Abstract. A rapid method for the identification and differentiation of Burkholderia pseudomallei and Burkholderia thailandensis colonies is described. It consists ...
Am. J. Trop. Med. Hyg., 66(6), 2002, pp. 759–761 Copyright © 2002 by The American Society of Tropical Medicine and Hygiene
SHORT REPORT: A RAPID METHOD FOR THE DIFFERENTIATION OF BURKHOLDERIA PSEUDOMALLEI AND BURKHOLDERIA THAILANDENSIS VANAPORN WUTHIEKANUN, NARISARA ANUNTAGOOL, NICHOLAS J. WHITE, AND STITAYA SIRISINHA Faculty of Tropical Medicine and Faculty of Science, Mahidol University, Bangkok, Thailand; Chulabhorn Research Institute, Bangkok, Thailand; University of Oxford, Oxford, England, United Kingdom
Abstract. A rapid method for the identification and differentiation of Burkholderia pseudomallei and Burkholderia thailandensis colonies is described. It consists of simultaneous use of 2 monoclonal antibody–based latex agglutination test systems. The anti-lipopolysaccharide test reacts with both species, whereas the anti-exopolysaccharide reacts only with B. pseudomallei. Compared with classical biochemical tests, the method is highly reproducible and accurate. It is particularly useful for the identification of the organisms in environmental specimens, which may contain both of these Burkholderia species. Thailand) and gives a positive reaction with both B. pseudomallei and B. thailandensis.16 The other latex agglutination test system is based on the use of a mixture of MAbs reactive specifically with the 200-kDa exopolysaccharide (anti-200 kDa) present only in B. pseudomallei and not in B. thailandensis (Laboratory of Immunology, Chulabhorn Research Institute, Bangkok, Thailand).7,17,18 Details of both latex agglutination test systems have been described previously.16,19,20 Briefly, the latex particles were coated with appropriate concentrations of purified MAbs in glycinebuffered saline. The adsorbed latex particles were then blocked with bovine serum albumin and resuspended to give a final latex suspension of 0.5–1%. In the assays, 1–2 colonies of bacteria were resuspended in 20–30 L of phosphatebuffered saline (for anti-200 kDa) or diluent buffer containing 2% n-octyl -D-glucopyranoside (for anti-LPS) on a glass slide and mixed with an equal volume of latex suspension. Positive results could be visualized within 1–2 minutes. The diagnostic value of these 2 test systems, particularly in identification of B. pseudomallei in hemoculture fluids, was found to be highly reproducible and provided satisfactory results compared with the culture method used as gold standard.16,19,20 In the present study with environmental specimens, 100 mL of sterile, distilled water was added to 100 g of soil and mixed. The mixture was allowed to sediment overnight, and the upper fluid layer (20 mL) was transferred to a sterile container and shaken. Aliquots (100 L) of the surface liquid and 3 serial 10-fold dilutions in sterile distilled water were then spread on the selective Ashdown agar21 (modified by using a higher concentration of gentamicin [8 mg/L] to minimize overgrowth of other soil dwelling bacteria) with a rotary plater so that individual colonies could be identified. Further aliquots of the surface liquid (100 L and 1 mL) were added to 9 mL of a selective/enrichment broth consisting of threonine-basal salt solution containing colistin 50 mg/L.22 This selective broth was previously found to give high recovery rates of B. pseudomallei.4 The organisms were also subcultured on modified Ashdown agar after 48 h. All plates and broths were incubated aerobically at 42°C. The plates were inspected daily for 6 days; B. pseudomallei and B. thailandensis were identified by characteristic colonial morphology on the Ashdown agar,21 positive oxidase reaction, resistance to colistin, and gentamicin as described previously.23 The utilization of L-arabinose was tested on an agar medium, and further confirmation was based on the results of API 50CH, as described previously.6 Suspect colonies were screened with
INTRODUCTION Burkholderia pseudomallei is an environmental saprophyte that causes the potentially fatal disease melioidosis, an infection that is endemic in areas of Southeast Asian countries and northern territory of Australia.1 The disease in humans varies from subclinical to severe acute septicemia, which may be fatal within 24–48 hours after hospital admission. It is acquired by wound exposure to contaminated soil and surface water or by inhalation of dust particles containing B. pseudomallei. Soil and water in the endemic areas of infection often contains both B. pseudomallei and a nonvirulent, closely related species, now known as B. thailandensis.2–4 The latter species was initially differentiated from its virulent counterpart by the ability to assimilate L-arabinose (ara+).5,6 It was shown subsequently that the 2 species could be distinguished from one another by monoclonal antibodies (MAbs) specific for the exopolysaccharide antigen present only in B. pseudomallei.7–9 The polyclonal rabbit antiserum currently available for diagnostic purposes cannot distinguish these 2 species.7,10 B. thailandensis can also be found in large quantities in the environment in nonendemic areas.3,11–13 The simultaneous presence of a mixture of these 2 closely related species in the same soil or water sample is a problem for environmental epidemiological surveys of melioidosis (authors’ unpublished data). Another complicating factor is variation in colonial morphology from different isolates of both B. pseudomallei and B. thailandensis. Different colony appearances may also be found within a single isolate.14 The techniques used for environmental surveys have varied widely between the use of animal inoculation, which is too costly, time-consuming, and laborious, and in addition, it cannot detect the nonvirulent B. thailandensis. Further, the use of artificial media may also lead to a failure to distinguish between B. pseudomallei and B. thailandensis. Currently, several biochemical test kits (e.g., API 20NE and API 50CH, Bio-Merieux, Marcy-l’Etoile, France), are available for biochemical distinction, but they are too costly to use for screening, particularly for epidemiological surveys15; and selective media have been developed, but these do not provide clear distinction and require some experience in interpretation. The alternative method described herein is based on a combination of 2 commercially available MAb-based latex agglutination test systems. One is specific against lipopolysaccharide (anti-LPS; National Center for Biotechnology, National Science and Technology Development Agency, Bangkok,
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both latex agglutination test systems by mixing 1–2 colonies with 30 L of latex suspension. The latex agglutination test results were read within 1–2 minutes and compared with those from the culture and biochemical tests used as a gold standard. Soil samples used in this study were collected from different provinces in the endemic northeastern part of Thailand. The sample collection was also expanded to include those from the neighboring Laos around Vientiane, carried out as part of epidemiology studies on melioidosis in both countries. The results presented in Table 1 demonstrate that these 2 latex agglutination test systems were in complete agreement with the conventional culture method in identifying B. pseudomallei and B. thailandensis simultaneously present in environmental samples. All 220 culture-confirmed Burkholderia isolates were positive with the anti-LPS system, whereas only the culture-proven B. pseudomallei isolates were positive with the anti-200 kDa system. The isolates positive with antiLPS and negative with anti-200 kDa were considered to be B. thailandensis, in complete agreement with the results from biochemical tests. The accuracy of the anti-200 kDa in distinguishing B. pseudomallei from B. thailandensis has been documented before by other investigators8 as well as by our own group.7 The combination of these 2 latex agglutination test systems was also 100% accurate in identifying simultaneously these organisms grown on the modified Ashdown agar at a stage when these 2 species could not yet be identified by other means. We therefore recommend replacing the conventional culture method with this new protocol in future epidemiological survey because it is less expensive, less time-consuming, and simple to perform. There is no need for laboratory personnel to have special skills or to have previous experience working with these organisms. All other colonies grown on the Ashdown agar—Burkholderia cepacia, Pseudomonas spp., or any other gram-negative organism—can be tested readily and quickly eliminated. By use of these 2 latex agglutinating test systems, we also prospectively identified B. pseudomallei colonies from clinical specimens collected at Sappasitprasong Hospital, located in the endemic Ubon Ratchatani province, during the year 1999–2000. The results presented in Table 2 were consistent with our previous field studies showing that both systems were accurate and reliable in detecting B. pseudomallei cultured from clinical specimens. The anti-200 kDa test could identify all culture-proven B. pseudomallei isolates and gave only one false-positive finding (out of 218 samples) with an isolate that was subsequently identified as a Pseudomonas spp. This Pseudomonas isolate could grow on the Ashdown medium, and its colonial appearance was different from those
TABLE 2 Comparative evaluation of anti-lipopolysaccharide (LPS) and anti200 kDa for identification of Burkholderia pseudomallei in clinical isolates Latex agglutination Anti-LPS
Anti-200 kDa
Organisms
Total
Positive
Negative
Positive
Negative
B. pseudomallei Others Gram-positive cocci Pseudomonas spp. Acinetobacter spp. Enterobacter spp. Klebsiella spp. Escherichia coli Chryseobacterium indofogenes Burkholderia cepacia Yeast No identification Pseudomonas aeruginosa Total
354
352
2
354
0
3 6 9 8 4 2
0 2 1 0 0 1
3 4 8 8 4 1
0 1 0 0 0 0
3 5 9 8 4 2
1 3 5 174 3 218
1 0 0 0 0 5
0 3 5 174 3 213
0 0 0 0 0 1
1 3 5 174 3 217
of typical Burkholderia species. This same isolate and 4 other gram-negative bacteria gave positive agglutination with the anti-LPS system. The anti-LPS test also failed to identify 2 culture-proven B. pseudomallei isolates. These 2 isolates, which gave positive agglutination with the anti-200 kDa test, were subsequently found to possess an unusual type of LPS, which we referred to as “atypical” LPS in our original report.24 We subsequently showed that 3–4% of nearly 1,000 Thai B. pseudomallei isolates exhibited this unusual, immunologically distinct, LPS.25 In contrast to the Thai isolates, 17% of the Australian B. pseudomallei isolates exhibited this unusual type of LPS.26 The results of the present series of clinical specimens showed the accuracy and reliability of the anti-200 kDa latex agglutination test system in the detection of B. pseudomallei and the diagnosis of melioidosis. Although this anti-200 kDa agglutination test does not react with B. thailandensis, this is not a problem in clinical practice because this organism is avirulent and therefore clinically insignificant.27,28 Therefore, with clinical specimens, the anti-LPS testing system is not mandatory. On the other hand, with environmental specimens where B. thailandensis is known to be present together with B. pseudomallei in some soil samples taken from the endemic areas of infection (authors’ unpublished data) both the anti200 kDa and anti-LPS should be used in order to identified the presence and to differentiate of these 2 species. Taken together, the results of this alternative protocol us-
TABLE 1 Identification of Burkholderia pseudomallei and Burkholderia thailandensis from soil samples by latex agglutination tests Latex agglutination Anti-lipopolysaccharide
Anti-200 kDa
Isolates from:
Burkholderia spp.
No. tested
Arabinose assimilation
Positive
Negative
Positive
Negative
Thailand
pseudomallei thailandensis pseudomallei thailandensis pseudomallei thailandensis
96 56 47 21 143 77
− + − + − +
96 56 47 21 143 77
0 0 0 0 0 0
96 0 47 0 143 0
0 56 0 21 0 77
Laos Total
DIFFERENTIATION OF B. PSEUDOMALLEI AND B. THAILANDENSIS
ing a combination of these 2 MAb-based latex agglutinating test systems were in complete agreement with those of the conventional culture and biochemical tests in identifying B. pseudomallei and B. thailandensis in both environmental and clinical specimens. This simple system is recommended, particularly for field surveys in which a large number of specimens needed to be identified. The results can be obtained quickly and can be readily performed anywhere, including small health centers and remote laboratories with minimum equipment in rural endemic areas. No special skills are needed, and it can be performed readily by any laboratory personnel. Acknowledgments: We thank the National Centre for Biotechnology, National Science and Technology Development Agency, Bangkok, Thailand, for supplying the anti-LPS latex agglutination test and Laboratory of Immunology, Chulabhorn Research Institute, Bangkok, for providing the anti-200 kDa latex agglutination kit. We thank Dr. Wipada Chaowagul of Sappasitprasong Hospital, Ubon Ratchatani, for her support. We thank Dr. Wironrong Cheragul, Dr. Jenny Short, and Sayan Langla for their assistance with this work. This study was part of the Wellcome Trust–Mahidol University, Oxford Tropical Medicine Research Programme. Financial support: This work was supported by the Wellcome Trust of Great Britain and Chulabhorn Research Institute, Thailand. Authors’ addresses: Vanaporn Wuthiekanun and Nicholas J. White, Wellcome Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand 10400. Narisara Anuntagool, Laboratory of Immunology, Chulabhorn Research Institute, Bangkok, Thailand 10210. Stitaya Sirisinha, Department of Microbiology, Faculty of Science, Mahidol University and Chulabhorn Research Institute, Bangkok, Thailand. Reprint requests: Stitaya Sirisinha, Laboratory of Immunology, Chulabhorn Research Institute, Bangkok 10210, Thailand, Telephone: 662-5740622-33 ext. 1403, Fax: 662-574-2027, E-mail: scssr@mahidol. ac.th.
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