Fluid Antigen Research Articles

Detection of specific IgG, IgM, IgE and IgG subclass antibodies for serological diagnosis of human cystic echinococcosis

Summary An enzyme linked immunosorbent assay (ELISA), based on sheep hydatid cyst fluid antigen was used for the detection of specific antibodies of IgG, IgM, IgE and IgG subclass in the serum samples of 62 clinically and radiologically diagnosed cystic echinococcosis (CE) patients, 8 clinically suspected cases of CE, 25 other parasitic disease controls and 25 healthy controls. The diagnostic sensitivity in the clinically and radiologically suggestive cases (n = 62) for IgG antibody detection was highest (93 %), followed by IgE, IgG4, IgG1, IgG2, IgM and IgG3 with 89 %, 87 %, 85 %, 76 %, 70 % and 55 % respectively. The detection of specific IgE, IgG1 and IgG4 antibody showed the higher diagnostic sensitivity and specificity to the extent that they can be safely used as better substitute to IgG. Even though, the diagnostic sensitivity of IgG was highest (93%) but was less specific (88 %) due to the frequent non-specific reactions in the sera of patients with other parasitic infections and healthy controls. None of the sera samples from healthy controls gave non-specific reaction with IgE, IgG1 and IgG4 and there was a considerably reduced cross-reaction with these antibodies. The most discriminatory and specific antibodies found in this study belonged to IgE, IgG1 and IgG4; therefore, these antibodies may serve as useful diagnostic markers for CE.

Flow-Through Assay for Detection of Antibodies Using Protein-A Colloidal Gold Conjugate as a Probe.

Flow-through assay (FTA) is a rapid, simple-to-perform, cost-effective, and user-friendly diagnostic test for monitoring infections in non-laboratory settings. It is mostly applied for antibody detection. FTA employing protein-A colloidal gold conjugate to detect antibodies against porcine cysticerci using cyst fluid and whole cyst antigens of Taenia solium metacestode is described here. Antibodies in the serum are captured by an antigen spotted onto a nitrocellulose membrane mounted on a flow-through device that serves as the antigen capture matrix. The bound antibodies are visualized by the addition of protein-A colloidal gold conjugate, which imparts a pink color. The test can be completed within 3 min at room temperature without any instrumentation. The sensitivity and specificity of the FTA are in agreement with ELISA.

Neuroinvasive cryptococcosis in an immunocompetent patient with a negative spinal fluid cryptococcus antigen.

58-year-old man presented with headache, nausea, vomiting, and gait disturbance. Brain MRI showed meningeal enhancement and herniation. Serum Cryptococcus antigen was positive but spinal fluid antigen and cultures were negative. A cerebellar biopsy revealed nonencapsulated Cryptococcus. He completed antifungal therapy. Serum Cryptococcus antigen titer decreased. He had a full neurological recovery.

Evaluation of reactivity to Echinococcus spp. among rural inhabitants in Poland.

A group of 172 rural inhabitants from eastern Poland (68 males and 104 females, mean age 49.0 ± 12.0 years) was examined for the presence of antibodies against Echinococcus granulosus and Echinococcus multilocularis. A population of 38 healthy urban dwellers from the city of Lublin (17 males and 21 females, mean age 36.2 ± 9.6 years) were examined as a control group. Sera of 22 rural inhabitants (12.8%) reacted positively to Echinococcus granulosus hydatid fluid antigen in the screening test. A cross-reactivity was observed with two serum samples that tested positive in ELISA for E. granulosus. Three serum samples were tested positive for E. multilocularis using the Em2plus ELISA assay and also positive for Western blot. None of the members of control group showed the presence of a seropositive reaction to Echinococcus spp. The reactivity to Echinococcus spp. among rural inhabitants decreased with age and this correlation was statistically significant (R = -0.197151, p = 0.009535). The percentage of positive findings was the highest (50.0%) in the youngest age group (14-20). No significant correlations were found between responses to interview questions (possession of domestic and farm animals, contact with wild animals, eating unwashed berries, drinking unboiled water) and the presence of seropositive reactions to Echinococcus spp. The presented results seem to indicate that echinococcosis is still a current problem in Poland that should not be neglected and, moreover, indicates the need for improvement in the routine laboratory diagnostics of Echinococcus spp. by standardizing the ELISA and Western blot tests.

Cryptococcal meningitis successfully treated with voriconazole and low-dose amphotericin B

A 79-year-old patient with diabetes mellitus was diagnosed with cryptococcal meningitis based on detection of Cryptococcus neoformans from cerebrospinal fluid culture. After administration of liposomal amphotericin B, the patient promptly developed acute renal failure as a side-effect. We therefore switched treatment to voriconazole with low-dose liposomal amphotericin B. Cerebrospinal fluid cultures and antigen titer for Cryptococcus became negative 266 days after initiating voriconazole. The present case suggests that voriconazole might be useful as induction therapy to reduce the risk of renal insufficiency and achieve successful outcomes.

Humoral response and evolution of Echinococcus infection in experimentally infected sheep.

There is little information on the humoral response of sheep experimentally infected with Echinococcus granulosus. Thus, the objective of this study was to evaluate this response and measure its evolution. Doses of 10, 100, 1000 and 10000 E. granulosus eggs were prepared and inoculated via intraruminal puncture. Blood samples were obtained before inoculation and every 48 h after inoculation, until they became seropositive. Thereafter, they were taken monthly for the first year and then every three months until 1700 days of observation had been completed. An ELISA test, with total hydatid fluid antigen, was used for immunodiagnosis. The average optical density of the 12 inoculated sheep was found to be above the mean cutoff value 10 days after inoculation, went on increasing until 180 days after inoculation and remained above the cutoff level until the end of the observation period. This confirms that the antibody response of sheep to E. granulosus infection occurs before production of hydatid fluid and that activation, mobilization and establishment of oncospheres in the tissues generates a persistent response from the host's immune system.

Reprograming of gut microbiome energy metabolism by the FUT2 Crohn's disease risk polymorphism.

Fucosyltransferase 2 (FUT2) is an enzyme that is responsible for the synthesis of the H antigen in body fluids and on the intestinal mucosa. The H antigen is an oligosaccharide moiety that acts as both an attachment site and carbon source for intestinal bacteria. Non-secretors, who are homozygous for the loss-of-function alleles of FUT2 gene (sese), have increased susceptibility to Crohn's disease (CD). To characterize the effect of FUT2 polymorphism on the mucosal ecosystem, we profiled the microbiome, meta-proteome and meta-metabolome of 75 endoscopic lavage samples from the cecum and sigmoid of 39 healthy subjects (12 SeSe, 18 Sese and 9 sese). Imputed metagenomic analysis revealed perturbations of energy metabolism in the microbiome of non-secretor and heterozygote individuals, notably the enrichment of carbohydrate and lipid metabolism, cofactor and vitamin metabolism and glycan biosynthesis and metabolism-related pathways, and the depletion of amino-acid biosynthesis and metabolism. Similar changes were observed in mice bearing the FUT2−/− genotype. Metabolomic analysis of human specimens revealed concordant as well as novel changes in the levels of several metabolites. Human metaproteomic analysis indicated that these functional changes were accompanied by sub-clinical levels of inflammation in the local intestinal mucosa. Therefore, the colonic microbiota of non-secretors is altered at both the compositional and functional levels, affecting the host mucosal state and potentially explaining the association of FUT2 genotype and CD susceptibility.

Diagnosis of Neurocysticercosis by Computed Tomography: How Much More Do Magnetic Resonance Imaging and Serology, in addition, Contribute?

Background: In the developing world the leading cause of seizures and epilepsy is neurocysticercosis caused by the larvae of Taenia solium . Neurocysticercosis is diagnosed by Computed Tomography (CT), Magnetic Resonance Imaging (MRI) or by serological tests on serum or cerebro spinal fluid. We evaluated the role of conventional MRI and serum anti bodies to cysticercal vasicular fluid antigen in patients with seizures diagnosed as neurocysticercosis by CT. Methods: Plain and gadolinium-enhanced MRI of brain in 51 patients, and ELISA to detect IgG antibodies in the serum to cysticercal vesicular fluid antigen in 44 patients, were performed. Results: The lesions observed were predominantly Single enhancing CT lesions (SECTL) in 36(70.5%) of the 51 patients studied; they were confined to the brain parenchyma in 50(98%) of the patients. There was no statistically significant difference in the number of lesions detected by CT or MRI in the brain parenchyma. IgG antibodies to cysticercal antigen could be detected in 11(24.8%) of the 44 patients tested by ELISA. Conclusion: In the patient population studied, MRI did not contribute much in the diagnosis of neurocysticercosis over CT in terms of number of lesions detected. Serology was found to be useful in the diagnosis of neurocysticercosis in only about a quarter of patients diagnosed by imaging modalities. Key words: Neurocysticercosis, MRI, CT, Antibodies to cysticercal vesicular fluid antigen.

Sero-epidemiological value of some hydatid cyst antigen in diagnosis of human cystic echinococcosis.

Cystic echinococcosis (CE) is a severe zoonotic disease which affects both human and animals. The disease has a considerable economic and social impact, because it has numerous complications leading to important disabilities and even death. CE is a widespread chronic endemic helminthic disease caused by infection with metacestodes of tapeworm Echinococcus granulosus. This study was conducted to diagnosis human CE by hydatid cyst antigens from camels and sheep. Hydatid fluid and protoscoleces crude antigens corresponding to camel and sheep were resolute by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and the protein bands of different antigens were exposed to infected patients serum CE through western blot (WB) assay. The camel hydatid fluid antigen revealed five polypeptide bands of 18-98.8kDa by SDS-PAGE while sheep hydatid fluid antigen revealed four polypeptide bands of 20-100kDa. Immune reactive bands were obtained through WB ranged from 25 to 125kDa. The study showed prominent immune reactive bands of 92, 52.2 and 35.7kDa which may helpful in diagnosis of human CE.

Immunodiagnosis of sheep infections with Echinococcus granulosus: in 35 years where have we come?

W. K. Yong and D. D. Heath published in 1979 a seminal paper in the first issue of Parasite immunology describing their efforts to determine whether the arc 5 precipitin band, formed when test human serum is reacted against electrophoresed hydatid cyst fluid antigen, would be a suitable immunodiagnostic test for the identification of sheep infected with Echinococcus granulosus. Although they found antibodies to arc 5 in the sera of hydatid-infected sheep, the sera of some sheep harbouring Taenia ovis and T. hydatigena also precipitated the hydatid cyst fluid arc 5 antigen, so they concluded arc 5 antibodies were not suitable for the specific immunodiagnosis of E. granulosus infection in sheep in New Zealand. Subsequent work has shown that the existence of multiple infections with different taeniid species, antigenic cross-reactivity between these related parasites and the low level of specific antibody response to infection continue to hinder efforts to improve the diagnosis of hydatid infection in sheep and other natural intermediate hosts, thereby preventing the development of any practical test. In particular, the poor antibody response of ruminants to naturally acquired hydatid infection may prove an insurmountable barrier in future efforts to develop a reliable and accurate immunological test.

Determination of Immunodominant Antigens of Dicrocoelium Dendriticum by Hyperimmune Sera

Dicrocoeliasis caused by the small liver fluke Dicrocoelium dendriticum (syn. D.lanceolatum), which live in the bile ducts and gall bladder of wild and domesticated mammals, particularly livestock farming. Parasitological methods are not reliable enough for diagnosis of this trematode infection. In the present study, the sera from rabbits immunized with somatic and excretory-secretory antigens of D.dendriticum were assayed with immunoblot technique. Adult worms were collected from infected livers of sheep at a local abattoir. The excretory-secretory product and somatic antigens were prepared through homogenization and incubation of trematodes, respectively. Polyclonal antibodies were raised in rabbits after inoculation of the two antigens with emulsified Freunds adjuvant followed by booster injection. Comparative study between D.dendriticum antigens and those of Fasciola, cysticercus tenuicollis and hydatid cyst fluids antigens was carried out. SDS-PAGE of antigens was performed and for immunoblotting analysis, hyperimmune sera were obtained from immunized rabbits of case and control groups. Immunoblot analysis of rabbits antisera against somatic and excretory-secretory antigens of D.dendriticum were showed six and four protein bands ranging from 25 to more than 170 and 25 to 70 kDa molecular weights, respectively. However cross-reaction with other specific proteins was observed in Fasciola excretory-secretory, hydatid cyst and cysticercus tenuicollis fluid antigens. The results demonstrated that the 25-27 kDa proteins were strongly recognized by both hyperimmune sera and was common in both somatic and excretory-secretory antigens. According to the results of this study performed with two antigens of D.dendriticum shows that the 25-27 kDa polypeptide as immunodominant protein, could be considered for the immunodiagnosis of dicrocoeliasis and it probably induces protective immunity against Dicrocoelium infection.

Chapter 29 - Rabies

Rabies is an acute encephalomyelitis of humans and animals caused by rabies virus, which is usually transmitted by an animal bite. Rabies has a very rich history. Worldwide, dogs are the most important vector, whereas wild animals, especially bats, are the main threat to humans in North America. After a delay at the site of entry, rabies virus spreads through the nervous system by fast axonal transport. Rabies can be effectively prevented after a recognized animal exposure with wound cleansing and administration of rabies vaccine and rabies immune globulin. Rabies typically develops after an incubation period of 20–90 days. Rabies has encephalitic and paralytic forms of disease and rabies is almost universally fatal after clinical onset. Hydrophobia is a characteristic clinical feature of encephalitic rabies. Pathologic changes include the presence of eosinophilic inclusions called Negri bodies in the cytoplasm of neurons and inflammatory changes. Imaging studies may be normal and do not show specific abnormalities. An antemortem laboratory diagnosis can be made with the detection of rabies virus antigen or RNA in tissues (e.g., skin) and/or body fluids (e.g., saliva) and with serologic testing. There is no known effective therapy for rabies, although there have been rare survivors.

Anion Exchange Chromatography for Purification of Antigen B of Cystic Echinococcosis

Cystic echinococcosis or hydatidosis causes severe economic loss and public health problem to both human beings and livestock in many temperate and tropical areas of the world. Immuno diagnosis has been found to be useful not only in primary diagnosis but also for follow up of patients after surgical or pharmacological treatment in man. Various immune diagnostic tests for hydatidosis in man and animals have been attempted using hydatid cyst fluid antigens with varied sensitivity and specificity. However these assays using crude hydatid antigens have been non-specific due to cross reaction with Cysticercus, Coenurus and other helminthic infections. In order to overcome these difficulties various novel tests using purified antigens are essential for confirmative diagnosis of hydatidosis in man and animals. One of the subunit of antigen B, 8 kDa protein has been shown to be hydatid specific. Various types of chromatography have been used for purification of Hydatid antigens. However, Anion exchange chromatography had been used in this study to get more antigen B 8 kDa than other chromatographic methods.

Point-Counterpoint: β- d -Glucan Testing Is Important for Diagnosis of Invasive Fungal Infections

ABSTRACT Invasive fungal infections are a significant cause of morbidity and mortality in patients who receive immunosuppressive therapy, such as solid organ and hematopoietic stem cell transplant (HSCT) recipients. Many of the fungi associated with these infections are angioinvasive and are best diagnosed by visualizing the organism in or culturing the organism from deep tissue. However, obtaining such tissue often requires an invasive procedure. Many HSCT recipients are thrombocytopenic, making such procedure too risky because of potential bleeding complications. Additionally, positive blood cultures are rare for patients with angioinvasive fungal infections, making this diagnostic strategy of little value. Undiagnosed fungal infections in these patient populations are a significant cause of mortality. Prophylactic use of antifungal agents, such as the echinocandins, during periods of neutropenia or graft-versus-host disease may prevent some fungal infections but increase the risk for others. Detection of fungal antigens in body fluids, including cryptococcus capsular polysaccharide, histoplasma antigen, galactomannan, and β- d -glucan, is viewed as being clinically useful for at least the presumptive diagnosis of invasive fungal infections. β- d -Glucan is an attractive antigen in that it is found in a broad range of fungal agents, including the commonly encountered agents Candida spp., Aspergillus spp., and Pneumocystis jirovecii. Cross-reactions with certain hemodialysis filters, beta-lactam antimicrobials, and immunoglobulins, which raise concerns about false-positive tests, have also been described. As a result, the use of this testing must be closely monitored. In this point-counterpoint, we have asked Elitza Theel, who directs the Infectious Disease Serology Laboratory at the Mayo Clinic, to address why she believes that this test has value in the diagnosis of invasive fungal infections. We have asked Christopher Doern, Director of Clinical Microbiology at Children's Medical Center of Dallas, why he questions the clinical value of β- d -glucan testing.

Serodiagnosis of human hydatidosis with an ELISA developed based on antigens derived from sheep hydatid cysts and comparison with a commercial human ELISA kit

ObjectiveTo explore the serodiagnosis of hydatid cyst in human using different antigens of sheep (hydatid fluid, Somatic and Excretory/secretory antigens of protoscolex) by ELISA and compares this result with commercial human ELISA kit. MethodsOne hundred blood samples from patients with history of severe abdominal pain and eosinophilia were obtained. Ten serum samples were obtained from surgically and pathologically confirmed cystic echinococcosis patients from Mashhad university hospital as positive control and 5 serum samples from infant under one year old as negative control. Blood samples were centrifuged at 3 000μg at 20 °C for 15 min and sera were stored at −20 °C. First, these samples were tested for the presence of antibody by commercial human ELISA. Then, ELISA was developed on microplates coated with hydatid fluid, Somatic and Excretory/secretory antigens of protoscolex of sheep. ResultsThe results of this study as analyzed by Kappa test showed that, hydatid fluid antigen could be used as a precise source of detection in indirect ELISA test. ConclusionsHydatid fluid in comparison with Excretory-secretory and somatic antigens showed more compatibility agreement in kappa test which can be used for further studies in development of any ELISA test for diagnosis of human hydatidosis.

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.

Serodiagnosis of Echinococcosis by ELISA Using Cystic Fluid from Uzbekistan Sheep

According to increase of travel, the cases of imported echinococcosis have been increasing in Korea. The present study was undertaken to develop a serodiagnostic system for echinococcosis in Korea. For diagnosis of echinococcosis, the fluid of Echinococcus granulosus hydatid cysts was collected from naturally infected sheep in Uzbekistan. Also serum samples of infected patients who were surgically confirmed were collected in a hospital in Tashkent, Uzbekistan. According to the absorbance of 59 echinococcosis positive and 39 negative control serum samples, the cut-off value was determined as 0.27. The sensitivity and specificity of ELISA with hydatid fluid antigen were 91.5% and 96%, respectively. The antigen cross-reacted with the serum of some cysticercosis or clonorchiasis patients. However, immunoblot analysis on the cystic fluid recognized antigenic proteins of 7-, 16-, and 24-kDa bands in their dominant protein quantity and strong blotting reactivity. In conclusion, the present ELISA system using hydatid cyst fluid antigen from Uzbekistan sheep is sensitive and specific for diagnosis of echinococcosis cases.

Evaluation of ELISA, neck muscle, tongue and eyelid examinations for the diagnosis of swine cysticercosis in a highly endemic area of north India

Swine cysticercosis is very common in the developing countries where pigs are raised. Undercooked measly pork consumption leads to taeniasis; Taenia carriers act as source of human and swine cysticercosis and neurocysticercosis. Diagnosis of swine cysticercosis is important to break the cycle of disease transmission. The present study compared the neck muscle, tongue and eye examinations, and serum ELISA with different preparations (crude lysate, cyst fluid, scolex and cyst wall antigens) of Taenia solium cyst for the diagnosis of swine cysticercosis. Total of 24 pigs initially identified by neck muscle, tongue and eyelid examinations were purchased from local slaughter house and subjected to MRI for confirmation of cysticercosis. Sera from 20 MRI confirmed infected pigs and 50 disease free controls were subjected to ELISA with T. solium cyst antigens. Neck muscle examination was 100% sensitive and 75% specific for the diagnosis of swine cysticercosis, whereas tongue and eye examinations were 70% and 25% sensitive, respectively. ELISA with crude lysate had 85% sensitivity and 98% specificity. ELISA with cyst fluid, scolex and cyst wall antigens showed 70%, 65%, and 45% sensitivity, respectively. The present study showed that neck muscle examination was highly sensitive but less specific, while ELISA with crude antigens had reasonable sensitivity and high specificity for diagnosis of swine cysticercosis. ELISA with crude lysate can be used as a screening tool for swine infection.

Seminal Fluid and the Generation of Regulatory T Cells for Embryo Implantation

T regulatory (Treg) cells are essential mediators of the maternal immune adaptation necessary for embryo implantation. In mice, insufficient Treg cell activity results in implantation failure, or constrains placental function and fetal growth. In women, Treg cell deficiency is linked with unexplained infertility, miscarriage, and pre-eclampsia. To devise strategies to improve Treg cell function, it is essential to define the origin of the Treg cells in gestational tissues, and the regulators that control their functional competence and recruitment. Male seminal fluid is a potent source of the Treg cell-inducing agents TGFβ and prostaglandin E, and coitus is one key factor involved in expanding the pool of inducible Treg cells that react with paternal alloantigens shared by conceptus tissues. In mice, coitus initiates a sequence of events whereby female dendritic cells cross-present seminal fluid antigens and activate T cells, which in turn circulate via the blood to be sequestered into the endometrium. Similar events may occur in the human genital tract, where seminal fluid induces immune cell changes that appear competent to prime Treg cells. Improved understanding of how seminal fluid influences Treg cells in women should ultimately assist in the development of new therapies for immune-mediated pathologies of pregnancy.

Seroprevalence of toxoplasmosis, toxocariasis and cysticercosis in a rural settlement, São Paulo State, Brazil

Background: The goal of this study was to estimate the seroprevalence of Toxocara spp., Toxoplasma gondii, and Taenia solium metacestode infection and determine some of the associated risk factors for people living in the Dona Carmen settlement, Pontal of Paranapanema, São Paulo, Brazil.Methods: Serum samples from 194 subjects were tested and participants answered a questionnaire. An enzyme-linked immunosorbent assay (ELISA) system based on Toxocara spp. excretory-secretory antigens obtained from the cultured second-stage larvae of Toxocara canis or vesicular fluid (VF) antigen from Taenia crassiceps metacestode was used to detect anti-Toxocara spp. IgG and IgE and anti-T. solium metacestode, respectively. For cysticercosis, the reactive ELISA samples were assayed by Western blotting using 18 kDa and 14 kDa proteins purified from VF. For T. gondii-specific IgG and IgM antibodies, anti-SAG-1, GRA-1, and GRA-7 epitope specificity was determined by ELISA.Results: Toxoplasma gondii IgG antibodies were found in 102/194 individuals (52·6%) with increased infections in females (P = 0·02) and those with ≤US$300 monthly income (P = 0·01). Positive IgM antibodies were detected in 21/194 individuals (10·8%). Antibodies specific to Toxocara spp. were found in 28/194 subjects (14·4%). All the individuals with Toxocara spp. also had T. gondii-specific IgG antibodies. Taenia solium metacestode antibodies were detected in 11 subjects (5·7%), but none were reactive based on Western blotting.Conclusion: In spite of environmental, educational, and socioeconomic factors favoring parasite infection, the seropositivity rates of T. gondii, Toxocara spp., and T. solium metacestode-specific IgG antibodies are similar to the rates found in studies conducted in different populations in Brazil.