Am J Trop Med HygAm. J. Trop. Med. HygtpmdThe American Journal of Tropical Medicine and Hygiene0002-96371476-1645The American Society of Tropical Medicine and Hygiene24710616404772710.4269/ajtmh.14-0025Short ReportArticlesEvaluation of a Latex Agglutination Assay for the Identification of Burkholderia pseudomallei and Burkholderia malleiDUVAL AND OTHERSLATEX AGGLUTINATION ASSAY FOR BURKHOLDERIA SPP. DETECTIONDuvalBrea D.ElrodMindy G.GeeJay E.ChantratitaNarisaraTandhavanantSarunpornLimmathurotsakulDirekHoffmasterAlex R.*Bacterial Special Pathogens Branch, Centers for Disease Control and Prevention, Atlanta, Georgia; Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Department of Tropical Hygiene, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand*Address correspondence to Alex R. Hoffmaster, Centers for Disease Control and Prevention, 1600 Clifton Rd. NE, MS-G34, Atlanta, GA 30333. E-mail: amh9@cdc.gov046201404620149061043104610120140232014©The American Society of Tropical Medicine and Hygiene2014This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cases of melioidosis and glanders are rare in the United States, but the etiologic agents of each disease (Burkholderia pseudomallei and Burkholderia mallei, respectively) are classified as Tier 1 select agents because of concerns about their potential use as bioterrorism agents. A rapid, highly sensitive, and portable assay for clinical laboratories and field use is required. Our laboratory has further evaluated a latex agglutination assay for its ability to identify B. pseudomallei and B. mallei isolates. This assay uses a monoclonal antibody that specifically recognizes the capsular polysaccharide produced by B. pseudomallei and B. mallei, but is absent in closely related Burkholderia species. A total of 110 B. pseudomallei and B. mallei were tested, and 36 closely related Burkholderia species. The latex agglutination assay was positive for 109 of 110 (99.1% sensitivity) B. pseudomallei and B. mallei isolates tested.

The Gram-negative bacteria Burkholderia pseudomallei and Burkholderia mallei are the etiologic agents of melioidosis and glanders, respectively. Melioidosis typically causes disease in humans and is endemic to Southeast Asia and northern Australia, whereas glanders is a disease most commonly seen in horses, mules, and donkeys in the Middle East, Africa, and India. Both bacteria are of concern because of their potential use as bioterrorism agents. The rarity of both diseases in the United States and other countries where the diseases are not endemic could delay proper diagnosis by physicians and laboratory staff during a bioterrorism event caused by responders' unfamiliarity with the diseases. Diagnostic confirmation of both diseases relies on microbiological culture. However, B. pseudomallei is commonly dismissed as a culture contaminant, and along with B. mallei may be misidentified by standard identification methods including API 20NE and other automated bacterial identification systems. Therefore, rapid diagnostic tools for bacterial identification are essential to provide an effective response by public health authorities in the event of a bioterrorism incident. The goal of this study was to evaluate a rapid assay for the identification of B. pseudomallei and B. mallei.

Latex agglutination assays have been used successfully in Southeast Asia and northern Australia to identify B. pseudomallei isolates and closely related species.1 Assays such as these are based on the use of monoclonal antibodies (MAbs) that recognize an exopolysaccharide present on the cell surface of B. pseudomallei and B. mallei.24 Nonetheless, these assays are normally evaluated with limited strains isolated from endemic areas, and its use for strains isolated from all other countries has not been adequately evaluated.24

Our laboratory has evaluated a rapid latex agglutination assay developed by Mahidol University (Bangkok, Thailand) using an inclusivity panel of 110 geographically and genetically diverse B. pseudomallei and B. mallei isolates, stored at The Centers for Disease Control and Prevention (CDC), Atlanta, GA. We also evaluated the assay with an exclusivity panel of 36 closely related Burkholderia species, which included agents that have not been previously tested by this or similar antigen detection assays. We focused on the closest phylogenetic relatives of B. pseudomallei including other Burkholderia species that have been associated with human disease such as Burkholderia oklahomensis and Burkholderia gladioli. Burkholderia oklahomensis has been reported to cause infections associated with deep tissue wounds,5,6 whereas B. gladioli can cause a range of diseases from fatal foodborne illness,7 to sepsis in newborns,8 and lung infections in patients with cystic fibrosis.9 This latex agglutination assay could be valuable in correctly identifying select agents and excluding closely related Burkholderia species that cause similar disease in humans.

The antibody-latex suspension based on the 4B11 monoclonal antibody was prepared by Mahidol University as previously described.24 The assay was performed also as previously described with slight modification.1 Briefly, isolates were subcultured twice on trypticase soy agar (TSA) containing 5% sheep's blood and incubated for 18–24 hours at 37°C. Single colonies were picked and added to 10 μL of the latex suspension on a ringed glass microscope slide. The glass slide containing the latex suspension with the suspended colony was subjected to gentle rocking for 2 minutes after which time the reaction was recorded as either positive (agglutination) or negative (no agglutination) (Figure 1). Burkholderia pseudomallei K96243 was used as the positive control in all experiments and Burkholderia thailandensis E264 (American Type Culture Collection [ATCC] type strain 700388) was used as the negative control each time isolates were tested, and all tests were performed in triplicate.

Burkholderia pseudomallei and Burkholderia thailandensis positive and negative reactions after incubation with the latex agglutination reagent.

Under our assay conditions, the latex agglutination test was positive on 109 of 110 (99.1% sensitivity) isolates tested on the inclusivity panel. This number included a total of 77 B. pseudomallei isolates, of which 76 (98.7% sensitivity) were positive and 33 B. mallei isolates of which all were positive (100% sensitivity) (Table 1). The B. pseudomallei isolate that tested negative in our assay, CDC2721686 (MSHR1655), was isolated from a patient with a chronic B. pseudomallei infection after being first diagnosed with melioidosis in 2000.10 This rare B. pseudomallei isolate was from the patient in an ongoing study consisting of 815 patients that were culture-positive for melioidosis in Darwin, Australia. Since 1989, this patient is the only survivor from this study to remain chronically colonized by B. pseudomallei. CDC2721686 (MSHR1655) was isolated 37 months after the initial melioidosis diagnosis and has undergone major genome-wide rearrangements resulting in a loss of function in many genes that are important in pathogenesis. Of particular interest to this study is the loss of function of wcbR, which encodes an essential fatty acid synthase required in capsular polysaccharide synthesis.11 We believe this would explain the inability of the latex agglutination assay to correctly identify this isolate. In addition to testing negative in our assay, when subjected to standard biochemical tests for the identification of B. pseudomallei, the isolate was non-motile, but otherwise normal under our assay conditions. When the latex agglutination assay was tested against an exclusivity panel of closely related Burkholderia species, 35 of 36 (97.2% specificity) yielded negative results (Table 2). The closely related Burkholderia that tested positive in our assay is a rare variant of B. thailandensis (CDC3015869, TX DOH) that has been previously described as containing B. pseudomallei capsule genes.12

Rapid diagnostic assays, such as the one we have evaluated, would have the most impact in clinical laboratories. This would allow for early identification of suspect isolates and thus on-site diagnosis instead of needing to submit samples to regional laboratories that would delay results. This assay does have several advantages over the current reference level testing. This assay is simple, does not require extra equipment, and can easily be performed. However, the extent to which this assay or similar antigen detection assays can be used on patient samples is yet to be determined.

Authors' addresses: Brea D. Duval, Mindy G. Elrod, Jay E. Gee, and Alex R. Hoffmaster, Centers for Disease Control and Prevention, Atlanta, GA, E-mails: BDuval@cdc.gov, MGElrod@cdc.gov, JGee1@cdc.gov, and AHoffmaster@cdc.gov. Narisara Chantratita, Sarunporn Tandhavanant, and Direk Limmathurotsakul, Mahidol University, Bangkok, Thailand, E-mails: Narisara@tropmedres.ac, Sarunporn@tropmedres.ac, and Direk@tropmedres.ac.

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Burkholderia pseudomallei and Burkholderia mallei inclusivity panel

SpeciesStrain identifierLocation of originResult
Burkholderia pseudomalleiCDC2721620FrancePositive
Burkholderia pseudomalleiCDC2721628MadagascarPositive
Burkholderia pseudomalleiCDC2721639KenyaPositive
Burkholderia pseudomalleiCDC0022138ThailandPositive
Burkholderia pseudomalleiBp92; CDC2721623AustraliaPositive
Burkholderia pseudomalleiHuman 88; PHLS 45ThailandPositive
Burkholderia pseudomalleiBp104; CDC2721624AustraliaPositive
Burkholderia pseudomalleiCDC2721635; PHLS 36SingaporePositive
Burkholderia pseudomalleiBp73; Ln31348MalaysiaPositive
Burkholderia pseudomalleiPHLS 208EcuadorPositive
Burkholderia pseudomalleiCDC2721102; F5013GeorgiaPositive
Burkholderia pseudomalleiBpG9709; CDC0032026IndiaPositive
Burkholderia pseudomalleiPHLS 19; CDC2721625SingaporePositive
Burkholderia pseudomalleiCDC2721676USAPositive
Burkholderia pseudomalleiBp2889; SID2889BangladeshPositive
Burkholderia pseudomalleiCDC2721630; 7605FrancePositive
Burkholderia pseudomalleiBp68; CDC2721641FijiPositive
Burkholderia pseudomalleiPHLS 17; CDC2721619IndonesiaPositive
Burkholderia pseudomalleiPHLS 38SingaporePositive
Burkholderia pseudomallei1106a; CDC0022030ThailandPositive
Burkholderia pseudomalleiBp53; CDC2721633ThailandPositive
Burkholderia pseudomalleiBp24; CDC2721620FrancePositive
Burkholderia pseudomalleiBpG9313; CDC0032029USAPositive
Burkholderia pseudomalleiCDC2721162AustraliaPositive
Burkholderia pseudomalleiCDC2721114; G6715USA (Ohio)Positive
Burkholderia pseudomalleiCDC2721626ThailandPositive
Burkholderia pseudomalleiCDC0032028USA (Ohio)Positive
Burkholderia pseudomalleiCDC721096; 81A442USA (New York)Positive
Burkholderia pseudomalleiCDC0032024Puerto RicoPositive
Burkholderia pseudomalleiThai NE Human 99ThailandPositive
Burkholderia pseudomalleiCDC1029240USA (Oregon)Positive
Burkholderia pseudomalleiCDC2721617AustraliaPositive
Burkholderia pseudomalleiBp14; CDC2721618PhilippinesPositive
Burkholderia pseudomalleiBpH1442; CDC0032025USA (Delaware)Positive
Burkholderia pseudomalleiMSHR640;CDC8724880AustraliaPositive
Burkholderia pseudomallei465a; CDC8724601AustraliaPositive
Burkholderia pseudomalleiMSHR99; CDC8724881AustraliaPositive
Burkholderia pseudomalleiCDC1756207AustraliaPositive
Burkholderia pseudomalleiCDC8724890AustraliaPositive
Burkholderia pseudomallei#711; CDC2721675USA (Washington)Positive
Burkholderia pseudomalleiCDC2734678; 620ThailandPositive
Burkholderia pseudomalleiCDC8724908AustraliaPositive
Burkholderia pseudomalleiCDC8724883AustraliaPositive
Burkholderia pseudomalleiCDC2734694; PM40ThailandPositive
Burkholderia pseudomalleiPM26; CDC2734683ThailandPositive
Burkholderia pseudomalleiPHLS 75MalaysiaPositive
Burkholderia pseudomalleiCDC8724901AustraliaPositive
Burkholderia pseudomalleiPM115; CDC2734709ThailandPositive
Burkholderia pseudomalleiCDC2721825ThailandPositive
Burkholderia pseudomalleiBp40SingaporePositive
Burkholderia pseudomalleiCDC8724894AustraliaPositive
Burkholderia pseudomalleiCDC2734661; SA923ThailandPositive
Burkholderia pseudomalleiPHLS 79MalaysiaPositive
Burkholderia pseudomalleiBpH1689; CDC0032024USA (Florida)Positive
Burkholderia pseudomalleiCDC2721184EcuadorPositive
Burkholderia pseudomalleiCDC2721634ThailandPositive
Burkholderia pseudomalleiCDC1756205AustraliaPositive
Burkholderia pseudomalleiCDC8724905AustraliaPositive
Burkholderia pseudomalleiCDC0022203ThailandPositive
Burkholderia pseudomalleiCDC2721637PakistanPositive
Burkholderia pseudomalleiCDC8724896ThailandPositive
Burkholderia pseudomalleiCDC8724889AustraliaPositive
Burkholderia pseudomalleiCDC8724898AustraliaPositive
Burkholderia pseudomalleiCDC2721686AustraliaNegative
Burkholderia pseudomalleiCDC8724899ThailandPositive
Burkholderia pseudomalleiCDC8724882AustraliaPositive
Burkholderia pseudomalleiCDC8724900AustraliaPositive
Burkholderia pseudomalleiCDC8724892AustraliaPositive
Burkholderia pseudomalleiCDC8724893AustraliaPositive
Burkholderia pseudomalleiCDC2721761VietnamPositive
Burkholderia pseudomalleiCDC8724885USAPositive
Burkholderia pseudomalleiCDC0022358ThailandPositive
Burkholderia pseudomalleiCDC8724877AustraliaPositive
Burkholderia pseudomalleiCDC1756206AustraliaPositive
Burkholderia pseudomalleiCDC8724895AustraliaPositive
Burkholderia pseudomalleiCDC8724903AustraliaPositive
Burkholderia pseudomalleiCDC8724878AustraliaPositive
Burkholderia malleiCDC2721277ChinaPositive
Burkholderia malleiCDC2734821ChinaPositive
Burkholderia malleiCDC2721278USA (New Mexico)Positive
Burkholderia malleiCDC0031066IndiaPositive
Burkholderia malleiCDC2734315TurkeyPositive
Burkholderia malleiCDC0031065TurkeyPositive
Burkholderia malleiCDC2734302TurkeyPositive
Burkholderia malleiCDC2734301TurkeyPositive
Burkholderia malleiCDC0031304USA (Maryland)Positive
Burkholderia malleiCDC2721273BurmaPositive
Burkholderia malleiKC 235; CDC2721274USA (Maryland)Positive
Burkholderia malleiKC0248; CDC4017733USAPositive
Burkholderia malleiCDC2721279USA (New York)Positive
Burkholderia malleiCDC2721280IranPositive
Burkholderia malleiCDC8724847UnknownPositive
Burkholderia malleiCDC2734305IndiaPositive
Burkholderia malleiCDC2734303; GB10IndiaPositive
Burkholderia malleiCDC8724837TurkeyPositive
Burkholderia malleiCDC8724838TurkeyPositive
Burkholderia malleiCDC8724839TurkeyPositive
Burkholderia malleiCDC8724841TurkeyPositive
Burkholderia malleiCDC2734300TurkeyPositive
Burkholderia malleiCDC2734301TurkeyPositive
Burkholderia malleiCDC2734317IndiaPositive
Burkholderia malleiCDC2721275ChinaPositive
Burkholderia malleiCDC2734299HungaryPositive
Burkholderia malleiCDC2734311EnglandPositive
Burkholderia malleiCDC0031063HungaryPositive
Burkholderia malleiCDC0031064IndiaPositive
Burkholderia malleiCDC2721276USAPositive
Burkholderia malleiCDC2721648BurmaPositive
Burkholderia malleiCDC2734312TurkeyPositive
Burkholderia malleiCDC2721280IranPositive

Burkholderia exclusivity panel

SpeciesStrain identifierLocation of originResult
Burkholderia thailandensisCDC3015869USA (Texas)Positive
Burkholderia thailandensisCDC2721621FranceNegative
Burkholderia thailandensisCDC2721627ThailandNegative
Burkholderia thailandensisCDC2721121USA (Louisiana)Negative
Burkholderia thailandensisCDC2721643UnknownNegative
Burkholderia thailandensisCDC2721701ThailandNegative
Burkholderia thailandensisCDC2721723ThailandNegative
Burkholderia thailandensisCDC2721744MalaysiaNegative
Burkholderia humptydooensisCDC2721687AustraliaNegative
Burkholderia oklahomensisCDC4002358USA (Oklahoma)Negative
Burkholderia oklahomensisCDC4021865USA (Oklahoma)Negative
Burkholderia oklahomensisCDC4021866USA (Oklahoma)Negative
Burkholderia vietnamiensisCDC2734483VietnamNegative
Burkholderia pyrrociniaATCC 15958UnknownNegative
Burkholderia caledonicaCDC8724197United KingdomNegative
Burkholderia caribensisCDC8724200MartiniqueNegative
Burkholderia ambifariaCDC8724201USA (Wisconsin)Negative
Burkholderia anthinaCDC8724199USA (Tennessee)Negative
Burkholderia cocovenenansCDC2734715IndonesiaNegative
Burkholderia ferrariaeCDC8724209BrazilNegative
Burkholderia hydrophilaCDC2721759ThailandNegative
Burkholderia fungorumATCC BAA-463UnknownNegative
Burkholderia glatheiCDC2734719GermanyNegative
Burkholderia graminisCDC2734716FranceNegative
Burkholderia hospitaCDC8724207BelgiumNegative
Burkholderia kururiensisCDC2734717ChinaNegative
Burkholderia nodosaCDC8724205BrazilNegative
Burkholderia phenaziniumATCC 33666UnknownNegative
Burkholderia phenoliruptrixCDC8724203USANegative
Burkholderia phymatumCDC8724208French GuianaNegative
Burkholderia phytofirmansCDC8724204GermanyNegative
Burkholderia sacchariCDC8724202BrazilNegative
Burkholderia silvatlanticaATCC BAA-1244BrazilNegative
Burkholderia rhizoxinicaDSM19002GermanyNegative
Burkholderia endofungorumDSM19003GermanyNegative
Burkholderia gladioliCDC3027208USA (California)Negative