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‣ Development of a Clinical Assay To Evaluate Toll-Like Receptor Function

Deering, Raquel P.; Orange, Jordan S.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /01/2006 Português
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Toll-like receptors (TLRS) recognize pathogen-associated molecular patterns to enable innate immune responses. A number of genetic defects influencing the function of these receptors have been identified and are associated with recurrent and/or severe infection. Our goal was to develop a reproducible assay of TLR function in order to evaluate patients with recurrent infection who would be suspected of having a genetic defect affecting TLR signaling. We chose to study peripheral blood mononuclear cells (PBMCS) to avoid potential influences of soluble factors contained in whole blood, and we utilized ligands for TLRS 1/2, 2/6, 3, 4, 5, 6, 7, and 9. Tumor necrosis factor (TNF) production in PBMC supernatants was measured by an enzyme-linked immunosorbent assay after TLR ligand stimulation and was dependent on gene transcription and NF-κB activation. Some variables affecting the assay were assessed, including the effects of: blood anticoagulant, serum-containing media, incubation time, ligand storage, blood storage time, and cell cryopreservation. By using optimized assay conditions, effective concentrations of individual ligands and mean responses to those ligands were established for healthy control donors. Finally, three patients with a mutation in the IKBKG gene...

‣ Evaluation of Various Methods of Maternal Placental Blood Collection for Immunology Studies

Othoro, Caroline; Moore, Julie M.; Wannemuehler, Kathleen; Nahlen, Bernard L.; Otieno, Juliana; Slutsker, Laurence; Lal, Altaf A.; Shi, Ya Ping
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /05/2006 Português
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The collection of maternal placental intervillous blood (IVB), without contamination of fetal blood and with an accurate mononuclear cell profile, is essential for immunological studies of placental malaria and other infectious diseases of the placenta. We have compared five documented methods of IVB collection: perfusion, incision, biopsy, tissue grinding, and puncture (prick) for fetal blood contamination and mononuclear cell profiles using flow cytometry. Twenty-five placentas were obtained from Plasmodium falciparum and human immunodeficiency virus-negative primigravid and secundigravid women delivering at Nyanza Provincial Hospital in Kisumu, western Kenya. Each of the five methods was performed on the same placenta. Fetal red blood cell contamination was significantly lower for the prick and perfusion methods (4.1% and 8.3%, respectively) than for incision (59.5%), biopsy (42.6%), and tissue grinding (19.9%). Significant variation was noted among the five methods in the percentages of monocytes, total T cells, CD4+ and CD8+ T cells, B cells, and NK cells. Further, a pairwise comparison of prick and perfusion, the two methods with low fetal blood contamination, showed that they were not different for fetal red blood cell contamination levels; however...

‣ Limited Diagnostic Capacities of Two Commercial Assays for the Detection of Leptospira Immunoglobulin M Antibodies in Laos

Blacksell, Stuart D.; Smythe, Lee; Phetsouvanh, Rattanaphone; Dohnt, Michael; Hartskeerl, Rudy; Symonds, Meegan; Slack, Andrew; Vongsouvath, Manivanh; Davong, Viengmone; Lattana, Olay; Phongmany, Simmaly; Keolouangkot, Valy; White, Nicholas J.; Day, Nicho
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /10/2006 Português
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The diagnostic utility of immunochromatographic (Leptotek) and enzyme-linked immunosorbent assay (ELISA; Panbio) tests for the detection of Leptospira immunoglobulin M antibodies was assessed in febrile adults admitted in Vientiane, Laos. Both tests demonstrated poor diagnostic accuracy using admission serum (Leptotek sensitivity of 47.3% and specificity of 75.5%: ELISA sensitivity of 60.9% and specificity of 65.6%) compared to the Leptospira “gold standard” microscopic agglutination test.

‣ Accuracy of Enzyme-Linked Immunosorbent Assay Using Crude and Purified Antigens for Serodiagnosis of Melioidosis▿

Chantratita, Narisara; Wuthiekanun, Vanaporn; Thanwisai, Aunchalee; Limmathurotsakul, Direk; Cheng, Allen C.; Chierakul, Wirongrong; Day, Nicholas P. J.; Peacock, Sharon J.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Português
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78.169653%
Five enzyme-linked immunosorbent assays developed to detect antibodies to different Burkholderia pseudomallei antigen preparations were evaluated as diagnostic tests for melioidosis in northeast Thailand. The highest diagnostic indices were observed for an affinity-purified antigen (sensitivity, 82%; specificity, 72%) and crude B. pseudomallei antigen (sensitivity, 81%; specificity, 70%), an improvement over the indirect hemagglutination assay (sensitivity, 73%; specificity, 64%).

‣ Evaluation of an Indirect Immunofluorescence Assay for Strongyloidiasis as a Tool for Diagnosis and Follow-Up▿

Boscolo, Marina; Gobbo, Maria; Mantovani, William; Degani, Monica; Anselmi, Mariella; Monteiro, Geraldo Badona; Marocco, Stefania; Angheben, Andrea; Mistretta, Manuela; Santacatterina, Maria; Tais, Stefano; Bisoffi, Zeno
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Português
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The diagnostic accuracy of an indirect immunofluorescence antibody test (IFAT) for Strongyloides stercoralis at different serum antibody titers was evaluated. To assess diagnostic sensitivity, sera from 156 patients with known strongyloidiasis were collected. Negative control sera were obtained from a composite group of 427 subjects (blood donors and hospitalized patients). With an area under the receiver-operating characteristic plot of 0.98, the IFAT showed a high level of diagnostic accuracy for strongyloidiasis. An antibody titer of ≥1:20, with 97% sensitivity and 98% specificity, was identified as the diagnostic threshold with the best overall performance. Cross-reactions were evaluated with 41 additional samples from patients with other known helminth infections, and the IFAT detected low-titer positivity in only one subject with filariasis. A positive IFAT result at an antibody dilution of ≥1:80 was virtually 100% specific, with 71% sensitivity. To test the usefulness of the IFAT as a monitoring tool, the changes in specific-antibody titers after treatment in a group of 155 patients were evaluated. Seroreversion or a decrease in antibody titer of twofold or more was observed in 60% of the patients. Response to treatment was directly correlated to the initial antibody titer...

‣ Evaluation of Dried Whole Blood Spots Obtained by Heel or Finger Stick as an Alternative to Venous Blood for Diagnosis of Human Immunodeficiency Virus Type 1 Infection in Vertically Exposed Infants in the Routine Diagnostic Laboratory▿

Patton, Janet C.; Akkers, Eveline; Coovadia, Ashraf H.; Meyers, Tammy M.; Stevens, Wendy S.; Sherman, Gayle G.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Português
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88.55945%
The diagnostic accuracy of the Roche Amplicor human immunodeficiency virus type 1 DNA PCR assay (version 1.5) on DNA extracted from pediatric heel prick dried blood spots using Roche MagNA Pure nucleic acid purification technology was evaluated. The methodologies transfer successfully from the labor-intensive research laboratory to the high-throughput automated routine laboratory.

‣ Quantification of 1,3-β-d-Glucan Levels in Children: Preliminary Data for Diagnostic Use of the β-Glucan Assay in a Pediatric Setting▿

Smith, P. Brian; Benjamin, Daniel K.; Alexander, Barbara D.; Johnson, Melissa D.; Finkelman, Malcolm A.; Steinbach, William J.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
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78.154824%
1,3-β-d-Glucan serum levels have demonstrated good diagnostic sensitivity and specificity for the diagnosis of candidiasis in adult patients, but normal levels for children have not been established. We found higher 1,3-β-d-glucan levels in children than those previously reported in adults.

‣ Epitope Length, Genospecies Dependency, and Serum Panel Effect in the IR6 Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Borrelia burgdorferi▿

Gomes-Solecki, Maria J. C.; Meirelles, Luciana; Glass, John; Dattwyler, Raymond J.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
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78.217207%
In the absence of erythema migrans, the basis for diagnosis of Lyme disease is the demonstration of an antibody response against Borrelia burgdorferi in an appropriate clinical setting. The C6 enzyme-linked immunosorbent assay, based on the IR6 region of VlsE, has become widely used in both the United States and Europe. We mapped the antigenic epitopes of IR6 to a shorter sequence that is equivalent in sensitivity and specificity to the full-length IR6 25-residue peptide. In addition, we observed significant differences in sensitivity between serum panels (60 to 100%), indicating that the selection of the serum panels can shape the apparent overall sensitivity of the assay. Contrary to prior reports, the assay sensitivity is greater when the IR6 peptide is derived from the sequence of the same infecting Borrelia genospecies. Using our North American panels and the two panels obtained from European Lyme disease patients, we determined that the IR6 assay that is based on a single genospecies of Borrelia spp. is not optimal for use as a universal diagnostic assay for Lyme disease.

‣ Rapid Detection of Hepatitis B Virus Surface Antigen by an Agglutination Assay Mediated by a Bispecific Diabody against Both Human Erythrocytes and Hepatitis B Virus Surface Antigen▿

Chen, Yu-Ping; Qiao, Yuan-Yuan; Zhao, Xiao-Hang; Chen, Hong-Song; Wang, Yan; Wang, Zhuozhi
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Português
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78.057256%
Bispecific antibodies have immense potential for use in clinical applications. In the present study, a bispecific diabody against human red blood cells (RBCs) and hepatitis B virus surface antigen (HBsAg) was used to detect HBsAg in blood specimens. The bispecific diabody was constructed by crossing over the variable region of the heavy chains and the light chains of anti-RBC and anti-HBsAg antibodies with a short linker, SRGGGS. In enzyme-linked immunosorbent assays, this bispecific diabody showed specific binding to both RBCs and HBsAg. When this bispecific diabody was mixed with human blood specimens in the presence of HBsAg, the dual binding sites of the diabody caused agglutination of human RBCs. This diabody-mediated agglutination assay was then used to test 712 clinical blood specimens and showed 97.7% sensitivity and 100% specificity when the results were compared with those of the conventional immunoassay, which was used as a reference. This autologous RBC agglutination assay provides a simple approach for rapid screening for HBsAg in blood specimens.

‣ Development of Recombinant Nucleoprotein-Based Diagnostic Systems for Lassa Fever▿

Saijo, Masayuki; Georges-Courbot, Marie-Claude; Marianneau, Philippe; Romanowski, Victor; Fukushi, Shuetsu; Mizutani, Tetsuya; Georges, Alain-Jean; Kurata, Takeshi; Kurane, Ichiro; Morikawa, Shigeru
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
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Diagnostic systems for Lassa fever (LF), a viral hemorrhagic fever caused by Lassa virus (LASV), such as enzyme immunoassays for the detection of LASV antibodies and LASV antigens, were developed using the recombinant nucleoprotein (rNP) of LASV (LASV-rNP). The LASV-rNP was expressed in a recombinant baculovirus system. LASV-rNP was used as an antigen in the detection of LASV-antibodies and as an immunogen for the production of monoclonal antibodies. The LASV-rNP was also expressed in HeLa cells by transfection with the expression vector encoding cDNA of the LASV-NP gene. An immunoglobulin G enzyme-linked immunosorbent assay (ELISA) using LASV-rNP and an indirect immunofluorescence assay using LASV-rNP-expressing HeLa cells were confirmed to have high sensitivity and specificity in the detection of LASV-antibodies. A novel monoclonal antibody to LASV-rNP, monoclonal antibody 4A5, was established. A sandwich antigen capture (Ag-capture) ELISA using the monoclonal antibody and an anti-LASV-rNP rabbit serum as capture and detection antibodies, respectively, was then developed. Authentic LASV nucleoprotein in serum samples collected from hamsters experimentally infected with LASV was detected by the Ag-capture ELISA. The Ag-capture ELISA specifically detected LASV-rNP but not the rNPs of lymphocytic choriomeningitis virus or Junin virus. The sensitivity of the Ag-capture ELISA in detecting LASV antigens was comparable to that of reverse transcription-PCR in detecting LASV RNA. These LASV rNP-based diagnostics were confirmed to be useful in the diagnosis of LF even in institutes without a high containment laboratory...

‣ Dominant Epitopes of the C6 Diagnostic Peptide of Borrelia burgdorferi Are Largely Inaccessible to Antibody on the Parent VlsE Molecule▿

Embers, Monica E.; Jacobs, Mary B.; Johnson, Barbara J. B.; Philipp, Mario T.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Português
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Lyme borreliosis (LB) is a disease for which antibody-based detection assays are often required for diagnosis. The variable surface molecule VlsE and IR6, one of its invariable regions, are commonly targeted by the antibody response in infected individuals. A series of enzyme-linked immunosorbent assays was performed to comparatively examine the antibody responses of North American LB patients (n = 37) to VlsE and invariable segments of this molecule. Both immunoglobulin M (IgM) and IgG responses to full-length VlsE and to peptides reproducing invariable regions 2, 4, and 6, as well as the invariable domains at the amino and carboxyl termini of VlsE, were assessed. The proportions and specificities of reactivity to the invariable segments were tested by using cognate peptides as competitors for VlsE binding by patient serum antibodies. IR6 epitopes (by the C6 peptide) were found to dominate the response to invariable segments. IR6 (C6)-specific antibodies were detected in 78% of the serum specimens, whereas <40% of patients generated antibodies that bound the N- or C-terminal domain and <12% of patients responded to either IR2 or IR4. Interestingly, 15 of 37 patients generated IgG antibodies that reacted with C6 but not with VlsE. Conversely...

‣ Single Antigen Detects both Immunoglobulin M (IgM) and IgG Antibodies Elicited by All Four Dengue Virus Serotypes▿ †

Hapugoda, Menaka D.; Batra, Gaurav; Abeyewickreme, W.; Swaminathan, S.; Khanna, N.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
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The resurgence of dengue (DEN) virus infections in the last few decades coupled with the lack of a preventive vaccine and specific antiviral drugs has jointly contributed to making this a significant global public health problem. Currently, symptomatic supportive treatment and fluid replacement therapy are the only means available to minimize DEN-induced mortality. As the clinical symptoms associated with DEN virus infections are indistinguishable from those of many other viral, bacterial, and parasitic infections, specific diagnostic tests assume critical importance in the unequivocal identification of DEN virus infections. We have designed a novel chimeric antigen based on envelope domain III (EDIII), a critical antigenic region of the major structural protein of DEN viruses. We fused EDIIIs corresponding to each of the four DEN virus serotypes using pentaglycyl linkers, overexpressed the resultant tetravalent chimeric protein in Escherichia coli, and affinity purified it in high yields, obtaining ∼30 mg protein of >95% purity per liter of culture. We show that this tetravalent antigen could specifically recognize anti-DEN virus antibodies of both the immunoglobulin M (IgM) and IgG classes. Using a large panel of IgM antibody capture-enzyme-linked immunosorbent assay- and hemagglutination inhibition-confirmed DEN virus-infected and uninfected patient sera (n = 289)...

‣ Use of Saccharomyces cerevisiae-Expressed Recombinant Nucleocapsid Protein To Detect Hantaan Virus-Specific Immunoglobulin G (IgG) and IgM in Oral Fluid▿

Petraitytė, Rasa; Jin, Li; Hunjan, Rashpal; Ražanskienė, Aušra; Žvirblienė, Aurelija; Sasnauskas, Kęstutis
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
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Hantaan virus is the causative agent of severe hemorrhagic fever with renal syndrome. Clinical surveillance for Hantaan virus infection is unreliable, and laboratory verification is essential. The detection of virus-specific immunoglobulin M (IgM) and IgG in serum is most commonly used for the diagnosis of hantavirus infection. Testing of oral fluid samples instead of serum offers many advantages for surveillance. However, commercial tests for hantavirus-specific antibodies are unavailable. For the detection of Hantaan virus in the oral fluid of humans, we have developed a monoclonal antibody-based capture enzyme-linked immunosorbent IgM assay (IgM capture ELISA) and indirect enzyme-linked immunosorbent IgG and IgM assays (indirect IgG and IgM ELISAs) for paired serum and oral fluid samples using the Saccharomyces cerevisiae yeast-expressed nucleocapsid protein of the Hantaan-Fojnica virus. The sensitivity and specificity of the oral fluid IgM capture ELISA in comparison with the results of the serum Hantaan virus IgM assay were 96.7% and of 94.9%, respectively. Thus, data on the overall performance of the oral fluid IgM capture ELISA are in close agreement with those of the serum IgM assay, and the method exhibits the potential to serve as an easily transferable tool for large-scale epidemiological studies. Data on the indirect IgM ELISA also showed close agreement with the serum IgM assay data; however...

‣ Utility of Composite Reference Standards and Latent Class Analysis in Evaluating the Clinical Accuracy of Diagnostic Tests for Pertussis▿

Baughman, Andrew L.; Bisgard, Kristine M.; Cortese, Margaret M.; Thompson, William W.; Sanden, Gary N.; Strebel, Peter M.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
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Numerous evaluations of the clinical sensitivity and specificity of PCR and serologic assays for Bordetella pertussis have been hampered by the low sensitivity of culture, the gold standard test, which leads to biased accuracy estimates. The bias can be reduced by using statistical approaches such as the composite reference standard (CRS) (e.g., positive if culture or serology positive; negative otherwise) or latent class analysis (LCA), an internal reference standard based on a statistical model. We illustrated the benefits of the CRS and LCA approaches by reanalyzing data from a 1995 to 1996 study of cough illness among 212 patients. The accuracy of PCR in this study was evaluated using three reference standards: culture, CRS, and LCA. Using specimens obtained 0 to 34 days after cough onset, estimates of the sensitivity of PCR obtained using CRS (47%) and LCA (34%) were lower than the culture-based estimate (62%). The CRS and LCA approaches, which utilized more than one diagnostic marker of pertussis, likely produced more accurate reference standards than culture alone. In general, the CRS approach is simple, with a well-defined disease status. LCA requires statistical modeling but incorporates more indicators of disease than CRS. When three or more indicators of pertussis are available...

‣ Usefulness of Four Different Echinococcus granulosus Recombinant Antigens for Serodiagnosis of Unilocular Hydatid Disease (UHD) and Postsurgical Follow-Up of Patients Treated for UHD▿

Hernández-González, Ana; Muro, Antonio; Barrera, Inmaculada; Ramos, Guillermo; Orduña, Antonio; Siles-Lucas, Mar
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
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Four different recombinant antigens derived from Echinococcus granulosus, designated B1t, B2t, E14t, and C317, were tested with enzyme-linked immunosorbent assays (ELISAs) for the detection of specific immunoglobulin G (IgG) in patients with unilocular hydatid disease (UHD). The results were compared to those obtained with hydatid fluid and were subjected to receiver operator characteristic analysis. The diagnostic performance of the above-listed proteins was defined with respect to their specificity, sensitivity, and predictive values (PV); the influence of cyst location; and usefulness in the follow-up of surgical treatment for UHD and in the determination of whether or not patients have been surgically cured of UHD. The best diagnostic results were obtained with the anti-B2t IgG ELISA, with 91.2% sensitivity, 93% specificity, and high positive and negative PV (89.4 and 94.2, respectively). In addition, this diagnostic tool proved to be useful for the follow-up of surgically treated UHD patients. The anti-B2t IgG ELISA may find an application in the serodiagnosis of UHD in clinical laboratories.

‣ Peripheral Blood Gamma Interferon Release Assays Predict Lung Responses and Mycobacterium tuberculosis Disease Outcome in Mice▿

Beamer, Gillian L.; Flaherty, David K.; Vesosky, Bridget; Turner, Joanne
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
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78.197563%
Current diagnostic tests for tuberculosis (TB) are not able to distinguish active disease from latent Mycobacterium tuberculosis infection, nor are they able to quantify the risk of a latently infected person progressing to active TB. There is interest, however, in adapting antigen-specific gamma interferon (IFN-γ) release assays (IGRAs) to predict disease outcome. In this study, we used the differential susceptibilities of inbred mouse strains to M. tuberculosis infection to evaluate the prognostic capabilities of IGRAs. Using lung and blood cultures, we determined that CBA/J, DBA/2, and C3H/HeJ mice (models of heightened risk of progression to active TB) produced less antigen-specific IFN-γ in response to M. tuberculosis culture filtrate proteins and early secreted antigenic target-6 than the relatively resistant C57BL/6 mouse strain. Additionally, reduced IFN-γ secretion in supernatants reflected a reduced frequency of IFN-γ-responding cells in the lung and blood and not a specific defect in IFN-γ secretion at the single-cell level. Importantly, detection of antigen-specific IFN-γ from blood cultures accurately reflected lung responses, indicating that blood can be an appropriate test tissue in humans. Furthermore, reduced antigen-specific IFN-γ production and low frequencies of IFN-γ-responding cells from peripheral blood predicted increased risk of TB disease progression across genetically diverse TB disease-susceptible mouse strains...

‣ Clinical and Diagnostic Developments of a Gamma Interferon Release Assay for Use in Bovine Tuberculosis Control Programs

Bass, K. E.; Nonnecke, B. J.; Palmer, M. V.; Thacker, T. C.; Hardegger, R.; Schroeder, B.; Raeber, A. J.; Waters, W. R.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/2013 Português
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Currently, the Bovigam assay is used as an official supplemental test within bovine tuberculosis control programs. The objectives of the present study were to evaluate two Mycobacterium bovis-specific peptide cocktails and purified protein derivatives (PPDs) from two sources, liquid and lyophilized antigen preparations. PPDs and peptide cocktails were also used for comparison of a second-generation gamma interferon (IFN-γ) release assay kit with the currently licensed first-generation kit (Bovigam; Prionics AG). Three strains of M. bovis were used for experimental challenge: M. bovis 95-1315, M. bovis Ravenel, and M. bovis 10-7428. Additionally, samples from a tuberculosis-affected herd (i.e., naturally infected) were evaluated. Robust responses to both peptide cocktails, HP (PC-HP) and ESAT-6/CFP10 (PC-EC), and the PPDs were elicited as early as 3 weeks after challenge. Only minor differences in responses to Commonwealth Serum Laboratories (CSL) and Lelystad PPDs were detected with samples from experimentally infected animals. For instance, responses to Lelystad M. avium-derived PPD (PPDa) exceeded the respective responses to the CSL PPDa in M. bovis Ravenel-infected and control animals. However, a 1:4 dilution of stimulated plasma demonstrated greater separation of PPDb from PPDa responses (i.e....

‣ Clinical Value of Multiplexed Bead-Based Immunoassays for Detection of Autoantibodies to Nuclear Antigens▿

Avaniss-Aghajani, Erik; Berzon, Sophia; Sarkissian, Arlen
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Português
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The advent of multiplexed bead assays in recent years has introduced a new dimension of testing for complex diseases such as lupus, which can involve multiple autoantibodies. The ability to rapidly identify multiple autoantibodies, with high sensitivity and specificity in an automated fashion, is highly attractive. The aim of this study was to assess the performance and clinical value of multiplexed bead-based (AtheNA Multi-Lyte ANA-II test system) immunoassays both by comparing the results with those achieved by indirect fluorescent-antibody assay (IFA) or conventional enzyme immunoassays (EIAs) and by independent identification of autoantibodies in well-characterized samples. To achieve this goal, 984 samples were tested for seven analytes (SS/A, SS/B, Sm, RNP, Scl-70, double-stranded DNA [dsDNA], and centromere B) in both traditional and bead-based assays. The average concordance for the different analytes was 91%, ranging from 81% (dsDNA) to 97% (centromere B). The average relative specificity and sensitivity for the analytes were also high, 92% and 81%, respectively. An examination of 93 “normal controls” demonstrated a 7% false-positive rate, which was comparable to IFA. Percentages of different autoantibodies found in patients with a variety of disease conditions (34 with calcinosis...

‣ Use of Whole-Blood Samples in In-House Bulk and Single-Cell Antigen-Specific Gamma Interferon Assays for Surveillance of Mycobacterium tuberculosis Infections▿

Palazzo, Raffaella; Spensieri, Fabiana; Massari, Marco; Fedele, Giorgio; Frasca, Loredana; Carrara, Stefania; Goletti, Delia; Ausiello, Clara M.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
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78.064883%
Tests based on the gamma interferon (IFN-γ) assay (IGA) are used as adjunctive tools for the diagnosis of Mycobacterium tuberculosis infection. Here we compared in-house and commercial whole-blood IGAs to identify a suitable assay for the surveillance of tuberculosis in population studies. The IGAs were selected on the basis of the ease with which they are performed and because they require a small amount of a biological sample and do not require cell purification. Since a “gold standard” for latently M. tuberculosis-infected individuals is not available, the sensitivities and the specificities of the IGAs were determined with samples from patients with clinically diagnosed active tuberculosis and in Mycobacterium bovis BCG-unvaccinated healthy controls. The in-house tests consisted of a bulk assay based on diluted whole blood and a single-cell assay based on IFN-γ intracellular staining. The commercial assays used were the QuantiFERON-TB-Gold (Q-TB) and the Q-TB in-tube tests. When the purified protein derivative was used as the antigen, in-house whole-blood intracellular staining was found to be highly discriminatory between active tuberculosis patients and BCG-vaccinated healthy controls, whereas the other IGAs did not discriminate between the two categories of patients. When M. tuberculosis-specific antigens were used...

‣ Risk and Protective Factors for Leprosy Development Determined by Epidemiological Surveillance of Household Contacts▿

Goulart, Isabela M. B.; Bernardes Souza, Dulcinéa O.; Marques, Carolina R.; Pimenta, Vânia L.; Gonçalves, Maria A.; Goulart, Luiz R.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
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Household contacts of leprosy patients are the group with the highest risk of developing the disease, and although many risk or prevention factors have been identified, they have not been employed in leprosy-monitoring programs. This investigation aimed to establish the relative risks or the preventive effects of the presence of BCG vaccination, the Mitsuda test, and the ML-Flow assay. Household contacts (1,396) were monitored for a 5-year period. Twenty-eight contacts (2%) developed leprosy and had their clinical and operational classifications established. All immunological tests were performed, and intradermal BCG vaccination was given after the BCG scar count. Of the affected contacts, 75% developed the disease in the first year, and 71.4% were classified as having paucibacillary forms. Contacts of lepromatous leprosy patients presented a 3.8-fold-higher risk of developing leprosy. BCG vaccination and the Mitsuda test showed a protective effect against leprosy of 0.27 (at least one scar) and 0.16 (>7 mm), respectively, and the positive ML-Flow test indicated a relative risk approximately sixfold higher for occurrence of the disease. All unfavorable combinations of two and three assays generated significant risk values that ranged from 5.76 to 24.47...