Evaluation of a latex agglutination assay for the identification of Burkholderia pseudomallei and Burkholderia mallei.

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 American Society of Tropical Medicine and Hygiene.

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.2–4 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.2–4

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.2–4 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.

Figure 1.

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

Table 1

Burkholderia pseudomallei and Burkholderia mallei inclusivity panel

Species	Strain identifier	Location of origin	Result
Burkholderia pseudomallei	CDC2721620	France	Positive
Burkholderia pseudomallei	CDC2721628	Madagascar	Positive
Burkholderia pseudomallei	CDC2721639	Kenya	Positive
Burkholderia pseudomallei	CDC0022138	Thailand	Positive
Burkholderia pseudomallei	Bp92; CDC2721623	Australia	Positive
Burkholderia pseudomallei	Human 88; PHLS 45	Thailand	Positive
Burkholderia pseudomallei	Bp104; CDC2721624	Australia	Positive
Burkholderia pseudomallei	CDC2721635; PHLS 36	Singapore	Positive
Burkholderia pseudomallei	Bp73; Ln31348	Malaysia	Positive
Burkholderia pseudomallei	PHLS 208	Ecuador	Positive
Burkholderia pseudomallei	CDC2721102; F5013	Georgia	Positive
Burkholderia pseudomallei	BpG9709; CDC0032026	India	Positive
Burkholderia pseudomallei	PHLS 19; CDC2721625	Singapore	Positive
Burkholderia pseudomallei	CDC2721676	USA	Positive
Burkholderia pseudomallei	Bp2889; SID2889	Bangladesh	Positive
Burkholderia pseudomallei	CDC2721630; 7605	France	Positive
Burkholderia pseudomallei	Bp68; CDC2721641	Fiji	Positive
Burkholderia pseudomallei	PHLS 17; CDC2721619	Indonesia	Positive
Burkholderia pseudomallei	PHLS 38	Singapore	Positive
Burkholderia pseudomallei	1106a; CDC0022030	Thailand	Positive
Burkholderia pseudomallei	Bp53; CDC2721633	Thailand	Positive
Burkholderia pseudomallei	Bp24; CDC2721620	France	Positive
Burkholderia pseudomallei	BpG9313; CDC0032029	USA	Positive
Burkholderia pseudomallei	CDC2721162	Australia	Positive
Burkholderia pseudomallei	CDC2721114; G6715	USA (Ohio)	Positive
Burkholderia pseudomallei	CDC2721626	Thailand	Positive
Burkholderia pseudomallei	CDC0032028	USA (Ohio)	Positive
Burkholderia pseudomallei	CDC721096; 81A442	USA (New York)	Positive
Burkholderia pseudomallei	CDC0032024	Puerto Rico	Positive
Burkholderia pseudomallei	Thai NE Human 99	Thailand	Positive
Burkholderia pseudomallei	CDC1029240	USA (Oregon)	Positive
Burkholderia pseudomallei	CDC2721617	Australia	Positive
Burkholderia pseudomallei	Bp14; CDC2721618	Philippines	Positive
Burkholderia pseudomallei	BpH1442; CDC0032025	USA (Delaware)	Positive
Burkholderia pseudomallei	MSHR640;CDC8724880	Australia	Positive
Burkholderia pseudomallei	465a; CDC8724601	Australia	Positive
Burkholderia pseudomallei	MSHR99; CDC8724881	Australia	Positive
Burkholderia pseudomallei	CDC1756207	Australia	Positive
Burkholderia pseudomallei	CDC8724890	Australia	Positive
Burkholderia pseudomallei	#711; CDC2721675	USA (Washington)	Positive
Burkholderia pseudomallei	CDC2734678; 620	Thailand	Positive
Burkholderia pseudomallei	CDC8724908	Australia	Positive
Burkholderia pseudomallei	CDC8724883	Australia	Positive
Burkholderia pseudomallei	CDC2734694; PM40	Thailand	Positive
Burkholderia pseudomallei	PM26; CDC2734683	Thailand	Positive
Burkholderia pseudomallei	PHLS 75	Malaysia	Positive
Burkholderia pseudomallei	CDC8724901	Australia	Positive
Burkholderia pseudomallei	PM115; CDC2734709	Thailand	Positive
Burkholderia pseudomallei	CDC2721825	Thailand	Positive
Burkholderia pseudomallei	Bp40	Singapore	Positive
Burkholderia pseudomallei	CDC8724894	Australia	Positive
Burkholderia pseudomallei	CDC2734661; SA923	Thailand	Positive
Burkholderia pseudomallei	PHLS 79	Malaysia	Positive
Burkholderia pseudomallei	BpH1689; CDC0032024	USA (Florida)	Positive
Burkholderia pseudomallei	CDC2721184	Ecuador	Positive
Burkholderia pseudomallei	CDC2721634	Thailand	Positive
Burkholderia pseudomallei	CDC1756205	Australia	Positive
Burkholderia pseudomallei	CDC8724905	Australia	Positive
Burkholderia pseudomallei	CDC0022203	Thailand	Positive
Burkholderia pseudomallei	CDC2721637	Pakistan	Positive
Burkholderia pseudomallei	CDC8724896	Thailand	Positive
Burkholderia pseudomallei	CDC8724889	Australia	Positive
Burkholderia pseudomallei	CDC8724898	Australia	Positive
Burkholderia pseudomallei	CDC2721686	Australia	Negative
Burkholderia pseudomallei	CDC8724899	Thailand	Positive
Burkholderia pseudomallei	CDC8724882	Australia	Positive
Burkholderia pseudomallei	CDC8724900	Australia	Positive
Burkholderia pseudomallei	CDC8724892	Australia	Positive
Burkholderia pseudomallei	CDC8724893	Australia	Positive
Burkholderia pseudomallei	CDC2721761	Vietnam	Positive
Burkholderia pseudomallei	CDC8724885	USA	Positive
Burkholderia pseudomallei	CDC0022358	Thailand	Positive
Burkholderia pseudomallei	CDC8724877	Australia	Positive
Burkholderia pseudomallei	CDC1756206	Australia	Positive
Burkholderia pseudomallei	CDC8724895	Australia	Positive
Burkholderia pseudomallei	CDC8724903	Australia	Positive
Burkholderia pseudomallei	CDC8724878	Australia	Positive
Burkholderia mallei	CDC2721277	China	Positive
Burkholderia mallei	CDC2734821	China	Positive
Burkholderia mallei	CDC2721278	USA (New Mexico)	Positive
Burkholderia mallei	CDC0031066	India	Positive
Burkholderia mallei	CDC2734315	Turkey	Positive
Burkholderia mallei	CDC0031065	Turkey	Positive
Burkholderia mallei	CDC2734302	Turkey	Positive
Burkholderia mallei	CDC2734301	Turkey	Positive
Burkholderia mallei	CDC0031304	USA (Maryland)	Positive
Burkholderia mallei	CDC2721273	Burma	Positive
Burkholderia mallei	KC 235; CDC2721274	USA (Maryland)	Positive
Burkholderia mallei	KC0248; CDC4017733	USA	Positive
Burkholderia mallei	CDC2721279	USA (New York)	Positive
Burkholderia mallei	CDC2721280	Iran	Positive
Burkholderia mallei	CDC8724847	Unknown	Positive
Burkholderia mallei	CDC2734305	India	Positive
Burkholderia mallei	CDC2734303; GB10	India	Positive
Burkholderia mallei	CDC8724837	Turkey	Positive
Burkholderia mallei	CDC8724838	Turkey	Positive
Burkholderia mallei	CDC8724839	Turkey	Positive
Burkholderia mallei	CDC8724841	Turkey	Positive
Burkholderia mallei	CDC2734300	Turkey	Positive
Burkholderia mallei	CDC2734301	Turkey	Positive
Burkholderia mallei	CDC2734317	India	Positive
Burkholderia mallei	CDC2721275	China	Positive
Burkholderia mallei	CDC2734299	Hungary	Positive
Burkholderia mallei	CDC2734311	England	Positive
Burkholderia mallei	CDC0031063	Hungary	Positive
Burkholderia mallei	CDC0031064	India	Positive
Burkholderia mallei	CDC2721276	USA	Positive
Burkholderia mallei	CDC2721648	Burma	Positive
Burkholderia mallei	CDC2734312	Turkey	Positive
Burkholderia mallei	CDC2721280	Iran	Positive

Table 2

Burkholderia exclusivity panel

Species	Strain identifier	Location of origin	Result
Burkholderia thailandensis	CDC3015869	USA (Texas)	Positive
Burkholderia thailandensis	CDC2721621	France	Negative
Burkholderia thailandensis	CDC2721627	Thailand	Negative
Burkholderia thailandensis	CDC2721121	USA (Louisiana)	Negative
Burkholderia thailandensis	CDC2721643	Unknown	Negative
Burkholderia thailandensis	CDC2721701	Thailand	Negative
Burkholderia thailandensis	CDC2721723	Thailand	Negative
Burkholderia thailandensis	CDC2721744	Malaysia	Negative
Burkholderia humptydooensis	CDC2721687	Australia	Negative
Burkholderia oklahomensis	CDC4002358	USA (Oklahoma)	Negative
Burkholderia oklahomensis	CDC4021865	USA (Oklahoma)	Negative
Burkholderia oklahomensis	CDC4021866	USA (Oklahoma)	Negative
Burkholderia vietnamiensis	CDC2734483	Vietnam	Negative
Burkholderia pyrrocinia	ATCC 15958	Unknown	Negative
Burkholderia caledonica	CDC8724197	United Kingdom	Negative
Burkholderia caribensis	CDC8724200	Martinique	Negative
Burkholderia ambifaria	CDC8724201	USA (Wisconsin)	Negative
Burkholderia anthina	CDC8724199	USA (Tennessee)	Negative
Burkholderia cocovenenans	CDC2734715	Indonesia	Negative
Burkholderia ferrariae	CDC8724209	Brazil	Negative
Burkholderia hydrophila	CDC2721759	Thailand	Negative
Burkholderia fungorum	ATCC BAA-463	Unknown	Negative
Burkholderia glathei	CDC2734719	Germany	Negative
Burkholderia graminis	CDC2734716	France	Negative
Burkholderia hospita	CDC8724207	Belgium	Negative
Burkholderia kururiensis	CDC2734717	China	Negative
Burkholderia nodosa	CDC8724205	Brazil	Negative
Burkholderia phenazinium	ATCC 33666	Unknown	Negative
Burkholderia phenoliruptrix	CDC8724203	USA	Negative
Burkholderia phymatum	CDC8724208	French Guiana	Negative
Burkholderia phytofirmans	CDC8724204	Germany	Negative
Burkholderia sacchari	CDC8724202	Brazil	Negative
Burkholderia silvatlantica	ATCC BAA-1244	Brazil	Negative
Burkholderia rhizoxinica	DSM19002	Germany	Negative
Burkholderia endofungorum	DSM19003	Germany	Negative
Burkholderia gladioli	CDC3027208	USA (California)	Negative

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.