Serological Diagnosis of Paracoccidioidomycosis through a Western Blot Technique

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.

Download Full-text

The sensitivity, specificity and efficiency values of some serological tests used in the diagnosis of paracoccidioidomycosis

Revista do Instituto de Medicina Tropical de São Paulo ◽

10.1590/s0036-46651991000400006 ◽

1991 ◽

Vol 33 (4) ◽

pp. 277-280 ◽

Cited By ~ 37

Author(s):

Gilda M.B. Del Negro ◽

Nilma M. Garcia ◽

Elaine G. Rodrigues ◽

M. Isabel N. Cano ◽

Mônica S.M.V. de Aguiar ◽

...

Keyword(s):

Yeast Cell ◽

Culture Filtrate ◽

Blood Donors ◽

Complement Fixation ◽

Paracoccidioides Brasiliensis ◽

Yeast Culture ◽

Double Immunodiffusion ◽

Serological Tests ◽

Sensitivity Specificity ◽

Negative Controls

This work reports on the results of double immunodiffusion (ID), counterimmunoelectrophoresis (CIE), complement fixation (CF) and indirect immunofluorescence (IIF) techniques in the serodiagnosis of paracoccidioidomycosis. The study was undertaken on four groups of individuals: 46 patients with untreated paracoccidioidomycosis, 22 patients with other deep mycoses, 30 with other infectious diseases (tuberculosis and cutaneous leishmaniasis) and 47 blood donors as negative controls. Data were obtained using Paracoccidioides brasiliensis antigens, i.e.,a yeast culture filtrate for ID, CIE and CF, and a yeast cell suspension for IIF. The sensitivity, specificity and efficiency values were measured according to GALEN & GAMBINO8.The gel precipitation tests (ID and CIE) showed the greatest sensitivity (91.3 and 95.6%, respectively), maximum specificity (100%) and the highest efficiency values when compared to the CF and IIF tests.

Download Full-text

Validity of a Serological Diagnostic Kit for SARS-CoV-2 Available in Iran

Archives of Iranian Medicine ◽

10.34172/aim.2020.75 ◽

2020 ◽

Vol 23 (9) ◽

pp. 629-632

Author(s):

Hamid Reza Shamsollahi ◽

Mostafa Amini ◽

Shaban Alizadeh ◽

Saharnaz Nedjat ◽

Ali Akbari-Sari ◽

...

Keyword(s):

Diagnostic Techniques ◽

Effective Control ◽

Molecular Technique ◽

Size Estimation ◽

Medical Sciences ◽

Serological Tests ◽

Standard Case ◽

Epidemic Size ◽

Low Sensitivity ◽

Negative Controls

Background: The Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) epidemic broke out in December 2019 and is now characterized as a pandemic. Effective control of this infectious disease requires access to diagnostic techniques, for both case finding and epidemic size estimation. The molecular technique is routinely used worldwide. Although it is the "standard" case detection and management method, it has its own shortcomings. Thus, some easy-to-use rapid serological tests have been developed. Methods: One hundred and fourteen positive RT-PCR-diagnosed patients were tested by VivaDiag Kit, a brand of rapid serological kits available in hospitals affiliated to Tehran University of Medical Sciences (TUMS), Tehran, Iran. Frozen serum specimens taken from healthy people in summer and fall 2019 were also tested as negative controls. Results: Test sensitivity was 47.9% (95% confidence interval [CI]: 38.8-56.9) for IgM and 47.0% (95% CI: 38.0–56.0) for IgG. There was no difference between IgG and IgM seropositivity except in one case. Specificity was calculated as 99.0% (95% CI: 96.4–99.9) for IgM and of 100.0% (95% CI: 0.98.2–100.0) for IgG. Sensitivity was higher in men and older participants. Conclusion: This test can be used for epidemiological investigations, especially for the estimation of the level of infection in the community, after it is properly corrected for sensitivity and specificity. The low sensitivity could be attributed to the technical limitations of the kit or low levels of antibodies after infection. The different sensitivity in age and sex groups supports the hypothesis that different people show different immune responses to this virus.

Download Full-text

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

Clinical and Vaccine Immunology ◽

10.1128/cvi.00130-10 ◽

2011 ◽

Vol 18 (4) ◽

pp. 604-608 ◽

Cited By ~ 20

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.

Download Full-text

Assessment of a serological diagnostic kit of SARS-CoV-2 availble in Iran

10.1101/2020.05.04.20090209 ◽

2020 ◽

Author(s):

Hamid Reza Shamsollahi ◽

Mostafa Amini ◽

Shaban Alizadeh ◽

Saharnaz Nedjat ◽

Ali Akbari-Sari ◽

...

Keyword(s):

Diagnostic Techniques ◽

Effective Control ◽

Molecular Technique ◽

Size Estimation ◽

Serological Tests ◽

Standard Case ◽

Technical Limitation ◽

Epidemic Size ◽

Low Sensitivity ◽

Negative Controls

AbstractBackgroundThe SARS-CoV-2 epidemic broke out in December 2019 and now is characterized as a pandemic. The effective control of this infectious disease requires access to diagnostic techniques, both for case finding and epidemic size estimation. The molecular technique is routinely being used worldwide. Although it is the “standard” case detection and management method, it has its own shortcomings. Thus, some easy-to-use rapid serological tests were developed.MethodsOne hundred and fourteen positive RT-PCR-diagnosed patients were tested by VivaDiag Kit, a brand of rapid serological kits available in hospitals affiliated to Tehran University of Medical Sciences (TUMS), Tehran, Iran. Frozen serum specimens taken from healthy people in summer and autumn 2019, were also tested as negative controls.ResultsThe test sensitivity was 47.9% (95% confidence interval [CI]: 38.8-56.9) for IgM and 47.0% (95% CI: 38.0-56.0) for IgG. There was no difference between IgG and IgM seropositivity except in one case. Specificity was calculated as 99.0% (95% CI: 96.4-99.9) for IgM and of 100.0% (95% CI: 0.98.2-100.0) for IgG. Sensitivity was higher in men and older participants.ConclusionThis test can be used for epidemiological investigations especially for the estimation of the level of infection in the community, after it is properly corrected for sensitivity and specificity. The low sensitivity could be attributed to the technical limitation of the kits or low levels of antibodies after infection. The different sensitivity in age and sex groups supports the hypothesis that different people show different immune responses to this virus.

Download Full-text

Evaluation of a New Immunochromatography Technology Test (LDBio Diagnostics) To Detect Toxoplasma IgG and IgM: Comparison with the Routine Architect Technique

Journal of Clinical Microbiology ◽

10.1128/jcm.01106-17 ◽

2017 ◽

Vol 55 (12) ◽

pp. 3395-3404 ◽

Cited By ~ 7

Author(s):

Caroline Mahinc ◽

Pierre Flori ◽

Edouard Delaunay ◽

Cécile Guillerme ◽

Sana Charaoui ◽

...

Keyword(s):

Western Blot ◽

Sensitivity And Specificity ◽

Blot Analysis ◽

False Positive ◽

Automated System ◽

University Hospitals ◽

Content Type ◽

Automated Screening ◽

Commercial Kits ◽

Fully Automated System

ABSTRACTA study comparing the ICT (immunochromatography technology)ToxoplasmaIgG and IgM rapid diagnostic test (LDBio Diagnostics, France) with a fully automated system, Architect, was performed on samples from university hospitals of Marseille and Saint-Etienne. A total of 767 prospective sera and 235 selected sera were collected. The panels were selected to test various IgG and IgM parameters. The reference technique,ToxoplasmaIgGII Western blot analysis (LDBio Diagnostics), was used to confirm the IgG results, and commercial kits Platelia Toxo IgM (Bio-Rad) and Toxo-ISAgA (bioMérieux) were used in Saint-Etienne and Marseille, respectively, as the IgM reference techniques. Sensitivity and specificity of the ICT and the Architect IgG assays were compared using a prospective panel. Sensitivity was 100% for the ICT test and 92.1% for Architect (cutoff at 1.6 IU/ml). The low-IgG-titer serum results confirmed that ICT sensitivity was superior to that of Architect. Specificity was 98.7% (ICT) and 99.8% (Architect IgG). The ICT test is also useful for detecting IgM without IgG and is both sensitive (100%) and specific (100%), as it can distinguish nonspecific IgM from specificToxoplasmaIgM. In comparison, IgM sensitivity and specificity on Architect are 96.1% and 99.6%, respectively (cutoff at 0.5 arbitrary units [AU]/ml). To conclude, this new test overcomes the limitations of automated screening techniques, which are not sensitive enough for IgG and lack specificity for IgM (rare IgM false-positive cases).

Download Full-text

Interlaboratory Optimization and Evaluation of a Serological Assay for Diagnosis of Human Baylisascariasis

Clinical and Vaccine Immunology ◽

10.1128/cvi.00387-13 ◽

2013 ◽

Vol 20 (11) ◽

pp. 1758-1763 ◽

Cited By ~ 15

Author(s):

Lisa N. Rascoe ◽

Cynthia Santamaria ◽

Sukwan Handali ◽

Sriveny Dangoudoubiyam ◽

Kevin R. Kazacos ◽

...

Keyword(s):

Western Blot ◽

Cross Reactivity ◽

Western Blot Assay ◽

Content Type ◽

National Reference ◽

Baylisascaris Procyonis ◽

Reference Centre ◽

Assay Performance ◽

Positive Serum ◽

Control And Prevention

ABSTRACTA Western blot assay using a recombinant protein, recombinantBaylisascaris procyonisRAG1 protein (rBpRAG1), was developed for the diagnosis of human baylisascariasis concurrently by the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia, and the National Reference Centre for Parasitology (NRCP) in Montreal, Canada. Assay performance was assessed by testing 275 specimens at the CDC and 405 specimens at the NRCP. Twenty specimens from 16 cases of baylisascariasis were evaluated. Eighteen were positive, with the assay correctly identifying 14 of 16 patients. The rBpRAG1 Western blot assay showed no cross-reactivity withToxocara-positive serum and had an overall sensitivity of 88% and a specificity of 98%.

Download Full-text

Evaluation of Recombinant Proteins of Burkholderia mallei for Serodiagnosis of Glanders

Clinical and Vaccine Immunology ◽

10.1128/cvi.00137-12 ◽

2012 ◽

Vol 19 (8) ◽

pp. 1193-1198 ◽

Cited By ~ 11

Author(s):

Vijai Pal ◽

Subodh Kumar ◽

Praveen Malik ◽

Ganga Prasad Rai

Keyword(s):

Sensitivity And Specificity ◽

Recombinant Proteins ◽

False Negative ◽

Enzyme Linked Immunosorbent Assay ◽

Burkholderia Mallei ◽

Content Type ◽

Whole Cells ◽

Negative Results ◽

Elisa Format ◽

Low Sensitivity

ABSTRACTGlanders is a contagious disease caused by the Gram-negative bacillusBurkholderia mallei. The number of equine glanders outbreaks has increased steadily during the last decade. The disease must be reported to the Office International des Epizooties, Paris, France. Glanders serodiagnosis is hampered by the considerable number of false positives and negatives of the internationally prescribed tests. The major problem leading to the low sensitivity and specificity of the complement fixation test (CFT) and enzyme-linked immunosorbent assay (ELISA) has been linked to the test antigens currently used, i.e., crude preparations of whole cells. False-positive results obtained from other diagnostic tests utilizing crude antigens lead to financial losses to animal owners, and false-negative results can turn a risk into a possible threat. In this study, we report on the identification of diagnostic targets using bioinformatics tools for serodiagnosis of glanders. The identified gene sequences were cloned and expressed as recombinant proteins. The purified recombinant proteins ofB. malleiwere used in an indirect ELISA format for serodiagnosis of glanders. Two recombinant proteins, 0375H and 0375TH, exhibited 100% sensitivity and specificity for glanders diagnosis. The proteins also did not cross-react with sera from patients with the closely related disease melioidosis. The results of this investigation highlight the potential of recombinant 0375H and 0375TH proteins in specific and sensitive diagnosis of glanders.

Download Full-text

A High-Performance Multiplex Immunoassay for Serodiagnosis of Flavivirus-Associated Neurological Diseases in Horses

BioMed Research International ◽

10.1155/2015/678084 ◽

2015 ◽

Vol 2015 ◽

pp. 1-13 ◽

Cited By ~ 29

Author(s):

Cécile Beck ◽

Philippe Desprès ◽

Sylvie Paulous ◽

Jessica Vanhomwegen ◽

Steeve Lowenski ◽

...

Keyword(s):

High Performance ◽

Neurological Diseases ◽

Expression System ◽

Cross Reactivity ◽

Encephalitis Virus ◽

Serological Tests ◽

Multiplex Immunoassay ◽

Tick Borne Encephalitis ◽

Tick Borne Encephalitis Virus ◽

Negative Controls

West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV) are flaviviruses responsible for severe neuroinvasive infections in humans and horses. The confirmation of flavivirus infections is mostly based on rapid serological tests such as enzyme-linked immunosorbent assays (ELISAs). These tests suffer from poor specificity, mainly due to antigenic cross-reactivity among flavivirus members. Robust diagnosis therefore needs to be validated through virus neutralisation tests (VNTs) which are time-consuming and require BSL3 facilities. The flavivirus envelope (E) glycoprotein ectodomain is composed of three domains (D) named DI, DII, and DIII, with EDIII containing virus-specific epitopes. In order to improve the serological differentiation of flavivirus infections, the recombinant soluble ectodomain of WNV E (WNV.sE) and EDIIIs (rEDIIIs) of WNV, JEV, and TBEV were synthesised using theDrosophilaS2 expression system. Purified antigens were covalently bonded to fluorescent beads. The microspheres coupled to WNV.sE or rEDIIIs were assayed with about 300 equine immune sera from natural and experimental flavivirus infections and 172 nonimmune equine sera as negative controls. rEDIII-coupled microspheres captured specific antibodies against WNV, TBEV, or JEV in positive horse sera. This innovative multiplex immunoassay is a powerful alternative to ELISAs and VNTs for veterinary diagnosis of flavivirus-related diseases.

Download Full-text

Systematic Review into Diagnostics for Post-Kala-Azar Dermal Leishmaniasis (PKDL)

Journal of Tropical Medicine ◽

10.1155/2013/150746 ◽

2013 ◽

Vol 2013 ◽

pp. 1-8 ◽

Cited By ~ 16

Author(s):

Emily R. Adams ◽

Inge Versteeg ◽

Mariska M. G. Leeflang

Keyword(s):

Diagnostic Accuracy ◽

Clinical Symptoms ◽

Statistical Modelling ◽

Study Quality ◽

Serological Tests ◽

Molecular Tests ◽

Kala Azar ◽

Valid Estimate ◽

Low Sensitivity ◽

Control Designs

Identification of post-kala-azar dermal leishmaniasis (PKDL) is important due to the long and toxic treatment and the fact that PKDL patients may serve as a reservoir for visceral leishmaniasis (VL). We summarized the published literature about the accuracy of diagnostic tests for PKDL. We searched Medline for eligible studies investigating the diagnostic accuracy of any test for PKDL. Study quality was assessed using QUADAS-2. Data were extracted from 21 articles including 43 separate studies. Twenty-seven studies evaluated serological tests (rK39 dipstick, ELISA, DAT, and leishmanin tests), six studies molecular tests, eight microscopy, and two cultures. Only a few of these studies reported a valid estimate of diagnostic accuracy, as most were case-control designs or used a reference standard with low sensitivity. The included studies were very heterogeneous, for example, due to a large variety of reference standards used. Hence, no summary estimates of sensitivity or specificity could be made. We recommend well-designed diagnostic accuracy trials that evaluate, side-by-side, all currently available diagnostics, including clinical symptoms, serological, antigen, molecular, and parasitological tests and possible use of statistical modelling to evaluate diagnostics when there is no suitable gold standard.

Download Full-text

Diagnosis and Treatment Algorythm in Lyme Neuroborreliosis

Acta Medica Marisiensis ◽

10.1515/amma-2015-0125 ◽

2016 ◽

Vol 62 (1) ◽

pp. 121-127

Author(s):

Brîndușa Țilea ◽

Rodica Bălașa ◽

Andrea Fodor ◽

Țilea Ioan

Keyword(s):

Borrelia Burgdorferi ◽

Bacterial Species ◽

Correct Diagnosis ◽

Clinical Manifestations ◽

Lyme Neuroborreliosis ◽

Early Administration ◽

Serological Tests ◽

Initiation Of Therapy ◽

Low Sensitivity ◽

Third Generation Cephalosporins

AbstractLyme neuroborreliosis is an infection of the nervous system caused by spirochetes of the Borrelia burgdorferi sensulato group. Neurological clinical manifestations usually present a steady evolution and are different in patients from Europe compared to those from America, possibly due to vector agents and different bacterial species. Various diagnostic markers were studied in consideration of a clear or possible diagnosis of the disease, because evolution and complications depend on early diagnosis and initiation of therapy. The isolation of the bacterium is difficult, microscopic examination and the bacterial dezoxiribonucleic acid amplification shows low sensitivity. However, the diagnosis of Lyme neuroborreliosis is mainly based on serological methods that have a satisfactory sensitivity and specificity. A correct diagnosis can be performed by strictly respecting clinical guidelines and protocols and carefully interpreting the serological tests. The presence of anti-borrelia burgdorferi antibodies in the cerebrospinal fluid with evidence of intrathecal antibody production is the gold standard diagnosis of Lyme neuroborreliosis. Early administration of antibiotic treatment (third generation cephalosporins, cyclins, aminopenicillins) can produce the remission of neurological symptoms, the eradication of spirochetes in acute phase of the disease, thus avoiding the development of the chronic disease.

Download Full-text