Melioidosis, an infection caused by Burkholderia pseudomallei, is endemic in Southeast ... of the bacteria in blood cultures in areas endemic for melioidosis.
Am. J. Trop. Med. Hyg., 61(4), 1999, pp. 658–662 Copyright q 1999 by The American Society of Tropical Medicine and Hygiene
RAPID IDENTIFICATION OF BURKHOLDERIA PSEUDOMALLEI IN BLOOD CULTURES BY LATEX AGGLUTINATION USING LIPOPOLYSACCHARIDE-SPECIFIC MONOCLONAL ANTIBODY TARARAJ DHARAKUL, SIRIRURG SONGSIVILAI, SAIJAI SMITHIKARN, CHARIN THEPTHAI, AND AMORNRAT LEELAPORN Laboratory of Cellular and Molecular Immunology, Department of Immunology, and Department of Microbiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand
Abstract. Melioidosis, an infection caused by Burkholderia pseudomallei, is endemic in Southeast Asia. The septicemic form of melioidosis is the leading cause of death due to community-acquired bacteremia in the northeastern part of Thailand. The delay in isolation and identification of the causative organism is a major contributing factor to the high mortality. The present study describes the evaluation of a latex agglutination test for rapid identification of the bacteria directly from blood cultures. The Bps-L1 monoclonal antibody recognized the lipopolysaccharide antigen of 96.8% of B. pseudomallei clinical isolates and was highly specific for B. pseudomallei. The diagnostic value of the latex agglutination test based on Bps-L1 monoclonal antibody was prospectively evaluated in an area endemic for melioidosis. The agglutination test kit was evaluated in 88 blood cultures with gram-negative bacteria identified with Gram staining. The sensitivity and specificity of the test kit were both 100%. These results indicated that the detection of B. pseudomallei lipopolysaccharide by specific monoclonal antibody in a latex agglutination format is clinically useful for the rapid identification of the bacteria in blood cultures in areas endemic for melioidosis. Melioidosis is an important infectious disease known to be endemic in Southeast Asia and the northern part of Australia. The causative agent, Burkholderia pseudomallei, is one of the most important causes of fatality in communityacquired septicemia in northeastern Thailand.1 The highest mortality occurs in patients with the septicemic form of melioidosis, which is characterized by dissemination of the bacteria in the circulation and isolation of the bacteria from blood and various organs. The clinical course of septicemic melioidosis often deteriorates rapidly and death occurs within the first few days after hospitalization. Rapid diagnosis and prompt treatment with appropriate antibiotics can reduce the mortality at least by half.2 Current clinical practice in areas endemic for melioidosis requires a combination of high clinical suspicion and bacterial isolation by culture. The gold standard method for laboratory diagnosis of melioidosis is the isolation of the bacteria, which generally takes 3–7 days. However, in many cases the results are obtained after the patient has died.1 Blood is the main clinical specimen subjected to bacterial isolation in septicemic melioidosis. Therefore, methods that can shorten the time required for the bacterial isolation, as well as for bacterial identification, are urgently needed. Our previous study demonstrated that the time required for growth of B. pseudomallei that was sufficient for isolation from blood culture was within 48 hr in 93% of the patients.3 The present study was therefore undertaken to evaluate the applicability of a method for rapid identification of B. pseudomallei in blood culture. A latex agglutination test kit based on a specific monoclonal antibody to the bacterial lipopolysaccharide was developed and the performance of the test was prospectively evaluated in an area endemic for melioidosis.
of Microbiology, Faculty of Medicine, Siriraj Hospital (Bangkok) and the Department of Clinical Pathology, Khonkaen Regional Hospital (Khonkaen). All of these isolates had the arabinose-negative phenotype. Fifty isolates of other medically important bacteria were used as controls. These included 1 clinical isolate each of Acinetobacter anitratus, Burkholderia cepacia, Escherichia coli, Enterobacter cloacae, Haemophilus influenzae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas putida, Serratia marcescens, Staphylococcus aureus, Streptococcus pneumoniae, group A, group B, and group D streptococci, as well as 17 isolates of Aeromonas hydrophila and 3 isolates of Aeromonas veronii obtained from the Department of Microbiology, Faculty of Medicine, Siriraj Hospital (Bangkok). In addition, 14 isolates of B. cepacia, and 1 isolate each of Stenotrophomonas maltophilia (DMS904/NCTC10257) and Pseudomonas fluorescens (DMS2589) type strains were provided by the National Institute of Health of Thailand. Stocks of the bacteria in 20% glycerol were aliquoted and kept at 2208C until use. Burkholderia pseudomallei–specific monoclonal antibody. The monoclonal antibody was produced using a standard hybridoma technique4 by fusion of the splenocytes with the P3X63-Ag8/653 mouse myeloma cell line using polyethylene glycol (PEG-4000; Accurate Chemical and Scientific Co., Westbury, NY). The antigen used for immunizing BALB/c mice was prepared from sonicated B. pseudomallei. Several hybridoma clones that recognized the lipopolysaccharide (LPS) of B. pseudomallei were produced, all of which produced antibodies of IgM isotype. One clone was selected for further characterization (Bps-L1). This monoclonal antibody recognized the LPS of B. pseudomallei as demonstrated by a characteristic ladder pattern of staining by Western blot analysis (Figure 1). The monoclonal antibody was produced in the CELLMAXy artificial capillary cell culture system (Cellco, Inc., Germantown, MD), according to the manufacturer’s recommendation. The culture supernatant fluid was concentrated by 50% ammonium sul-
MATERIALS AND METHODS
Bacterial strains. A total of 126 clinical isolates of B. pseudomallei, including 65 isolates from blood and 61 isolates from other sites, were obtained from the Department
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LATEX AGGLUTINATION KIT FOR DIAGNOSIS OF MELIOIDOSIS
TABLE 1 Reactivities of the monoclonal antibody Bps-L1 with 126 clinical isolates of Burkholderia pseudomallei from blood and other sites
FIGURE 1. Western blot analysis of the Burkholderia pseudomallei crude lipopolysaccharide with Bps-L1 monoclonal antibody. Lane 1, Escherichia coli; lanes 2–5, non-agglutinable B. pseudomallei isolates; lane 6, agglutinable B. pseudomallei; lane 7, molecular weight markers. kD 5 kilodaltons.
fate precipitation and the IgM antibodies were further purified by Sephadex G-200 gel filtration chromatography. Western blot analysis. The supernatant fluid of the bacterial culture was treated with 100 mg/ml of proteinase K (Sigma Chemical Co., St. Louis, MO) at 568C for 1 hr and the enzyme was inactivated at 958C for 10 min. The proteinase K-treated bacterial lysate was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDSPAGE) and blotted onto a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany). The blotted nitrocellulose membrane was blocked with skim milk and was subsequently incubated with the culture supernatant fluid of Bps-L1 monoclonal antibody. The reaction was detected using alkaline phosphatase–conjugated goat anti-mouse IgM (Kirkegaard and Perry Laboratories, Gaithersburg, MD) o-dianisidine and b-naphthyl phosphate as substrates.4 Silver staining of the LPS. The crude bacterial lysate containing LPS was prepared by treatment with proteinase K and subjected to SDS-PAGE as above. The gel was incubated in 30% ethanol and 10% acetic acid overnight, followed by 30% ethanol for 30 min twice and 3 changes of deionized water. The gel was then incubated with 0.1% fresh silver nitrate for 30 min in the dark. The reaction was developed with 2.5% sodium carbonate and 0.02% formaldehyde until bands began to appear.4 Passive adsorption of the IgM monoclonal antibody onto latex particles. Purified IgM monoclonal antibody (200 mg/ml) was passively adsorbed onto 0.38-mm polystyrene latex particles (Interfacial Dynamics Corp., Portland, OR) for 2 hr at room temperature. The adsorbed latex particles were washed 3 times in glycine-buffered saline. The remaining protein binding sites on the surface of the latex were blocked with a 1% (w/v) solution of bovine serum albumin (BSA). The coated latex particles were then washed and resuspended in glycine-buffered saline containing 0.1% BSA and 0.1% sodium azide to give a final concentration of 0.5% latex suspension. The latex suspension was stored at
Source of B. pseudomallei clinical isolates
Number tested
Blood Sputum Urine Pus Total
65 17 11 33 126
Number of isolates tested positive with the Bps-L1 coated latex particles (%)
65 16 10 31 122
(100) (94.1) (90.9) (93.9) (96.8)
48C for up to 1 year and brought to room temperature before use. Latex agglutination assay. One drop of the bacterial culture was mixed with an equal volume of a diluent buffer containing 2% n-octyl b-D-glucopyranoside (b-OG) on a clean glass slide using a wooden applicator. A drop of the latex reagent was added to the mixture and the slide was rotated gently. Agglutination could be seen in the dark background within 2 min. The agglutination reaction was scored from 11 to 41. Typically, a 41 reaction was observed as large floccules formed instantly with clear background fluid. A negative reaction was characterized by a uniform milky fluid with no visible floccules. Field evaluation of the Bps-L1 latex agglutination test kit for the detection of B. pseudomallei antigen in blood cultures. The Bps-L1-based latex agglutination test kit was prospectively evaluated in an area endemic for melioidosis. The evaluation was performed at the clinical microbiology laboratory at Burirum General Hospital (Burirum, Thailand) by the local laboratory personnel who routinely processed the blood culture specimens. This laboratory used a manual blood culture system that was widely used in Thailand. Briefly, blood from patients with suspected bacteremia was inoculated into brain heart infusion broth and cultured at 378C. The cultures were examined daily by the laboratory personnel for turbidity and the specimens with turbid culture fluid were subjected to Gram staining. All specimens that showed gram-negative staining were then tested using the latex agglutination test kit and the results were recorded. The result of standard bacterial identification by biochemical reactions, which were usually obtained 1–2 days later, were also independently recorded. At the end of the study period, the results of both tests were analyzed. RESULTS
Burkholderia pseudomallei LPS-specific monoclonal antibody. The reactivity of the monoclonal antibody BpsL1 was evaluated using 126 B. pseudomallei clinical isolates (Table 1). This antibody reacted with 122 (96.8%) of 126 B. pseudomallei isolates, and reacted with all isolates from blood (100%), but not with 1, 1, and 2 specimens from sputum, urine, and pus, respectively. The 4 B. pseudomallei clinical isolates that were not agglutinated by the Bps-L1 monoclonal antibody were further characterized. The identity of these isolates was confirmed by polymerase chain reaction amplification of the 16S ribosomal RNA gene5 and by standard biochemical identification. The reactivities of the monoclonal antibody was con-
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FIGURE 2. Silver staining of the Burkholderia pseudomallei lipopolysaccharide (LPS), demonstrating the distinct LPS pattern of the 4 nonagglutinable B. pseudomallei isolates. Lane 1, molecular weight markers; lanes 2, 3, and 8, agglutinable B. pseudomallei isolates; lanes 4–7, non-agglutinable B. pseudomallei isolates. kD 5 kilodaltons.
firmed in Western blot analysis. The Bps-L1 antibody reacted with the LPS of 122 B. pseudomallei isolates, seen as a ladder of bands approximately 31–45 kD (Figure 1), but did not react with these 4 isolates. Silver staining of the LPS of these 4 isolates showed the pattern that was distinctively different from that of other B. pseudomallei isolates recognized by Bps-L1 (Figure 2). The specificity of the Bps-L1 monoclonal antibody was evaluated by latex agglutination using 50 isolates of other medically important bacteria. This antibody demonstrated no cross-reactivity with any of the other bacteria tested (Table 2). TABLE 2 Reactivities Burkholderia pseudomallei and other baterial species with the Bps-L1 coated latex particles Organisms identified
Burkholderia pseudomallei Other bacteria Acinetobacter anitratus Aeromonas hydrophila Aeromonas veronii Burkholderia cepacia Enterobacter cloacae Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa Pseudomonas fluorescens (DMS2589) Pseudomonas putida Stenotrophomonas maltophilia (DMS904/NCTC10257) Haemophilus influenzae Serratia marcescens Staphylococcus aureus Streptococcus pneumoniae Group A streptococcus Group B streptococcus Group D streptococcus Total
No. tested
No. positive (%)
126
122 (96.8%)
1 17 3 15 1 1 1 1 1 1
0 0 0 0 0 0 0 0 0 0
1 1 1 1 1 1 1 1 50
0 0 0 0 0 0 0 0 0 (0%)
Increased sensitivity of the latex agglutination test by treatment with detergents. Approximately 108 colonyforming units (CFU)/ml of B. pseudomallei was required for agglutination by Bps-L1-coated latex particles. This amount of bacteria was obtained after 20 hr of culture with an initial bacterial count of , 10 CFU/ml. The sensitivity of the latex agglutination test can be enhanced by inducing the release of B. pseudomallei LPS by treatment with heat or detergents. Two anionic detergents, SDS and sarkosyl, and 5 nonionic detergents, 3-[(3-cholamidopropyl) dimethylammonio]-1propane sulfonate (CHAPS), Triton X-114, Triton X-100, Tween 20, and b-OG, all at a final concentration of 1% were evaluated for their ability to release LPS. CHAPS, Triton X114, and b-OG enhanced the agglutination activity from 6 to 41, similar to that observed after boiling the bacterial culture for 5 min. However, Triton X-114 caused autoagglutination of the coated latex in the absence of bacteria. Further evaluation of CHAPS and b-OG indicated that 2.0% CHAPS or 1.0% b-OG was optimal in significantly enhancing the release of LPS and increasing the sensitivity of the latex agglutination from negative to 41. When 2% b-OG was used, the detection limit of the latex agglutination test kit was approximately 107 CFU/ml of B. pseudomallei. Development of the latex agglutination test kit. The latex agglutination test kit was developed based on Bps-L1 monoclonal antibody. The kit contained 100 tests each of the latex reagent and diluent buffer (2% b-OG in normal saline), and the positive and negative control samples. The test kit was stable and maintained its activity for at least 12 months. The test was performed by mixing 1 drop (30–50 ml) of blood culture fluid with 30 ml of diluent buffer containing detergent for 1 min on a clean glass slide, followed by adding 30 ml of the latex reagent and mixing; the agglutination reaction was clearly visible within 2 min. Evaluation of the Bps-L1 latex agglutination test kit for the detection of B. pseudomallei antigen in blood cultures in clinical microbiology laboratory in an area endemic for melioidosis. During the study period, all positive
LATEX AGGLUTINATION KIT FOR DIAGNOSIS OF MELIOIDOSIS
TABLE 3 Field evaluation of the latex agglutination kit for rapid identification of Burkholderia pseudomallei in blood culture No. tested
No. positive (%)
Burkholderia pseudomallei
Organisms identified
32
32 (100%)
Other gram-negative bacteria Burkholderia cepacia Acinetobacter anitratus Enterobacter cloacae Escherichia coli Klebsiella pneumoniae Providencia rettgeri Pseudomonas aeruginosa Salmonella group E Salmonella paratyphi Total
1 3 1 35 8 1 4 1 2 56
0 0 0 0 0 0 0 0 0 0 (0%)
blood cultures were subjected to Gram staining. A total of 88 blood culture specimens gave gram-negative bacterial staining. Thirty-two of these were confirmed to be B. pseudomallei by a standard microbiologic technique, and the other 56 were other gram-negative bacteria. The latex agglutination test was performed on all samples before the biochemical identification. The result of the agglutination test is summarized in Table 3. All B. pseudomallei cultures (100%) tested positive by the latex agglutination test kit. All but 1 of the specimens showed $ 31 agglutination, and 1 specimen showed a 11 agglutination reaction. None of other gram-negative bacteria showed a positive agglutination reaction. Therefore, the field evaluation of the test kit in the microbiology laboratory in the endemic area indicated that the sensitivity and specificity of the test kit were both 100%. DISCUSSION
The latex agglutination test using polyclonal anti-B. pseudomallei antiserum was previously reported for the detection of bacteria in blood culture and in clinical specimens.6,7 Although the polyclonal antibody contained high titer anti-LPS activity, it also contained antibodies to other components of the bacteria that may contribute to the cross-reactivity unless pre-absorption was performed. The purpose of the present study was to develop and evaluate the test kit based on a specific monoclonal antibody to B. pseudomallei. This antibody would detect bacterial antigen in the blood after initial cultures as a supplement to the standard bacterial identification method and hasten the diagnosis of melioidosis. The use of the monoclonal antibody provided several advantages, including high specificity with no cross-reactivity that may be present when polyclonal antibody to whole bacteria was used, the minimal batch-to-batch variation, and the ability to scale up production of the test kit. Recently, a latex agglutination test based on a monoclonal antibody against an exopolysaccharide of B. pseudomallei was also shown to react with B. pseudomallei isolated from several countries, including Thailand, but its application in an actual clinical situation was not evaluated.8 In the present study, the monoclonal antibody directed against LPS, which is a well-known major structural antigenic component of the bacteria, was used. The LPS of B. pseudomallei has been extensively studied
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and it is generally accepted that it represents a highly species-specific antigen that can be used for diagnostic purposes.9–11 The test kit based on this LPS-specific monoclonal antibody was developed and its performance was independently evaluated in a clinical microbiology laboratory in an area endemic for melioidosis. The monoclonal antibody described in this study recognizes the LPS of approximately 97% of the B. pseudomallei isolates. This finding supported the general conclusion that B. pseudomallei circulating in Thailand have only a single major serotype. The homogeneity of B. pseudomallei LPS was demonstrated in 2 previous studies.11,12 However, another study suggested the presence of 2 bacterial serotypes based on the O antigen.13 Interestingly, the 4 isolates that demonstrated the distinct ladder pattern of LPS and were not recognized by the monoclonal antibody in this study may represent another population of B. pseudomallei. Burkholderia pseudomallei with a distinct LPS pattern was also reported in 4 of 123 clinical isolates from Thailand,12 although the immunoreactivity was not described. In addition, an atypical B. pseudomallei isolate that contained only O-polysaccharide (O-PS) I and was not recognized by the polyclonal and monoclonal antibodies to O-PS II was also reported.9 Further study is required to elucidate the identity of these isolates. In addition, it should be noted that the test was evaluated in a clinical setting in Thailand where melioidosis is endemic. Although B. pseudomallei isolates are rather homogeneous and little differences are found among isolates from different geographic regions, the performance of this latex agglutination test kit in other countries remains to be investigated. Field evaluation of the Bps-L1 latex agglutination test kit showed a sensitivity and specificity of 100% after initial Gram staining; only gram-negative bacterial cultures were subjected to the agglutination test. This procedure appeared to be economical and effective in detecting B. pseudomallei in cultures. In addition, a minimal extra work load was required in incorporating the latex agglutination test in routine laboratory procedures. Identification of B. pseudomallei by the latex agglutination test can be done in less than 5 min, and diagnosis of melioidosis can be provided at least 1 day earlier. The earlier diagnosis will result in earlier treatment with appropriate antibiotics that may be able to save the lives of the critically ill patients. A previous study has shown that appropriate antibiotic therapy in septicemic melioidosis saved lives after the first 48 hr.2 The use of ceftazidime as a first-line drug before culture results are known appears attractive in a highly endemic area such as the northeastern part of Thailand; however, the high cost of such treatment prevent its wide use. The availability of this latex agglutination test kit may help hasten the diagnosis of septicemic melioidosis and appropriate treatment can therefore be provided earlier. In summary, the present study demonstrated the clinical applicability of the latex agglutination test kit that uses a monoclonal antibody (Bps-L1) to B. pseudomallei LPS. This test kit was evaluated for the detection of B. pseudomallei antigen in blood culture; the test was highly sensitive and specific. Studies are underway to evaluate this test kit on a larger scale in several laboratories in areas endemic for melioidosis. In addition, more sensitive assays based on this
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monoclonal antibody are being developed for the earlier detection of B. pseudomallei antigen in blood cultures, as well as in other specimens, such as urine or pus, which contain less of the bacterial antigen. Acknowledgments: We thank Nuanchan Piyasangthong, Stitaya Sirisinha, Surang Dejsirilert, and Somporn Srifuangfung for providing bacterial isolates; Kavi Rattanabannangkoon and Prakaithip Thongkum for technical assistance; and Somchai Jitrathai and Chaiya Rengpimai for help in the field evaluation of the latex agglutination test kit. Financial support: This work was supported by a grant from the National Science and Technology Development Agency of Thailand. Authors’ addresses: Tararaj Dharakul, Sirirurg Songsivilai, Saijai Smithikarn, and Charin Thepthai, Department of Immunology, Faculty of Medicine, Siriraj Hospital, Mahidol University, 2 Prannok Road, Bangkok 10700, Thailand. Amornrat Leelaporn, Department of Microbiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, 2 Prannok Road, Bangkok 10700, Thailand. Reprint requests: Tararaj Dharakul, Department of Immunology, Faculty of Medicine, Siriraj Hospital, Mahidol University, 2 Prannok Road, Bangkok 10700, Thailand. REFERENCES
1. Chaowagul W, White NJ, Dance DAB, Wattanagoon Y, Naigowit P, Davis TME, Looareesuwan S, Pitakwatchara N, 1989. Melioidosis: a major cause of community-acquired septicemia in northeastern Thailand. J Infect Dis 159: 890–899. 2. White NJ, Dance DAB, Chaowagul W, Wattanagoon Y, Wuthiekanun V, Pitakwatchara N, 1989. Halving of mortality of severe melioidosis by ceftazidime. Lancet ii: 697–700. 3. Tiangpitayakorn C, Songsivilai S, Piyasangthong N, Dharakul T, 1997. Speed of detection of Burkholderia pseudomallei in blood cultures and its correlation with the clinical outcome. Am J Trop Med Hyg 57: 96–99.
4. Harrow E, Lane D, 1988. Antibodies. A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. 5. Dharakul T, Songsivilai S, Viriyachitra S, Luangwedchakarn V, Tassaneetritap B, Chaowagul W, 1996. Detection of Burkholderia pseudomallei DNA in patients with septicemic melioidosis. J Clin Microbiol 34: 609–614. 6. Smith MD, Wuthieckanun V, Walsh AL, Pitt TL, 1993. Latex agglutination test for identification of Pseudomonas pseudomallei. J Clin Pathol 46: 374–375. 7. Smith MD, Wuthieckanun V, Walsh AL, Teerawattanasook N, Desakorn V, Suputtamongkol Y, Pitt TL, White NJ, 1995. Latex agglutination for rapid detection of Pseudomonas pseudomallei antigen in urine of patients with melioidosis. J Clin Pathol 48: 174–176. 8. Steinmetz I, Reganzerowski A, Brenneke B, Haussler S, Simpson A, White NJ, 1999. Rapid identification of Burkholderia pseudomallei by latex agglutination based on an exopolysaccharide-specific monoclonal antibody. J Clin Microbiol 37: 225–228. 9. Perry MB, MacLean LL, Schollaardt T, Bryan LE, Ho M, 1995. Structural characterization of the lipopolysaccharide O antigens of Burkholderia pseudomallei. Infect Immun 63: 3348– 3352. 10. Ho M, Schollaardt T, Smith MD, Perry M, Brett PJ, Chaowagul W, Bryan LE, 1997. Specificity and functional activity of antiBurkholderia pseudomallei polysaccharide antibodies. Infect Immun 65: 3648–3653. 11. Pitt TL, Aucken H, Dance DAB, 1992. Homogeneity of lipopolysaccharide antigens in Pseudomonas pseudomallei. J Infect 25: 139–146. 12. Anuntagool N, Intachote P, Wuthiekanun V, White NJ, Sirisinha S, 1998. Lipopolysaccharide from nonvirulent Ara1 Burkholderia pseudomallei isolates is immunologically indistinguishable from lipopolysaccharide from virulent Ara- clinical isolates. Clin Diag Lab Immunol 5: 225–229. 13. Dodin A, Fournier J, 1970. Antigenes precipitants et antigenes agglutinants de Pseudomonas pseudomallei (B. de Whitmore). Ann Inst Pasteur 119: 211–221.
