Evaluation of the comparative accuracy of the complement fixation test, Western blot and five enzyme-linked immunosorbent assays for serodiagnosis of glanders

Elschner, Mandy C.; Laroucau, Karine; Singha, Harisankar; Tripathi, Bhupendra Nath; Saqib, Muhammad; Gardner, Ian A.; Saini, Sheetal; Kumar, Subodh; El-Adawy, Hosny; Melzer, Falk; Khan, Iahtasham; Malik, Praveen; Sauter‐Louis, Carola; Neubauer, Heinrich

doi:10.1371/journal.pone.0214963

Cited by 40 publications

(63 citation statements)

References 26 publications

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“…These samples were considered true positives because they were also positive in both CFT and B. mallei ELISA. Moreover, same sensitivity and specificity was observed in western blot as compared to ELISA ( Table 2) (18). The results show that glycosylated CtxB-BCAL2737a but the not unglycosylated form can be recognized by antibodies present in these serum samples (Fig.…”

Section: Glanders-infected Horse Serum Samples Contain Anti-o-glycan supporting

confidence: 70%

“…Successful control of glanders infection requires reliable diagnostic tools. Although numerous methods (9-11, 13, 16, 18, 35) have been described for diagnosis of glanders, some have limitations (9)(10)(11)18). Protein glycosylation, a post-translational modification that covalently attaches a glycan to a protein, is prevalent in Archaea and bacteria (36,37).…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, indirect ELISAs based on the intracellular motility A protein (BimA) (15,16), the type 6 secreted TssB (17) and Hcp1 (18) proteins, the heat shock protein GroEL (19) and a semi-purified fraction of B. mallei have been developed (11). However, these methods require more investigation using large-scale sample surveys and optimization to improve specificity and sensitivity (18). Previous work has identified a O-glycosylation (ogc) gene cluster in B. cenocepacia encoding enzymes for the synthesis of a lipid-linked trisaccharide, which consists of a β-Gal-(1,3)-α-GalNAc-(1,3)-β-GalNAc (20) and is incorporated in membrane-exported Burkholderia proteins by the PglL O-glycosyltransferase (O-Tase) (21).…”

Section: Complement Fixation Test (Cft) Is the Most Accurate And Relimentioning

confidence: 99%

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A glycoengineered antigen exploiting a conserved proteinO-glycosylation pathway in theBurkholderiagenus for diagnosis of glanders infections

Wang

Glaser

Scott

et al. 2020

Preprint

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We recently described a protein O-glycosylation pathway conserved in all species of the Burkholderia genus that results in synthesis and incorporation of a trisaccharide glycan to membrane-exported proteins. Here, we exploited this system to construct and evaluate a diagnostic tool for glanders. Burkholderia mallei, the causative agent of glanders, is a highly infectious and fatal zoonotic pathogen that mainly infects horses, mules, donkeys and occasionally humans. A highly sensitive and specific diagnostic tool is crucial for the control, elimination and eradication of B. mallei infections. We constructed plasmids carrying synthetic genes encoding a modified, previously unannotated Burkholderia glycoprotein containing three glycosylation sequons fused to the cholera toxin B-subunit. The resulting proteins were glycosylated in the B. cenocepacia K56-2 parental strain, but not in glycosylation-deficient mutants, as determined by SDS-PAGE and fluorescent lectin blots. One of these glycoproteins was used as an antigen in ELISA and western blots to screen a panel of serum samples collected from glanders-infected and healthy horses previously investigated by complement fixation test and indirect ELISA based on a semi-purified fraction of B. mallei. We show that ELISA and western blot assays based on our glycoprotein antigen provide 100 % specificity, with a sensitivity greater than 88%. The glycoprotein antigen was recognized by serum samples collected from patients infected with B. pseudomallei, B. mallei, B. multivorans and B. cenocepacia. Our results indicate that protein O-glycosylation in Burkholderia can be exploited as a biomarker for diagnosis of Burkholderia-associated infections.IMPORTANCEGlanders is a severe zoonotic disease caused by the Gram-negative bacterium Burkholderia mallei, which affects horses, mules and donkeys, as well as humans. B. mallei is also considered a category B biothreat agent. Due to insufficient pathognomonic symptoms in the early stages of glanders, diagnosis can be difficult. Complement fixation is the most accurate and reliable serological test prescribed by the World Organization for Animal Health; however, this test has a considerable number of false-positive results. We have recently described a conserved protein O-glycosylation pathway present in all species of the Burkholderia genus; we also demonstrated that Burkholderia-infected humans develop anti-glycan antibodies. Here, we exploited this system to construct and evaluate a synthetic glycoengineered protein antigen as a diagnostic tool for glanders. Our results show 100 % specificity in the detection of antibodies from infected horses, indicating that protein O-glycosylation in Burkholderia can be exploited as a biomarker for diagnosis of Burkholderia-associated infections.

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Section: Glanders-infected Horse Serum Samples Contain Anti-o-glycan supporting

confidence: 70%

Section: Discussionmentioning

confidence: 99%

Section: Complement Fixation Test (Cft) Is the Most Accurate And Relimentioning

confidence: 99%

See 1 more Smart Citation

A glycoengineered antigen exploiting a conserved proteinO-glycosylation pathway in theBurkholderiagenus for diagnosis of glanders infections

Wang

Glaser

Scott

et al. 2020

Preprint

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“…The Hcp1 indirect ELISA was performed as described previously (Elschner et al, ). Sample/positive ratio (S/P%) was calculated for each sample by (OD Sample‐OD Negative Control)/(OD Positive Control–OD Negative Control) ×100.…”

Section: Methodsmentioning

confidence: 99%

Serological surveillance and clinical investigation of glanders among indigenous equines in India from 2015 to 2018

Singha

Karuppusamy

Tripathi

et al. 2020

Transbound Emerg Dis

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Equine glanders is an infectious and notifiable bacterial disease caused by Burkholderia mallei. The disease has been reported in South American, African and Asian countries including India. Here, we present the outcome of glanders serosurveillance carried out between January 2015 and December 2018 to know the status of equine glanders among different states in India. A total of 102,071 equid sera from 299 districts of twenty-one states and one union territory were tested for glanders. Samples were screened with Hcp1 indirect ELISA followed by confirmatory diagnosis by CFT. During this four-year surveillance, a total of 932 glanders-positive cases were detected from 120 districts of 12 states. The study also revealed increasing trend of glanders from 2016 onwards with maximum occurrence in northern India. Overall seroprevalence ranged between 0.62% (95% CI, 0.52-0.72) and 1.145% (95% CI, 1.03-1.25). Seasonal shifting from winter to summer (March to June) coincided with highest number glanders | 1337 SINGHA et Al.

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“…However, due to animal welfare concerns, this test is not currently recommended [ 26 ]. The complement fixation test (CFT) is considered a suitable screening test for the diagnosis of equine glanders [ 4 , 11 , 12 , 25 , 27 , 28 , 30 ]. CFT is mandated by the World Organization for Animal Health (OIE) as a confirmatory test for international trade [ 26 ].…”

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confidence: 99%

Seroprevalence of equine glanders in horses in the central and eastern parts of Mongolia

et al. 2020

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Glanders is a contagious and fatal equine disease caused by the gram-negative bacterium Burkholderia mallei. B. mallei is prevalent among horse populations in Asia, the Middle East, and South America. More than four million horses have been registered in Mongolia in 2020. However, the resent prevalence of glanders has not been well investigated. In this study, we aimed to investigate the seropositivity of B. mallei in horse populations in Mongolia using the complement fixation test (CFT) and Rose Bengal plate agglutination test (RBT). We randomly collected blood samples from horses in central and eastern Mongolia between 2018 and 2019. Of 337 horses, 26 (7.7%) and 28 (8.3%) were seropositive using RBT and CFT, respectively. Interestingly, seropositivity in horses resulting from crossbreeding of Mongolian native horses with thoroughbred horses was higher than that in Mongolian native horses. Our observations suggest that equine glanders are still endemic to Mongolia.

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Evaluation of the comparative accuracy of the complement fixation test, Western blot and five enzyme-linked immunosorbent assays for serodiagnosis of glanders

Cited by 40 publications

References 26 publications

A glycoengineered antigen exploiting a conserved proteinO-glycosylation pathway in theBurkholderiagenus for diagnosis of glanders infections

A glycoengineered antigen exploiting a conserved proteinO-glycosylation pathway in theBurkholderiagenus for diagnosis of glanders infections

Serological surveillance and clinical investigation of glanders among indigenous equines in India from 2015 to 2018

Seroprevalence of equine glanders in horses in the central and eastern parts of Mongolia

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