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Rapid Immunofluorescence Microscopy for Diagnosis of Melioidosis

Faculty of Tropical Medicine, Wellcome Trust-Oxford University-Mahidol University Tropical Medicine Research Program,¹ Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand,² Menzies School of Health Research, Charles Darwin University, and Northern Territory Clinical School, Flinders University, Darwin, Australia,³ Centre for Clinical Vaccinology and Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, Churchill Hospital, Oxford, United Kingdom⁴

Received 26 November 2004/ Returned for modification 4 January 2005/ Accepted 14 January 2005

	ABSTRACT

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An immunofluorescent (IF) method that detects Burkholderia pseudomallei in clinical specimens within 10 min was devised. The results of this rapid method and those of an existing IF method were prospectively compared with the culture results for 776 specimens from patients with suspected melioidosis. The sensitivities of both IF tests were 66%, and the specificities were 99.5 and 99.4%, respectively.

	TEXT

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Melioidosis, the disease caused by Burkholderia pseudomallei, is endemic in Southeast Asia. Melioidosis accounts for approximately one-fifth of all community-acquired cases of septicemia in areas of endemicity, such as northeast Thailand, where it is associated with a mortality rate of about 50% (1, 5). Rapid diagnosis in rural Thailand is important, since the antibiotics empirically prescribed for the treatment of patients presenting with sepsis of unknown cause may not include the relatively expensive antibiotics, ceftazidime or a carbapenem, that are required for the treatment of melioidosis. Culture of B. pseudomallei from any specimen is diagnostic and represents the "gold standard," but a delay of 24 to 48 h or more between the time of specimen plating and bacterial growth plus presumptive identification often occurs. A simple, rapid test performed directly with clinical samples may influence the time taken to begin effective treatment and the subsequent outcome. We have previously described a direct immunofluorescent (IF) technique for the detection of B. pseudomallei in clinical samples (3) and have used this technique in our diagnostic and research laboratory in a provincial hospital for more than a decade. It is relatively labor intensive and takes more than 2 h to complete; here, we report on the results of a simplified method that can be completed in 10 min.

A prospective study was conducted between June 2002 and October 2004 by a study team at Sappasithiprasong Hospital, Ubon Ratchathani, northeast Thailand. Patients with suspected melioidosis were actively sought during twice-daily rounds of the medical and intensive care wards. Specimens for microbiological examination were taken from all patients and were cultured by standard procedures (4).

The methodology for preparation and storage of the IF conjugate was performed as described previously (2), except that bacteria were killed with formalin rather than heat. Two direct IF methods were applied in parallel to clinical samples of respiratory secretions, pus from sterile sites, urine, and blood culture fluid to detect B. pseudomallei. The standard direct IF technique was performed as described previously (3), except that 5% skim milk in phosphate-buffered saline was used as the blocking agent. The rapid method was a one-step technique in which 1 drop (10 µl) of specimen was mixed on a clean glass microscopic slide with an equal volume of conjugate and a coverslip applied. The white blood cells present in pus were lysed prior to examination by the addition of an equal volume of distilled water, and respiratory secretions were mixed with an equal volume of sterile distilled water before examination. Conjugate was used at a 1:200 dilution in blocking buffer. The slides were examined with a fluorescent microscope with a x100 oil-immersion lens. A positive result by either test was recorded when the periphery of the bacilli showed a strong apple-green fluorescence. A slide known to be positive (for a clinical isolate of B. pseudomallei) was prepared and examined in each test run.

Testing was performed directly on 776 specimens (respiratory secretions, urine, or pus from sterile sites) from 646 patients with suspected melioidosis. B. pseudomallei was cultured from 154 samples taken from 120 patients. Of these, 108 samples (70%) from 84 patients were positive by either the standard or the rapid IF method (Table 1); 96 samples were positive by both IF methods, 6 were positive by the standard IF method only, and 6 were positive by the rapid IF method only. Of the 46 specimens that were not positive by either IF method, 9 (20%) grew viable colonies only after enrichment in a selective broth, compared to 2 of the 108 specimens positive by either method (P < 0.001). The sensitivity of the standard IF method was 66%, and that of the rapid IF method was also 66%.

The IF techniques were also evaluated for their abilities to presumptively identify B. pseudomallei from specimens in blood culture bottles. A total of 241 blood cultures were taken from 227 patients with suspected melioidosis. Five milliliters of blood was inoculated into aerobic BacT/Alert FA bottles (BioMérieux, Durham, N.C.), which were incubated for 7 days at 37°C. The bottles were inspected daily, and the contents subcultured onto blood agar if the indicator changed color, and routinely on days 1, 2, and 7. B. pseudomallei was isolated from 42 of 241 blood culture specimens. The other organisms isolated included Escherichia coli (n = 34), Pseudomonas spp. (n = 31), Enterobacter spp. (n = 14), Acinetobacter spp. (n = 11), Klebsiella spp. (n = 11), Salmonella spp. (n = 9), other gram-negative rods (n = 6), Staphylococcus aureus (n = 22), coagulase-negative staphylococci (n = 24), Streptococcus spp. (n = 10), other gram-positive cocci (n = 2), gram-positive rods (n = 6), and fungal organisms (n = 19). Positive bottles were simultaneously examined by the standard and rapid IF methods. All samples culture positive for B. pseudomallei were positive by both the standard and the rapid IF methods; there were no false-positive test results. Thus, the sensitivity and specificity of both the standard and the rapid IF methods were 100%.

There were a small number of discordant results between the two IF methods, but the use of both methods in parallel increased the sensitivity from only 66 to 70%. The overall sensitivity of the direct IF method reported previously (3) was 73%. The lower result here may reflect the effect of earlier empirical treatment for melioidosis or presentation for care earlier in the course of the illness, both of which result in lower bacterial loads and diagnostic yields. The small number of false-positive results may reflect the presence of nonviable organisms affected by prior antibiotic administration. We conclude that the rapid IF method has a sensitivity and a specificity equivalent to those of the previously reported standard IF method.

	ACKNOWLEDGMENTS

 
We are grateful for the support of the medical, nursing, and laboratory staff at Sappasithiprasong Hospital, including Wipada Chaowagul and Nittaya Teerawattanasook. We also thank Ty Pitt, Central Public Health Laboratory, London, United Kingdom, for significant technical assistance. We acknowledge the assistance of staff of the Wellcome Trust-Mahidol University-Oxford University Tropical Medicine Research Program, including Nongluk Getchalarat, Jintana Suwannapreuk, and Sukallaya Paengmee.

A.C.C. is supported by an Australian National Health and Medical Council Training Scholarship, and S.J.P. is supported by a Wellcome Trust Career Development Award in Clinical Tropical Medicine. This study was funded by the Wellcome Trust of Great Britain.

None of the authors has a conflict of interest.

	FOOTNOTES

 
* Corresponding author. Mailing address: Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Rd., Bangkok 10400, Thailand. Phone: 66 2 354 9172. Fax: 66 2 354 9169. E-mail: lek{at}tropmedres.ac.

	REFERENCES

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1. Chaowagul, W., N. J. White, D. A. Dance, Y. Wattanagoon, P. Naigowit, T. M. Davis, S. Looareesuwan, and N. Pitakwatchara. 1989. Melioidosis: a major cause of community-acquired septicemia in northeastern Thailand. J. Infect. Dis. 159:890-899.[Medline]
2. Samuel, D., R. J. Patt, and R. A. Abuknesha. 1988. A sensitive method of detecting proteins on dot and Western blots using a monoclonal antibody to FITC. J. Immunol. Methods 107:217-224.[CrossRef][Medline]
3. Walsh, A. L., M. D. Smith, V. Wuthiekanun, Y. Suputtamongkol, V. Desakorn, W. Chaowagul, and N. J. White. 1994. Immunofluorescence microscopy for the rapid diagnosis of melioidosis. J. Clin. Pathol. 47:377-379.[Abstract/Free Full Text]
4. Walsh, A. L., and V. Wuthiekanun. 1996. The laboratory diagnosis of melioidosis. Br. J. Biomed. Sci. 53:249-253.[Medline]
5. White, N. J. 2003. Melioidosis. Lancet 361:1715-1722.[CrossRef][Medline]

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