types of tumors suggested the utility of these antibodies as a new tool for clinical diagnosis, although their function in the pathogenesis of the various syndromes is still unclear. This paper intends to review the characteristics of the anti-neuronal antibodies so far identified, their correlation with clinical syndromes, and the function of antigens. In addition, the paper will offer some insights on the immunological mechanisms of neuronal damage and on treatment options.     

Anti-glomerular basement membrane vasculitis

Antiglomerular basement membrane disease (anti-GBM) is a rare life-threatening autoimmune vasculitis that involves small vessels and it is characterized by circulating autoantibodies directed against type IV collagen antigens expressed in glomerular and alveolar basement membrane. The typical clinical manifestations are the rapidly progressive glomerulonephritis and the alveolar hemorrhage. The diagnosis is usually confirmed by the detection of anti-GBM circulating antibodies. If not rapidly recognized, anti-GBM disease can lead to end stage kidney disease (ESKD). An early diagnosis and prompt treatment with immunosuppressive therapies and plasmapheresis are crucial to prevent a poor outcome. In this review, we discuss the primary form of anti-GBM (the so called Goodpasture syndrome) but also cases associated with other autoimmune diseases such as antineutrophil-cytoplasmic-antibody (ANCA) vasculitis, membranous nephropathy, IgA nephritis and systemic lupus erythematosus (SLE), as well as the few cases of anti-GBM vasculitis complicating kidney transplantation in the Alport syndrome.     

A comparison of the IFA and the ELISA for the demonstration of antibodies against schistosome gut-associated polysaccharide antigens in schistosomiasis

The applicability of two immunodiagnostic techniques was studied for the detection of antibodies against schistosome gut-associated polysaccharide antigens in human schistosomiasis mansoni: the immunofluorescent antibody reaction (IFA) using Rossman's fixed paraffin sections of adult worms and the enzyme-linked immunosorbent assay (ELISA) with a trichloroacetic acid soluble fraction of total adult worm antigens (AWA-TCA).With the IFA, gut-associated polysaccharide antigens could be demonstrated with an anti-IgM conjugate in a high percentage of the sera tested, although false-negative reactions were occasionally recorded. The use of an anti-IgG conjugate resulted in the demonstration of antibodies against additional antigens in the parenchyma of the worm and on the tegument. Specific IgM antibodies were present in higher concentrations in the sera from children than in those from adults.Using AWA-TCA as the antigen preparation in the ELISA, only antibodies against the circulating anodic antigen (CAA) could be demonstrated. Pretreatment of the ELISA-plates with poly-L-lysine to couple AWA-TCA was not necessary. The ELISA was sucessfully applied with anti-Ig, anti-IgG and anti-IgM conjugates. With anti-Ig conjugate the test was very sensitive and gave less false-negative reactions than the IFA. There was a significant difference between Ig, IgG, and IgM titres of children and adults. The use of an immunogalactosidase assay with a fluorogenic substrate in the ELISA, resulted in a test which was able to detect antibodies at ten times higher dilutions than with the immunoperoxidase assay.This investigation received financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases