Development of Rapid Enzyme-Linked Immunosorbent Assays for Detection of Antibodies to Burkholderia pseudomallei

Burkholderia pseudomallei, the causative agent of melioidosis, is an environmental bacillus found in northeast Thailand. The mortality rate of melioidosis is ∼40%. An indirect hemagglutination assay (IHA) is used as a reference serodiagnostic test; however, it has low specificity in areas where the background seropositivity of healthy people is high. To improve assay specificity and reduce the time for diagnosis, four rapid enzyme-linked immunosorbent assays (ELISAs) were developed using two purified polysaccharide antigens (O-polysaccharide [OPS] and 6-deoxyheptan capsular polysaccharide [CPS]) and two crude antigens (whole-cell [WC] antigen and culture filtrate [CF] antigen) ofB. pseudomallei. The ELISAs were evaluated using serum samples from 141 culture-confirmed melioidosis patients from Thailand along with 188 healthy donors from Thailand and 90 healthy donors from the United States as controls. The areas under receiver operator characteristic curves (AUROCC) using Thai controls were high for the OPS-ELISA (0.91), CF-ELISA (0.91), and WC-ELISA (0.90), while those of CPS-ELISA (0.84) and IHA (0.72) were lower. AUROCC values using U.S. controls were comparable to those of the Thai controls for all ELISAs except IHA (0.93). Using a cutoff optical density (OD) of 0.87, the OPS-ELISA had a sensitivity of 71.6% and a specificity of 95.7% for Thai controls; for U.S. controls, specificity was 96.7%. An additional 120 serum samples from tuberculosis, scrub typhus, or leptospirosis patients were evaluated in all ELISAs and resulted in comparable or higher specificities than using Thai healthy donors. Our findings suggest that antigen-specific ELISAs, particularly the OPS-ELISA, may be useful for serodiagnosis of melioidosis in areas where it is endemic and nonendemic.

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Evidence of Burkholderia pseudomallei-Specific Immunity in Patient Sera Persistently Nonreactive by the Indirect Hemagglutination Assay

Clinical and Vaccine Immunology ◽

10.1128/cvi.00077-11 ◽

2011 ◽

Vol 18 (8) ◽

pp. 1288-1291 ◽

Cited By ~ 7

Author(s):

Patrick N. A. Harris ◽

Natasha L. Williams ◽

Jodie L. Morris ◽

Natkunam Ketheesan ◽

Robert E. Norton

Keyword(s):

Enzyme Immunoassay ◽

Specific Antibody ◽

Burkholderia Pseudomallei ◽

Serological Test ◽

Lymphocyte Response ◽

Hemagglutination Assay ◽

Cutoff Value ◽

Content Type ◽

Serial Testing ◽

Indirect Hemagglutination

ABSTRACTThe indirect hemagglutination assay (IHA) is the most frequently used serological test to confirm exposure toBurkholderia pseudomallei. Patients with culture-confirmed disease often have a nonreactive IHA at presentation and occasionally fail to seroconvert on serial testing. We investigated whether using antigens derived from the cultured isolates of persistently IHA-nonreactive patient sera improved the sensitivity of the IHA. In addition, we assessed the antigen-specific lymphocyte response in this group of patients to a panel ofB. pseudomalleiantigens, including those derived from their own cultured isolates. Eleven patients with culture-proven melioidosis were identified as having persistently IHA-nonreactive sera. A modified IHA using erythrocytes sensitized with patient isolate-derived antigen tested against convalescent-phase serum was performed. The majority (82%) of sera showed a negative (≤1:5) result, one was borderline (1:20), and one was positive at the cutoff value (1:40). IHA-nonreactive sera were also tested by enzyme immunoassay (EIA), with 73% (8/11) demonstrating IgG positivity. In addition, lymphocytes isolated from persistently IHA-nonreactive patient sera demonstrated significantly increased proliferation in response toB. pseudomalleiantigens compared to controls. These studies demonstrate the presence ofB. pseudomallei-specific antibody by EIA andB. pseudomallei-specific lymphocytes in patient sera categorized as persistently nonreactive according to the IHA. New immunoassays are required and should incorporateB. pseudomalleiantigens that are immunoreactive for this subset of IHA-nonreactive patient sera.

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Surprisingly Low Seroprevalence of Burkholderia pseudomallei in Exposed Healthy Adults in the Darwin Region of Tropical Australia Where Melioidosis Is Highly Endemic

Clinical and Vaccine Immunology ◽

10.1128/cvi.00021-13 ◽

2013 ◽

Vol 20 (5) ◽

pp. 759-760 ◽

Cited By ~ 10

Author(s):

Gemma L. James ◽

Ben Delaney ◽

Linda Ward ◽

Kevin Freeman ◽

Mark Mayo ◽

...

Keyword(s):

Burkholderia Pseudomallei ◽

Healthy Adults ◽

Northeast Thailand ◽

Hemagglutination Assay ◽

Seropositivity Rate ◽

Content Type ◽

Indirect Hemagglutination ◽

Tropical Australia

ABSTRACTIn the Darwin region of Australia where melioidosis is highly endemic, only 11/354 (3%) healthy residents were seropositive by indirect hemagglutination assay, despite extensive exposure toBurkholderia pseudomallei. None developed melioidosis, but some described a prior self-limiting illness. This seropositivity rate is much lower than that seen in northeast Thailand, where melioidosis is similarly highly endemic, potentially reflecting important differences between these two locations in the epidemiology of melioidosis.

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Neutrophil Extracellular Traps Exhibit Antibacterial Activity against Burkholderia pseudomallei and Are Influenced by Bacterial and Host Factors

Infection and Immunity ◽

10.1128/iai.00806-12 ◽

2012 ◽

Vol 80 (11) ◽

pp. 3921-3929 ◽

Cited By ~ 51

Author(s):

Donporn Riyapa ◽

Surachat Buddhisa ◽

Sunee Korbsrisate ◽

Jon Cuccui ◽

Brendan W. Wren ◽

...

Keyword(s):

Burkholderia Pseudomallei ◽

Capsular Polysaccharide ◽

Neutrophil Extracellular Traps ◽

Polymorphonuclear Neutrophils ◽

Dependent Manner ◽

Predisposing Factor ◽

Bacterial Killing ◽

Content Type ◽

Extracellular Traps ◽

Type Iii Protein Secretion

ABSTRACTBurkholderia pseudomalleiis the causative pathogen of melioidosis, of which a major predisposing factor is diabetes mellitus. Polymorphonuclear neutrophils (PMNs) kill microbes extracellularly by the release of neutrophil extracellular traps (NETs). PMNs play a key role in the control of melioidosis, but the involvement of NETs in killing ofB. pseudomalleiremains obscure. Here, we showed that bactericidal NETs were released from human PMNs in response toB. pseudomalleiin a dose- and time-dependent manner.B. pseudomallei-induced NET formation required NADPH oxidase activation but not phosphatidylinositol-3 kinase, mitogen-activated protein kinases, or Src family kinase signaling pathways.B. pseudomalleimutants defective in the virulence-associated Bsa type III protein secretion system (T3SS) or capsular polysaccharide I (CPS-I) induced elevated levels of NETs. NET induction by such mutants was associated with increased bacterial killing, phagocytosis, and oxidative burst by PMNs. Taken together the data imply that T3SS and the capsule may play a role in evading the induction of NETs. Importantly, PMNs from diabetic subjects released NETs at a lower level than PMNs from healthy subjects. Modulation of NET formation may therefore be associated with the pathogenesis and control of melioidosis.

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Key Role of Capsular Polysaccharide in the Induction of Systemic Infection and Abortion by Hypervirulent Campylobacter jejuni

Infection and Immunity ◽

10.1128/iai.00001-17 ◽

2017 ◽

Vol 85 (6) ◽

Cited By ~ 8

Author(s):

Orhan Sahin ◽

Samantha A. Terhorst ◽

Eric R. Burrough ◽

Zhangqi Shen ◽

Zuowei Wu ◽

...

Keyword(s):

Guinea Pig ◽

Campylobacter Jejuni ◽

Capsular Polysaccharide ◽

Systemic Infection ◽

The United States ◽

Content Type ◽

Model Following ◽

Capsule Production ◽

Guinea Pig Model

ABSTRACT Campylobacter jejuni is a zoonotic pathogen, and a hypervirulent clone, named clone SA, has recently emerged as the predominant cause of ovine abortion in the United States. To induce abortion, orally ingested Campylobacter must translocate across the intestinal epithelium, spread systemically in the circulation, and reach the fetoplacental tissue. Bacterial factors involved in these steps are not well understood. C. jejuni is known to produce capsular polysaccharide (CPS), but the specific role that CPS plays in systemic infection and particularly abortion in animals remains to be determined. In this study, we evaluated the role of CPS in bacteremia using a mouse model and in abortion using a pregnant guinea pig model following oral challenge. Compared with C. jejuni NCTC 11168 and 81-176, a clone SA isolate (IA3902) resulted in significantly higher bacterial counts and a significantly longer duration of bacteremia in mice. The loss of capsule production via gene-specific mutagenesis in IA3902 led to the complete abolishment of bacteremia in mice and abortion in pregnant guinea pigs, while complementation of capsule expression almost fully restored these phenotypes. The capsule mutant strain was also impaired for survival in guinea pig sera and sheep blood. Sequence-based analyses revealed that clone SA possesses a unique CPS locus with a mosaic structure, which has been stably maintained in all clone SA isolates derived from various hosts and times. These findings establish CPS as a key virulence factor for the induction of systemic infection and abortion in pregnant animals and provide a viable candidate for the development of vaccines against hypervirulent C. jejuni.

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First report of Chlamydia psittaci seroprevalence in black-headed gulls (Larus ridibundus) at Dianchi Lake, China

Open Life Sciences ◽

10.1515/biol-2018-0030 ◽

2018 ◽

Vol 13 (1) ◽

pp. 250-252

Author(s):

Hua Chang ◽

Jiangqiang Han ◽

Yan Yang ◽

Gang Duan ◽

Fengcai Zou ◽

...

Keyword(s):

Human Health ◽

Potential Risk ◽

Wild Bird ◽

Chlamydia Psittaci ◽

Dianchi Lake ◽

Serum Samples ◽

Present Survey ◽

Hemagglutination Assay ◽

Larus Ridibundus ◽

Indirect Hemagglutination

AbstractChlamydiosis is an important zoonosis which can transmit from birds to humans, and investigation first reported the seroprevalence of Chlamydia psittaci in black-headed gulls (Larus ridibundus) at the Dianchi Lake, China. A total of 1029 serum samples collected from black-headed gulls between 2012-2015 were analyzed. The gulls were randomly caught and blood collected at Dianchi Lake, China. All the samples were analyzed for the presence of antibodies to C. psittaci by indirect hemagglutination assay (IHA). In this survey, the total infection rate was 11.86% (122/1029). The results of the present survey documented the existence of relatively high C. psittaci seroprevalence in black-headed gulls, which have a potential risk to the wild bird health and human health. Comprehensive practical control approaches and measures should be executed.

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Role of Antilipopolysaccharide Antibodies in Serum Bactericidal Activity against Salmonella enterica Serovar Typhimurium in Healthy Adults and Children in the United States

Clinical and Vaccine Immunology ◽

10.1128/cvi.00289-13 ◽

2013 ◽

Vol 20 (10) ◽

pp. 1491-1498 ◽

Cited By ~ 36

Author(s):

Estela Trebicka ◽

Susan Jacob ◽

Waheed Pirzai ◽

Bryan P. Hurley ◽

Bobby J. Cherayil

Keyword(s):

United States ◽

Bactericidal Activity ◽

Salmonella Enterica ◽

The United States ◽

Healthy Children ◽

Healthy Individuals ◽

Serum Samples ◽

Content Type ◽

Serovar Typhimurium

ABSTRACTRecent observations from Africa have rekindled interest in the role of serum bactericidal antibodies in protecting against systemic infection withSalmonella entericaserovar Typhimurium. To determine whether the findings are applicable to other populations, we analyzed serum samples collected from healthy individuals in the United States. We found that all but 1 of the 49 adult samples tested had robust bactericidal activity againstS. Typhimurium in a standardin vitroassay. The activity was dependent on complement and could be reproduced by immunoglobulin G (IgG) purified from the sera. The bactericidal activity was inhibited by competition with soluble lipopolysaccharide (LPS) fromS. Typhimurium but not fromEscherichia coli, consistent with recognition of a determinant in the O-antigen polysaccharide. Sera from healthy children aged 10 to 48 months also had bactericidal activity, although it was significantly less than in the adults, correlating with lower levels of LPS-specific IgM and IgG. The lone sample in our collection that lacked bactericidal activity was able to inhibit killing ofS. Typhimurium by the other sera. The inhibition correlated with the presence of an LPS-specific IgM and was associated with decreased complement deposition on the bacterial surface. Our results indicate that healthy individuals can have circulating antibodies to LPS that either mediate or inhibit killing ofS. Typhimurium. The findings contrast with the observations from Africa, which linked bactericidal activity to antibodies against anS. Typhimurium outer membrane protein and correlated the presence of inhibitory anti-LPS antibodies with human immunodeficiency virus infection.

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Distribution of Serological Response to Burkholderia pseudomallei in Swine from Three Provinces of Vietnam

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph17145203 ◽

2020 ◽

Vol 17 (14) ◽

pp. 5203

Author(s):

Michael H. Norris ◽

Hang Thi Thu Tran ◽

Morgan A. Walker ◽

Andrew P. Bluhm ◽

Diansy Zincke ◽

...

Keyword(s):

Human Disease ◽

Burkholderia Pseudomallei ◽

Capsular Polysaccharide ◽

Severe Disease ◽

Population Estimates ◽

Serological Response ◽

Moist Soil ◽

Serum Samples ◽

Two Populations ◽

Hoa Binh

(1) Background: Burkholderia pseudomallei is an environmentally mediated saprophytic pathogen that can cause severe disease in humans. It is well known that B. pseudomallei survives in tropical moist soil environments worldwide, but melioidosis is gaining recognition as a public and veterinary health issue in Vietnam. The contribution of animals to human disease is unknown, necessitating further investigation. (2) Methods: Swine sera were collected from two populations, one grazing and one commercially farmed, from three provinces in Vietnam. ELISAs utilizing B. pseudomallei capsular polysaccharide (CPS), outer polysaccharide (OPS), and Hcp1 protein were used to screen serum samples. Positive samples were mapped to the commune level. Seroprevalence calculations and pig population estimates were used to approximate number of swine exposures per commune. (3) Results: Grazing pigs from Hoa Binh had significantly higher seropositivity levels (11.4%, 95% CI: 9.7–13.1) compared to farmed pigs from Ha Tinh and Nghe An (4%, 95% CI: 3.3–4.7). Average swine seropositivity rates were ~6.3% (95% CI: 5–7.9), higher than previously identified in Vietnam (~0.88%). (4) Conclusions: Initial serological sampling identified a significant number of seropositive and potential melioidosis infections occurring in swine in Vietnam. This work is a critical step in understanding the role swine may play in the epidemiology of human melioidosis in Vietnam.

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USA300 and USA500 Clonal Lineages of Staphylococcus aureus Do Not Produce a Capsular Polysaccharide Due to Conserved Mutations in the cap5 Locus

mBio ◽

10.1128/mbio.02585-14 ◽

2015 ◽

Vol 6 (2) ◽

Cited By ~ 42

Author(s):

Susan Boyle-Vavra ◽

Xue Li ◽

Md Tauqeer Alam ◽

Timothy D. Read ◽

Julia Sieth ◽

...

Keyword(s):

United States ◽

Staphylococcus Aureus ◽

Capsular Polysaccharide ◽

The United States ◽

Whole Genome Sequence ◽

Content Type ◽

Capsule Production ◽

Usa300 Strain

ABSTRACTThe surface capsular polysaccharide (CP) is a virulence factor that has been used as an antigen in several successful vaccines against bacterial pathogens. A vaccine has not yet been licensed againstStaphylococcus aureus, although two multicomponent vaccines that contain CP antigens are in clinical trials. In this study, we evaluated CP production in USA300 methicillin-resistantS. aureus(MRSA) isolates that have become the predominant community-associated MRSA clones in the United States. We found that all 167 USA300 MRSA and 50 USA300 methicillin-susceptibleS. aureus(MSSA) isolates were CP negative (CP−). Moreover, all 16 USA500 isolates, which have been postulated to be the progenitor lineage of USA300, were also CP−. Whole-genome sequence analysis of 146 CP−USA300 MRSA isolates revealed they all carry acap5locus with 4 conserved mutations compared with strain Newman. Genetic complementation experiments revealed that three of these mutations (in thecap5promoter,cap5Dnucleotide 994, andcap5Enucleotide 223) ablated CP production in USA300 and that Cap5E75 Asp, located in the coenzyme-binding domain, is essential for capsule production. All but three USA300 MSSA isolates had the same fourcap5mutations found in USA300 MRSA isolates. Most isolates with a USA500 pulsotype carried three of these four USA300-specific mutations, suggesting the fourth mutation occurred in the USA300 lineage. Phylogenetic analysis of thecaploci of our USA300 isolates as well as publicly available genomes from 41 other sequence types revealed that the USA300-specificcap5mutations arose sequentially inS. aureusin a common ancestor of USA300 and USA500 isolates.IMPORTANCEThe USA300 MRSA clone emerged as a community-associated pathogen in the United States nearly 20 years ago. Since then, it has rapidly disseminated and now causes health care-associated infections. This study shows that the CP-negative (CP−) phenotype has persisted among USA300 isolates and is a universal and characteristic trait of this highly successful MRSA lineage. It is important to note that a vaccine consisting solely of CP antigens would not likely demonstrate high efficacy in the U.S. population, where about half of MRSA isolates comprise USA300. Moreover, conversion of a USA300 strain to a CP-positive (CP+) phenotype is unlikelyin vivoorin vitrosince it would require the reversion of 3 mutations. We have also established that USA300 MSSA isolates and USA500 isolates are CP−and provide new insight into the evolution of the USA300 and USA500 lineages.

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

Clinical and Vaccine Immunology ◽

10.1128/cvi.00289-06 ◽

2006 ◽

Vol 14 (1) ◽

pp. 110-113 ◽

Cited By ~ 34

Author(s):

Narisara Chantratita ◽

Vanaporn Wuthiekanun ◽

Aunchalee Thanwisai ◽

Direk Limmathurotsakul ◽

Allen C. Cheng ◽

...

Keyword(s):

Immunosorbent Assay ◽

Diagnostic Tests ◽

Burkholderia Pseudomallei ◽

Enzyme Linked Immunosorbent Assay ◽

Northeast Thailand ◽

Hemagglutination Assay ◽

Assay Sensitivity ◽

Diagnostic Indices ◽

Indirect Hemagglutination ◽

Purified Antigen

ABSTRACT 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%).

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Burkholderia pseudomallei: Challenges for the Clinical Microbiology Laboratory

Journal of Clinical Microbiology ◽

10.1128/jcm.01636-16 ◽

2016 ◽

Vol 54 (12) ◽

pp. 2866-2873 ◽

Cited By ~ 14

Author(s):

Peera Hemarajata ◽

Jonathan D. Baghdadi ◽

Risa Hoffman ◽

Romney M. Humphries

Keyword(s):

Burkholderia Pseudomallei ◽

The United States ◽

Clinical Syndrome ◽

Clinical Microbiology Laboratory ◽

Department Of Agriculture ◽

Content Type ◽

Clinical Laboratories ◽

Response Network ◽

Control And Prevention ◽

Inspection Service

Melioidosis is a potentially fatal infection caused by the bacterium Burkholderia pseudomallei . Clinical diagnosis of melioidosis can be challenging since there is no pathognomonic clinical syndrome, and the organism is often misidentified by methods used routinely in clinical laboratories. Although the disease is more prevalent in Thailand and northern Australia, sporadic cases may be encountered in areas where it is not endemic, including the United States. Since the organism is considered a tier 1 select agent according to the Centers for Disease Control and Prevention and the U.S. Department of Agriculture Animal and Plant Health Inspection Service, clinical laboratories must be proficient at rapidly recognizing isolates suspicious for B. pseudomallei , be able to safely perform necessary rule-out tests, and to refer suspect isolates to Laboratory Response Network reference laboratories. In this minireview, we report a case of melioidosis encountered at our institution and discuss the laboratory challenges encountered when dealing with clinical isolates suspicious for B. pseudomallei or clinical specimens from suspected melioidosis cases.

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