scholarly journals Development and Evaluation of a Latex Agglutination Test for the Serodiagnosis of Paracoccidioidomycosis

2011 ◽  
Vol 18 (4) ◽  
pp. 604-608 ◽  
Author(s):  
Fabíola Silveira-Gomes ◽  
Dayse Nogueira Sarmento ◽  
Thifany Mendes Pinto ◽  
Rosiane Ferreira Pimentel ◽  
Lívia Barreto Nepomuceno ◽  
...  
Keyword(s):  
Latex Particle ◽  
Paracoccidioides Brasiliensis ◽  
Cross Reactivity ◽  
Latex Agglutination ◽  
Dimorphic Fungus ◽  
Double Immunodiffusion ◽  
Serum Samples ◽  
Content Type ◽  
Healthy Donors ◽  
Low Costs

ABSTRACTParacoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Latin America. It is caused by the dimorphic fungusParacoccidioides brasiliensis. The immunodiffusion (ID) test is one of the most widely used techniques for PCM serologic diagnosis due to the simplicity and low costs of its execution. However, it requires trained and qualified people to execute it. The purpose of this study was to evaluate a latex particle agglutination (LA) test for the detection of anti-P. brasiliensisantibodies by using pooled crude exoantigens from the fungus. Fifty-one serum samples obtained from patients with PCM were tested. Positivity was observed in 84% (43/51) of these patients, and the agglutination patterns varied from small clumps with a cloudy background to large clumps with a clear background. The antibody titer reactivity ranged from 1:2 to 1:64. Cross-reactivity was observed in sera from patients with aspergillosis, histoplasmosis, and nonfungal disease. Serum samples obtained from healthy donors were not reactive. The sensitivity and specificity of the LA test were 84% and 81%, respectively. When comparing the LA test with the double-immunodiffusion test, we found an agreement of 92%. Further work is needed to improve the performance of the LA assay before it can be proposed as a reliable diagnostic tool, mainly in laboratories with little infrastructure.

scholarly journals Effects of Pretreating Serum Samples on the Performance of a Latex Agglutination Test for Serodiagnosis of Paracoccidioidomycosis

2011 ◽  
Vol 19 (3) ◽  
pp. 386-390 ◽  
Author(s):  
Fabíola Silveira-Gomes ◽  
Silvia Helena Marques-da-Silva
Keyword(s):  
Sensitivity And Specificity ◽  
Bacterial Infections ◽  
Paracoccidioides Brasiliensis ◽  
Latex Agglutination ◽  
Agglutination Test ◽  
Latex Agglutination Test ◽  
Serum Samples ◽  
Content Type ◽  
Human Sera ◽  
Normal Human

ABSTRACTParacoccidioidomycosis (PCM) is a fungal disease caused byParacoccidioides brasiliensis, and Brazil is one of the principal countries where it is endemic. Diagnosis is based on the observation of buddingP. brasiliensisyeast in clinical specimens from patients; however, the sensitivity of the visualization of fungi is low, indicating that serological tests are used for early diagnosis. The double-immunodiffusion test (ID) is the “gold standard” test for serology in PCM, although the execution of this test requires the availability of laboratorial infrastructure. We report the improved performance of a latex agglutination test (LAT) by pretreating 30 serum samples from PCM patients and 71 controls (histoplasmosis and aspergillosis patients, patients with bacterial infections, and normal human sera) with a dilution buffer incubated at 37°C for 30 min. The sensitivity and specificity of the LAT test in the nonpretreated samples were 73% and 79%, respectively. However, when samples were pretreated, the sensitivity and specificity of the test increased to 90%. In this study, we did not observe cross-reactivity with histoplasmosis patient sera, but some reactions to sera from patients with aspergillosis and bacterial infections were noted. Normal human sera were not reactive in our tests. These results indicate the need for the elimination of heterologous reactions so that we can adequately use this method for screening cases of PCM.

scholarly journals Subtractive Phage Display Selection from Canine Visceral Leishmaniasis Identifies Novel Epitopes That Mimic Leishmania infantum Antigens with Potential Serodiagnosis Applications

2013 ◽  
Vol 21 (1) ◽  
pp. 96-106 ◽  
Author(s):  
Lourena E. Costa ◽  
Mayara I. S. Lima ◽  
Miguel A. Chávez-Fumagalli ◽  
Daniel Menezes-Souza ◽  
Vivian T. Martins ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Phage Display ◽  
Leishmania Infantum ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Phage Display Technology ◽  
Predictive Values ◽  
Content Type ◽  
Phage Elisa

ABSTRACTVisceral leishmaniasis (VL) is a zoonotic disease that is endemic to Brazil, where dogs are the main domestic parasite reservoirs, and the percentages of infected dogs living in regions where canine VL (CVL) is endemic have ranged from 10% to 62%. Despite technological advances, some problems have been reported with CVL serodiagnosis. The present study describes a sequential subtractive selection through phage display technology from polyclonal antibodies of negative and positive sera that resulted in the identification of potential bacteriophage-fused peptides that were highly sensitive and specific to antibodies of CVL. A negative selection was performed in which phage clones were adhered to purified IgGs from healthy andTrypanosoma cruzi-infected dogs to eliminate cross-reactive phages. The remaining supernatant nonadhered phages were submitted to positive selection against IgG from the blood serum of dogs that were infected withLeishmania infantum. Phage clones that adhered to purified IgGs from the CVL-infected serum samples were selected. Eighteen clones were identified and their reactivities tested by a phage enzyme-linked immunosorbent assay (phage-ELISA) against the serum samples from infected dogs (n= 31) compared to those from vaccinated dogs (n= 21), experimentally infected dogs with cross-reactive parasites (n= 23), and healthy controls (n= 17). Eight clones presented sensitivity, specificity, and positive and negative predictive values of 100%, and they showed no cross-reactivity withT. cruzi- orEhrlichia canis-infected dogs or with dogs vaccinated with two different commercial CVL vaccines in Brazil. Our study identified eight mimotopes ofL. infantumantigens with 100% accuracy for CVL serodiagnosis. The use of these mimotopes by phage-ELISA proved to be an excellent assay that was reproducible, simple, fast, and inexpensive, and it can be applied in CVL-monitoring programs.

scholarly journals Heterologous Expression, Purification, and Immunological Reactivity of a Recombinant HSP60 from Paracoccidioides brasiliensis

2002 ◽  
Vol 9 (2) ◽  
pp. 374-377 ◽  
Author(s):  
Daniela A. Cunha ◽  
Roseli M. Zancopé-Oliveira ◽  
M. Sueli ◽  
S. Felipe ◽  
Silvia M. Salem-Izacc ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Paracoccidioides Brasiliensis ◽  
High Sensitivity ◽  
Cross Reactivity ◽  
Antibody Responses ◽  
Healthy Individuals ◽  
Immunological Reactivity ◽  
Serum Samples ◽  
Recombinant Antigens

ABSTRACT The complete coding cDNA of HSP60 from Paracoccidioides brasiliensis was overexpressed in an Escherichia coli host to produce high levels of recombinant protein. The protein was purified by affinity chromatography. A total of 169 human serum samples were tested for reactivity by Western blot analysis with the purified HSP60 recombinant protein. Immunoblots indicated that the recombinant P. brasiliensis HSP60 was recognized by antibodies in 72 of 75 sera from paracoccidioidomycosis patients. No cross-reactivity was detected with individual sera from patients with aspergillosis, sporotrichosis, cryptococcosis, and tuberculosis. Reactivity to HSP60 was observed in sera from 9.52% of control healthy individuals and 11.5% of patients with histoplasmosis. The high sensitivity and specificity (97.3 and 92.5%, respectively) for HSP60 suggested that the recombinant protein can be used singly or in association with other recombinant antigens to detect antibody responses in P. brasiliensis-infected patients.

scholarly journals Expression of Antibodies Directed to Paracoccidioides brasiliensis Glycosphingolipids during the Course of Paracoccidioidomycosis Treatment

2006 ◽  
Vol 14 (2) ◽  
pp. 150-156 ◽  
Author(s):  
Silvio Bertini ◽  
Arnaldo L. Colombo ◽  
Helio K. Takahashi ◽  
Anita H. Straus
Keyword(s):  
Immune Response ◽  
Paracoccidioides Brasiliensis ◽  
Immunoglobulin M ◽  
Antifungal Treatment ◽  
Primary Immune Response ◽  
Enzyme Linked Immunosorbent Assay ◽  
Carbohydrate Structure ◽  
Dimorphic Fungus ◽  
Serum Samples ◽  
Antibody Titers

ABSTRACT Paracoccidioidomycosis (PCM) is a granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis. The immunoglobulin classes and isotypes of antibodies directed to acidic glycosphingolipids (GSLs) and glucosylceramide of P. brasiliensis were determined by enzyme-linked immunosorbent assay of sera from 31 PCM patients. The reactivities of 38 serum samples were analyzed by considering the stage of treatment: before antifungal treatment (n = 10), during 1 to 4 months of treatment (T1-4; n = 9), during 5 to 12 months of treatment (T5-12; n = 9), and posttreatment (PT; n = 10). Sera from healthy subjects (n = 12) were used as controls. Only the GSL Pb-1 antigen, which presents the carbohydrate structure Galfβ1-6(Manα1-3)Manβ1, was reactive with the PCM patient sera. The PCM patient sera did not react with Pb-2, which lacks the Galf residue and which is considered the biosynthetic precursor of Pb-1, indicating that the Galf residue is essential for antibody reactivity. The Pb-1 glycolipid from nontreated patients elicited a primary immune response with immunoglobulin M (IgM) production and subsequent switching to IgG1 production. The IgG1 titer increased after the start of antifungal treatment (T1-4 group), and general decreases in the anti-Pb-1 antibody titers were observed after 5 months of treatment (T5-12 and PT groups). The Pb-1 antigen, an acidic GSL with terminal Galf residue, has potential application as an elicitor of the host immune response in patients with PCM.

scholarly journals Cross-Reactivity Using Chimeric Trypanosoma cruzi Antigens: Diagnostic Performance in Settings Where Chagas Disease and American Cutaneous or Visceral Leishmaniasis Are Coendemic

2019 ◽  
Vol 57 (8) ◽  
Author(s):  
Ramona Tavares Daltro ◽  
Leonardo Maia Leony ◽  
Natália Erdens Maron Freitas ◽  
Ângelo Antônio Oliveira Silva ◽  
Emily Ferreira Santos ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Chagas Disease ◽  
Diagnostic Performance ◽  
Latent Class ◽  
Cross Reactivity ◽  
Chimeric Proteins ◽  
Serum Samples ◽  
Perfect Agreement ◽  
Content Type ◽  
Chronic Chagas Disease

ABSTRACT Chimeric T. cruzi antigens have been proposed as a diagnostic tool for chronic Chagas disease (CD) in both settings where Chagas disease is endemic and those where it is not endemic. Antibody response varies in accordance to each T. cruzi strain, presenting challenges to the use of antigens lacking demonstrated cross-reactivity with Leishmania spp. Our group expressed four chimeric proteins (IBMP-8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4) and previously assessed their diagnostic performance to determine cross-reactivity with Leishmania spp. Here, we validated our findings using serum samples from different Brazilian geographic areas reporting endemic Chagas disease, endemic visceral or American cutaneous leishmaniasis (ACL), or both. Overall, 829 serum samples were evaluated using commercial and IBMP enzyme-linked immunosorbent assays. Due to the absence of a reference assay to diagnosis CD, latent class analysis (LCA) was performed through the use of a statistical model. The incidence of cross-reactivity for ACL-positive samples varied from 0.35% (IBMP-8.3) to 0.70% (IBMP-8.1 and IBMP-8.2). Regarding visceral leishmaniasis (VL)-positive samples, the IBMP-8.2 and IBMP-8.3 antigens cross-reacted with six (3.49%) and with only one sample (0.58%), respectively. No cross-reactivity with either ACL or VL was observed for the IBMP-8.4 antigen. Similarly, no cross-reactions were found when VL-positive samples were assayed with IBMP-8.1. The agreement among the results obtained using IBMP antigens ranged from 97.3% for IBMP-8.2 and 99% for IBMP-8.1 and IBMP-8.3 to 100% for IBMP-8.4, demonstrating almost perfect agreement with LCA. Accordingly, in light of the negligible cross-reactivity with both ACL and VL, we suggest the use of IBMP antigens in regions where T. cruzi and Leishmania spp. are coendemic.

scholarly journals Identification of Allergenic Component Tyr p 8 from Tyrophagus putrescentiae and Cross-Reactivity with Der p 8

2013 ◽  
Vol 20 (4) ◽  
pp. 506-512 ◽  
Author(s):  
En-Chih Liao ◽  
Yi-Hsueh Lin ◽  
Chih-Liang Chiu ◽  
Ting-Chu Lin ◽  
Jaw-Ji Tsai
Keyword(s):  
Sequence Homology ◽  
Positive Response ◽  
Specific Binding ◽  
Major Allergen ◽  
Specific Ige ◽  
Cross Reactivity ◽  
House Dust Mites ◽  
Tyrophagus Putrescentiae ◽  
Serum Samples ◽  
Content Type

ABSTRACTGroup 8 mite allergens exhibit sequence homology to glutathioneS-transferases (GSTs), such as that fromDermatophagoides pteronyssinus(Der p 8). GSTs have been identified as important allergens in studies of allergens from house dust mites, cockroaches, and fungi. Our objective was to purify the native group 8 allergen fromTyrophagus putrescentiae(nTyr p 8) and generate recombinant Tyr p 8 (rTyr p 8) for immunological characterization. The allergenicity was determined by antibody recognition, IgE inhibition, and triggering of the basophil-sensitized release of histamine, usingT. putrescentiaehypersensitivity sera. The results showed that the mRNA transcript of nTyr p 8 is 657 bp long, contains 218 amino acids with a molecular mass of 26 kDa, and exhibits 83% sequence homology to Der p 8. Serum samples from the allergic patients with an IgE-positive response toT. putrescentiaewere analyzed to determine their IgE response to rTyr p 8. The results showed that the sera of 48 subjects (45.3%) had specific IgE against rTyr p 8. However, sera of only 19 subjects (17.9%) had specific IgE against rTyr p 8 afterD. pteronyssinusabsorption. Histamine release was observed fromT. putrescentiae-allergic subjects in the presence of rTyr p 8. Both the nTyr p 8 andT. putrescentiaecrude extract had been demonstrated to possess GST enzymatic activity. Although the specific binding of serum IgE to rTyr p 8 was only 17.9%, which indicates that rTyr p 8 was not a major allergen, the positive response to rTyr p 8 was due to the cross-reactivity with Der p 8. The group 8 mite allergen might be of use in the design of a suitable allergen for diagnosis and for the development of novel immunotherapies.

scholarly journals Mycobacterium avium subsp. paratuberculosis Antibody Response, Fecal Shedding, and Antibody Cross-Reactivity to Mycobacterium bovis in M. avium subsp. paratuberculosis-Infected Cattle Herds Vaccinated against Johne's Disease

2014 ◽  
Vol 21 (5) ◽  
pp. 698-703 ◽  
Author(s):  
Deepanker Tewari ◽  
Ernest Hovingh ◽  
Rick Linscott ◽  
Edmond Martel ◽  
John Lawrence ◽  
...  
Keyword(s):  
Mycobacterium Bovis ◽  
Mycobacterium Avium ◽  
Cross Reactivity ◽  
Johne's Disease ◽  
Antibody Responses ◽  
Johne’S Disease ◽  
Serum Samples ◽  
Content Type ◽  
Fecal Shedding ◽  
Cattle Herds

ABSTRACTVaccination for Johne's disease with killed inactivated vaccine in cattle herds has shown variable success. The vaccine delays the onset of disease but does not afford complete protection. Johne's disease vaccination has also been reported to interfere with measurements of cell-mediated immune responses for the detection of bovine tuberculosis. Temporal antibody responses and fecal shedding ofMycobacterium aviumsubsp.paratuberculosis, the causative agent of Johne's disease, were measured in 2 dairy cattle herds using Johne's disease vaccine (Mycopar) over a period of 7 years. Vaccination against Johne's disease resulted in positive serumM. aviumsubsp.paratuberculosisantibody responses in both herds, and the responses persisted in vaccinated cattle up to 7 years of age. Some vaccinated animals (29.4% in herd A and 36.2% in herd B) showed no serological reactivity toM. aviumsubsp.paratuberculosis.M. aviumsubsp.paratuberculosis-specific antibody responses were also detected in milk from Johne's disease-vaccinated animals, but fewer animals (39.3% in herd A and 49.4% in herd B) had positive results with milk than with serum samples. With vaccination againstM. aviumsubsp.paratuberculosis, fecal shedding in both dairy herds was reduced significantly (P< 0.001). In addition, when selected Johne's disease-vaccinated and -infected animals were investigated for serological cross-reactivity toMycobacterium bovis, no cross-reactivity was observed.

scholarly journals Serological Diagnosis of Paracoccidioidomycosis through a Western Blot Technique

2012 ◽  
Vol 19 (4) ◽  
pp. 616-619 ◽  
Author(s):  
M. C. Z. Perenha-Viana ◽  
I. A. A. Gonzales ◽  
S. R. Brockelt ◽  
L. N. C. Machado ◽  
T. I. E. Svidzinski
Keyword(s):  
Western Blot ◽  
Positive Reaction ◽  
Paracoccidioides Brasiliensis ◽  
Double Immunodiffusion ◽  
Serological Tests ◽  
Content Type ◽  
Mycological Examination ◽  
Low Sensitivity ◽  
Effective Diagnosis ◽  
Negative Controls

ABSTRACTParacoccidioidomycosis (PCM) is a serious infectious disease that progresses toward death if untreated. Its confirmatory diagnosis is made by the detection of the fungusParacoccidioides brasiliensisin a direct mycological examination or by histopathology. However, these techniques are of low sensitivity. Serological tests seem to be more promising. The objective of this study was to test Western blot (WB) analysis using sera from patients suspected of PCM to determine whether it represents a safe and sensitive serological technique for a rapid and effective diagnosis for this disease. Sera from 517 patients were analyzed through WB analysis and double-immunodiffusion (DID) techniques using a crude exoantigen ofP. brasiliensis339. DID gave positive reactions for 140 sera (27%) and WB for 250 sera (48.4%). All sera that had a positive reaction by DID also had a positive result with a 43-kDa glycoprotein by WB analysis. Among the 377 samples that were negative by DID, 29.1% were reactive in WB analysis. For the cutoff dilution used (1:400), a positive reaction was not observed with any of the 102 sera from patients with other diseases in regions where such diseases are endemic and 30 healthy individuals tested as negative controls. These results prove WB analysis to be a sensitive technique and suggest its inclusion among routine laboratory assays as a safe method for PCM diagnosis.

scholarly journals The Pathogenic Fungus Paracoccidioides brasiliensis Exports Extracellular Vesicles Containing Highly Immunogenic α-Galactosyl Epitopes

2011 ◽  
Vol 10 (3) ◽  
pp. 343-351 ◽  
Author(s):  
Milene C. Vallejo ◽  
Alisson L. Matsuo ◽  
Luciane Ganiko ◽  
Lia C. Soares Medeiros ◽  
Kildare Miranda ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Fungal Pathogens ◽  
Histoplasma Capsulatum ◽  
Paracoccidioides Brasiliensis ◽  
Pathogenic Fungus ◽  
Yeast Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dimorphic Fungus ◽  
Content Type

ABSTRACTExosome-like vesicles containing virulence factors, enzymes, and antigens have recently been characterized in fungal pathogens, such asCryptococcus neoformansandHistoplasma capsulatum. Here, we describe extracellular vesicles carrying highly immunogenic α-linked galactopyranosyl (α-Gal) epitopes inParacoccidioides brasiliensis. P. brasiliensisis a dimorphic fungus that causes human paracoccidioidomycosis (PCM). For vesicle preparations, cell-free supernatant fluids from yeast cells cultivated in Ham's defined medium-glucose were concentrated in an Amicon ultrafiltration system and ultracentrifuged at 100,000 ×g. P. brasiliensisantigens were present in preparations from phylogenetically distinct isolates Pb18 and Pb3, as observed in immunoblots revealed with sera from PCM patients. In an enzyme-linked immunosorbent assay (ELISA), vesicle components containing α-Gal epitopes reacted strongly with anti-α-Gal antibodies isolated from both Chagas' disease and PCM patients, withMarasmius oreadesagglutinin (MOA) (a lectin that recognizes terminal α-Gal), but only faintly with natural anti-α-Gal. Reactivity was inhibited after treatment with α-galactosidase. Vesicle preparations analyzed by electron microscopy showed vesicular structures of 20 to 200 nm that were labeled both on the surface and in the lumen with MOA. InP. brasiliensiscells, components carrying α-Gal epitopes were found distributed on the cell wall, following a punctuated confocal pattern, and inside large intracellular vacuoles. Lipid-free vesicle fractions reacted with anti-α-Gal in ELISA only when not digested with α-galactosidase, while reactivity with glycoproteins was reduced after β-elimination, which is indicative of partial O-linked chain localization. Our findings open new areas to explore in terms of host-parasite relationships in PCM and the role playedin vivoby vesicle components and α-galactosyl epitopes.

scholarly journals Seroreactive Profiling of Filoviruses in Chinese Bats Reveals Extensive Infection of Diverse Viruses

2020 ◽  
Vol 94 (7) ◽  
Author(s):  
Chang Zhang ◽  
Zhongyi Wang ◽  
Jianqiu Cai ◽  
Xiaomin Yan ◽  
Fuqiang Zhang ◽  
...  
Keyword(s):  
Southern China ◽  
Hot Spot ◽  
Emerging Infectious Diseases ◽  
Neutralization Assay ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antigenic Relationship ◽  
Serum Samples ◽  
Content Type ◽  
Fruit Bat

ABSTRACT Southern China is a hot spot of emerging infectious diseases, in which diverse species of bats dwell, a large group of flying mammals considered natural reservoirs for zoonotic viruses. Recently, divergent filoviruses (FiVs) have been identified in bats within this region, which pose a potential risk to public health, but the true infection situation in bats remains largely unclear. Here, 689 archived bat serum samples were analyzed by enzyme-linked immunosorbent assay (ELISA), Western blotting, and neutralization assay to investigate the seroprevalence and cross-reactivity of four divergent FiVs and two other viruses (rabies virus and Tuhoko pararubulavirus 1) of different families within the order Mononegavirales. Results showed no cross-antigenicity between FiVs and other mononegaviruses but different cross-reactivity among the FiVs themselves. The total FiV seroreactive rate was 36.3% (250/689), with infection by the indigenous Chinese FiV DH04 or an antigenically related one being the most widely and the most highly prevalent. Further viral metagenomic analysis of fruit bat tissues also identified the gene sequence of a novel FiV. These results indicate the likely prevalence of other so far unidentified FiVs within the Chinese bat population, with frugivorous Rousettus leschenaultii and Eonycteris spelaea bats and insectivorous Myotis horsfieldii and Miniopterus schreibersii bats being their major reservoirs. IMPORTANCE Bats are natural hosts of many FiVs, from which diverse FiVs were serologically or virologically detected in Africa, Europe, and East Asia. Recently, very divergent FiVs were identified in the Chinese bat population, but their antigenic relationship with other known FiVs remains unknown. Here, we conducted serological characterization and investigation of Chinese indigenous FiVs and prototypes of other viruses in bats. Results indicated that Chinese indigenous FiVs are antigenically distant to other FiVs, and infection of novel or multiple FiVs occurred in Chinese bats, with FiV DH04 or an antigenically related one being the most widely and the most highly prevalent. Additionally, besides Rousettus leschenaultii and Eonycteris spelaea bats, the insectivorous Myotis horsfieldii and M. schreibersii bats are highly preferential hosts of FiVs. Seroreactive and viral metagenomic results indicated that more as yet unknown bat-borne FiVs circulate in Southern China, and to uncover them further, investigation and timely surveillance is needed.

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