Evaluation of Recombinant Proteins of Burkholderia mallei for Serodiagnosis of Glanders

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.

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In Vivo-Expressed Proteins of Virulent Leptospira interrogans Serovar Autumnalis N2 Elicit Strong IgM Responses of Value in Conclusive Diagnosis

Clinical and Vaccine Immunology ◽

10.1128/cvi.00509-15 ◽

2015 ◽

Vol 23 (1) ◽

pp. 65-72 ◽

Cited By ~ 8

Author(s):

Veerapandian Raja ◽

Santhanam Shanmughapriya ◽

Murugesan Kanagavel ◽

Sergey C. Artiushin ◽

Sridhar Velineni ◽

...

Keyword(s):

Sensitivity And Specificity ◽

Recombinant Proteins ◽

Natural Infection ◽

Enzyme Linked Immunosorbent Assay ◽

Leptospira Interrogans ◽

Content Type ◽

Igm Elisa ◽

Conclusive Diagnosis

ABSTRACTLeptospirosis is a serious zoonosis that is underdiagnosed because of limited access to laboratory facilities in Southeast Asia, Central and South America, and Oceania. Timely diagnosis of locally distributed serovars of high virulence is crucial for successful care and outbreak management. Using pooled patient sera, an expression gene library of a virulentLeptospira interrogansserovar Autumnalis strain N2 isolated in South India was screened. The identified genes were characterized, and the purified recombinant proteins were used as antigens in IgM enzyme-linked immunosorbent assay (ELISA) either singly or in combination. Sera (n= 118) from cases of acute leptospirosis along with sera (n= 58) from healthy subjects were tested for reactivity with the identified proteins in an ELISA designed to detect specific IgM responses. We have identified nine immunoreactive proteins, ArgC, RecA, GlpF, FliD, TrmD, RplS, RnhB, Lp28.6, and Lrr44.9, which were found to be highly conserved among pathogenic leptospires. Apparently, the proteins ArgC, RecA, GlpF, FliD, TrmD, and Lrr44.9 are expressed during natural infection of the host and undetectable inin vitrocultures. Among all the recombinant proteins used as antigens in IgM ELISA, ArgC had the highest sensitivity and specificity, 89.8% and 95.5%, respectively, for the conclusive diagnosis of leptospirosis. The use of ArgC and RecA in combination for IgM ELISA increased the sensitivity and specificity to 95.7% and 94.9%, respectively. ArgC and RecA thus elicited specific IgM responses and were therefore effective in laboratory confirmation ofLeptospirainfection.

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Use of a Recombinant Burkholderia Intracellular Motility A Protein for Immunodiagnosis of Glanders

Clinical and Vaccine Immunology ◽

10.1128/cvi.05185-11 ◽

2011 ◽

Vol 18 (9) ◽

pp. 1456-1461 ◽

Cited By ~ 13

Author(s):

Subodh Kumar ◽

Praveen Malik ◽

Shailendra Kumar Verma ◽

Vijai Pal ◽

Vandana Gautam ◽

...

Keyword(s):

Indirect Elisa ◽

High Specificity ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Healthy Human ◽

Content Type ◽

Elisa Format ◽

Low Sensitivity ◽

Rapid Tool ◽

Intracellular Motility

ABSTRACTGlanders, caused by the Gram-negative, nonmotile bacteriumBurkholderia mallei, is a contagious and highly fatal disease of equines. During the last decade, the number of glanders outbreaks has increased steadily. The disease also has high zoonotic significance andB. malleiis listed biological warfare agent. The complement fixation test (CFT) is a routinely used and internationally recognized test to screen equine sera for the glanders. However, discrepant results have been observed using the CFT. The low sensitivity and specificity of the CFT and enzyme-linked immunosorbent assay (ELISA) have been linked to the use of crude test antigens. We expressed a novel recombinantBurkholderiaintracellular motility A (rBimA) protein inEscherichia colifor the diagnosis of equine glanders. Purified rBimA was used in an indirect ELISA format. All of the 21 true-positive serum samples used in the study tested positive, whereas only 17 of the 1,524 potentially negative sera tested positive by indirect ELISA, thus exhibiting 100% sensitivity and 98.88% specificity. Also, rBimA protein did not react with melioidosis patient and normal healthy human serum samples, showing its high specificity. The developed assay can be used as a simple and rapid tool for diagnosis of glanders in equine serum samples. An Indian patent (1328/DEL/2010) has been filed for the reagent.

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Sensitive and Specific Serodiagnosis of Leishmania infantum Infection in Dogs by Using Peptides Selected from Hypothetical Proteins Identified by an Immunoproteomic Approach

Clinical and Vaccine Immunology ◽

10.1128/cvi.00023-13 ◽

2013 ◽

Vol 20 (6) ◽

pp. 835-841 ◽

Cited By ~ 22

Author(s):

Miguel A. Chávez-Fumagalli ◽

Vivian T. Martins ◽

Miriam C. S. Testasicca ◽

Daniela P. Lage ◽

Lourena E. Costa ◽

...

Keyword(s):

Sensitivity And Specificity ◽

Leishmania Infantum ◽

False Negative ◽

Hypothetical Proteins ◽

Content Type ◽

Negative Results ◽

False Negative Results ◽

Serological Studies ◽

Potential Tool ◽

Higher Sensitivity

ABSTRACTIn Brazil, the percentage of infected dogs living in areas where canine visceral leishmaniasis (CVL) is endemic ranges from 10 to 62%; however, the prevalence of infection in dogs is probably higher than figures reported from serological studies. In addition, problems with the occurrence of false-positive or false-negative results in the serodiagnosis of CVL have been reported. The present work analyzed the potential of synthetic peptides mapped from hypothetical proteins for improvement of the serodiagnosis ofLeishmania infantuminfection in dogs. From 26 identified leishmanial proteins, eight were selected, considering that no homologies between these proteins and others from trypanosomatide sequence databases were encountered. The sequences of these proteins were mapped to identify linear B-cell epitopes, and 17 peptides were synthesized and tested in enzyme-linked immunosorbent assays (ELISAs) for the serodiagnosis ofL. infantuminfection in dogs. Of these, three exhibited sensitivity and specificity values higher than 75% and 90%, respectively, to differentiateL. infantum-infected animals fromTrypanosoma cruzi-infected animals and healthy animals. SolubleLeishmaniaantigen (SLA) showed poor sensitivity (4%) and specificity (36%) to differentiateL. infantum-infected dogs from healthy andT. cruzi-infected dogs. Lastly, the three selected peptides were combined in different mixtures and higher sensitivity and specificity values were obtained, even when sera fromT. cruzi-infected dogs were used. The study's findings suggest that these three peptides can constitute a potential tool for more sensitive and specific serodiagnosis ofL. infantuminfection in dogs.

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Serodiagnosis of Equine Leptospirosis by Enzyme-Linked Immunosorbent Assay Using Four Recombinant Protein Markers

Clinical and Vaccine Immunology ◽

10.1128/cvi.00649-13 ◽

2014 ◽

Vol 21 (4) ◽

pp. 478-483 ◽

Cited By ~ 16

Author(s):

Cuilian Ye ◽

Weiwei Yan ◽

Patrick L. McDonough ◽

Sean P. McDonough ◽

Hussni Mohamed ◽

...

Keyword(s):

Sensitivity And Specificity ◽

Recombinant Proteins ◽

Indirect Elisa ◽

Zoonotic Diseases ◽

Agglutination Test ◽

Enzyme Linked Immunosorbent Assay ◽

Leptospira Interrogans ◽

Protein Markers ◽

Content Type ◽

The World

ABSTRACTLeptospirosis, caused byLeptospiraspp., is one of the most common zoonotic diseases in the world. We tested four recombinant proteins ofLeptospira interrogans, namely, rLipL21, rLoa22, rLipL32, and rLigACon4-8, to evaluate their potential for use as antigens for the diagnosis of equine leptospirosis. We employed equine sera (n= 130) that were microscopic agglutination test (MAT) negative and sera (n= 176) that were MAT positive for the 5 serovars that most commonly cause equine leptospirosis. The sensitivity and specificity of ELISA compared to MAT were 82.39% and 86.15%, respectively, for LigACon4-8, 77.84% and 92.31%, respectively, for Loa22, 77.84% and 86.15%, respectively, for LipL32, and 84.66% and 83.85%, respectively, for LipL21. When one of the two antigens was test positive, the sensitivity and specificity of ELISA were 93.75% and 78.46%, respectively, for rLigACon4-8 and LipL32, 93.18% and 76.15%, respectively, for rLigACon4-8 and LipL21, 89.77% and 80.77%, respectively, for rLigACon4-8 and Loa22, 91.48% and 78.46%, respectively, for LipL21 and Loa22, 93.75% and 76.92%, respectively, for LipL21 and LipL32, and 90.34% and 80.77%, respectively, for Loa22 and LipL32. In conclusion, we have developed an indirect ELISA utilizing rLigACon4-8, rLoa22, rLipL32, and rLipL21 as diagnostic antigens for equine leptospirosis. The use of four antigens in the ELISA was found to be sensitive and specific, the assay was easy to perform, and the results concurred with the results of the standardLeptospiraMAT.

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Diagnostic Performance of Ankle-Brachial Pressure Index in Lower Extremity Arterial Disease

The Surgery Journal ◽

10.1055/s-0041-1731444 ◽

2021 ◽

Vol 07 (03) ◽

pp. e132-e137

Author(s):

Mohammed Alagha ◽

Thomas M. Aherne ◽

Ahmed Hassanin ◽

Adeel S. Zafar ◽

Doireann P. Joyce ◽

...

Keyword(s):

Lower Extremity ◽

Sensitivity And Specificity ◽

False Negative ◽

Arterial Disease ◽

Initial Assessment ◽

Reference Standard ◽

Lower Extremity Arterial Disease ◽

Negative Results ◽

Positive Results ◽

Arterial Imaging

Abstract Introduction Ankle-brachial pressure indices (ABIs) continue to form the basis of diagnostics for lower extremity arterial disease (LEAD). However, there remains a paucity of data to support its accuracy. This study aims to evaluate its diagnostic sensitivity and specificity using established arterial-imaging modalities as a benchmark. Methods In this retrospective study, a regional, prospectively maintained, vascular laboratory database was interrogated to identify referred patients with arterial disease who underwent concomitant assessment with ABI and lower limb arterial duplex ultrasound (DUS). Duplex acted as the reference standard. Those who had peripheral computed tomography angiogram (CTA) within 3 months of initial assessment were included in a subgroup analysis to correlate ABI with CTA. The primary end point was the sensitivity and specificity of ABI compared with DUS as the reference standard. Results Concomitant assessment was performed in 438 limbs (250 patients) over a 27-month period. The ABI was normal (0.9 to 1.4) in 196 limbs (44.9%) and abnormal in the remaining 241 limbs (55.1%). False-positive results occurred in 83 out of 241 limbs (34.4%), and false-negative results occurred in 54 limbs out of 196 (27.5%). True-positive results were 158 out of 241 limbs (65.6%), whereas true-negative results were 142 out of 196 limbs (72.4%). ABI using DUS as a benchmark identified a sensitivity for peripheral artery disease of 72.3% and a specificity of 69.3%. Concomitant CTA imaging was available in 200 limbs. The sensitivity and specificity of ABI correlated with CTA were 65.5 and 68.8%, respectively. Conclusion ABIs have a moderate predictive value in the diagnosis of LEAD. Normal range outcomes cannot be taken to infer the absence of LEAD and, as such, further arterial imaging in the form of DUS or angiography should be strongly considered in those with suspected underlying disease requiring intervention. Further noninvasive tests such as exercise studies or pulse volume waveforms should be considered, if diagnostic uncertainty exists, in those requiring nonoperative intervention and risk factor control.

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Evaluation of the Carba NP Test for Rapid Detection of Carbapenemase-Producing Enterobacteriaceae and Pseudomonas aeruginosa

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00878-13 ◽

2013 ◽

Vol 57 (9) ◽

pp. 4578-4580 ◽

Cited By ~ 147

Author(s):

Nathalie Tijet ◽

David Boyd ◽

Samir N. Patel ◽

Michael R. Mulvey ◽

Roberto G. Melano

Keyword(s):

Pseudomonas Aeruginosa ◽

Positive Predictive Value ◽

Negative Predictive Value ◽

Predictive Value ◽

Rapid Detection ◽

False Negative ◽

Bacterial Extract ◽

Content Type ◽

Negative Results ◽

False Negative Results

ABSTRACTThe Carba NP test was evaluated against a panel of 244 carbapenemase- and non-carbapenemase-producingEnterobacteriaceaeandPseudomonas aeruginosaisolates. We confirmed the 100% specificity and positive predictive value of the test, but the sensitivity and negative predictive value were 72.5% and 69.2%, respectively, and increased to 80% and 77.3%, respectively, using a more concentrated bacterial extract. False-negative results were associated with mucoid strains or linked to enzymes with low carbapenemase activity, particularly OXA-48-like, which has emerged globally in enterobacteria.

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Humoral Immune Responses in a Human Case of Glanders

Clinical and Vaccine Immunology ◽

10.1128/cvi.05567-11 ◽

2012 ◽

Vol 19 (5) ◽

pp. 814-816 ◽

Cited By ~ 2

Author(s):

David M. Waag ◽

Marilyn J. England ◽

David DeShazer

Keyword(s):

Immune Responses ◽

Burkholderia Pseudomallei ◽

Human Case ◽

Burkholderia Mallei ◽

Baseline Serum ◽

Content Type ◽

Whole Cells ◽

Humoral Immune ◽

Diagnostic Antigen ◽

Humoral Immune Responses

ABSTRACTWithin 2 months of acquiring glanders, a patient developed 8-, 16-, and 4-fold increases, respectively, in specific IgA, IgG, and IgM serological titers againstBurkholderia mallei. Within 14 months of infection, the titers decreased to the baseline. Serum from this patient was also highly reactive againstBurkholderia pseudomalleiwhole cells.Burkholderia malleiwhole cells did not react with sera from patients with other diseases. Therefore, an assay using aB. malleicellular diagnostic antigen may be useful for the serodiagnosis of glanders.

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Establishment of a New PNA-FISH Method for Aspergillus fumigatus Identification: First Insights for Future Use in Pulmonary Samples

Microorganisms ◽

10.3390/microorganisms8121950 ◽

2020 ◽

Vol 8 (12) ◽

pp. 1950

Author(s):

Laura Cerqueira ◽

Sara Moura ◽

Carina Almeida ◽

Maria João Vieira ◽

Nuno Filipe Azevedo

Keyword(s):

Aspergillus Fumigatus ◽

False Negative ◽

Clinical Samples ◽

Suitable Alternative ◽

Fish Method ◽

Negative Results ◽

Specificity And Sensitivity ◽

Pure Cultures ◽

Low Sensitivity ◽

Pna Fish

Aspergillus fumigatus is the main causative agent of Invasive Aspergillosis. This mold produces conidia that when inhaled by immunocompromized hosts can be deposited in the lungs and germinate, triggering disease. In this paper, the development of a method using peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) is described. The PNA-FISH probe was tested in several strains and a specificity and sensitivity of 100% was obtained. Detection of A. fumigatussensu stricto was then achieved in artificial sputum medium (ASM) pre-inoculated with 1 × 102 conidia·mL−1–1 × 103 conidia·mL−1, after a germination step of 24 h. The PNA-FISH method was evaluated in 24 clinical samples (10 sputum, 8 bronchoalveolar lavage (BAL), and 6 bronchial lavage (BL)) that were inoculated with 1 × 104 conidia·mL−1 in sputum; 1 × 103 conidia·mL−1 in BL and BAL, for 24 h. Despite a specificity of 100%, the sensitivity was 79%. This relatively low sensitivity can be explained by the fact that hyphae can yield “fungal ball“ clusters, hindering pipetting procedures and subsequent detection, leading to false negative results. Nonetheless, this study showed the potential of the PNA-FISH method for A. fumigatussensu stricto detection since it takes only 1 h 30 m to perform the procedure with a pre-enrichment step of 6 h (pure cultures) and 24 h (clinical samples), and might provide a suitable alternative to the existing methods for studies in pure cultures and in clinical settings.

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Routine intraoperative angiography during aneurysm surgery

Journal of Neurosurgery ◽

10.3171/jns.2002.96.6.0988 ◽

2002 ◽

Vol 96 (6) ◽

pp. 988-992 ◽

Cited By ~ 134

Author(s):

Veronica L. Chiang ◽

Phillipe Gailloud ◽

Kieran J. Murphy ◽

Daniele Rigamonti ◽

Rafael J. Tamargo

Keyword(s):

Surgical Treatment ◽

False Negative ◽

Anterior Communicating Artery ◽

Parent Vessel ◽

Vessel Occlusion ◽

Intraoperative Angiography ◽

Content Type ◽

Negative Results ◽

Parent Vessel Occlusion ◽

Fine Print

Object. The routine use of intraoperative angiography as an aid in the surgical treatment of aneurysms is uncommon. The advantages of the ability to visualize residual aneurysm or unintended occlusion of parent vessels intraoperatively must be weighed against the complications associated with repeated angiography and prolonged vascular access. The authors reviewed the results of their routine use of intraoperative angiography to determine its safety and efficacy. Methods. Prospectively gathered data from all aneurysm cases treated surgically between January 1996 and June 2000 were reviewed. A total of 303 operations were performed in 284 patients with aneurysms; 24 patients also underwent postoperative angiography. Findings on intraoperative angiographic studies prompted reexploration and clip readjustment in 37 (11%) of the 337 aneurysms clipped. Angiography revealed parent vessel occlusion in 10 cases (3%), residual aneurysm in 22 cases (6.5%), and both residual lesion and parent vessel occlusion in five cases (1.5%). When compared with subsequent postoperative imaging, false-negative results were found on two intraoperative angiograms (8.3%) and a false-positive result was found on one (4.2%). Postoperative angiograms obtained in both false-negative cases revealed residual anterior communicating artery aneurysms. Both of these aneurysms also subsequently rebled, requiring reoperation. In the group that underwent intraoperative angiography, in 303 operations eight patients (2.6%) suffered complications, of which only one was neurological. Conclusions. In the surgical treatment of intracranial aneurysms, the use of routine intraoperative angiography is safe and helpful in a significant number of cases, although it does not replace careful intraoperative inspection of the surgical field.

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Pearls and Pitfalls of Interpretation in CT Colonography

Canadian Association of Radiologists Journal ◽

10.1177/0846537119892881 ◽

2020 ◽

Vol 71 (2) ◽

pp. 140-148

Author(s):

Michael Schonberger ◽

Philippe Lefere ◽

Abraham H. Dachman

Keyword(s):

Computed Tomography ◽

Sensitivity And Specificity ◽

False Positive ◽

Ct Colonography ◽

False Negative ◽

High Sensitivity ◽

Negative Results ◽

False Negative Results ◽

The Common

The accuracy of computed tomography (CT) colonography (CTC) requires that the radiologist be well trained in the recognition of pitfalls of interpretation. In order to achieve a high sensitivity and specificity, the interpreting radiologist must be well versed in the causes of both false-positive and false-negative results. In this article, we review the common and uncommon pitfalls of interpretation in CTC.

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