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Laboratory Diagnosis

The only way in which the diagnosis can be made with certainty is by demonstration of the organism. Bacilli may be recovered from the blood, urine, sputum, pus or skin lesions of patients with melioidosis. The organism has been found in cerebrospinal fluid and can be recognized either by direct smear or culture. (In artificially infected laboratory animals, multiple small nodules or abscesses form in the internal organs; the animals develop a permanent discharge from the nose and eyes, and excrete the organisms in their urine and feces.)

Burkholderia pseudomallei is a small, pleomorphic, gram-negative, rod-shaped bacterium that has bipolar staining properties best appreciated by Gram or Giemsa stain. Typical bacilli may be sparse in acute suppurative lesions, where they can be both extra- and intracellular. They are more readily seen within cells in the subacute and chronic granulomatous forms of the disease, where they appear in macrophages and giant cells as globi of criss-crossed bacilli.

B. pseudomallei is not fastidious, but indolent in culture. Cultures are usually positive within 48 hours (but may take longer) after incubation at 37 or 42C on most routine culture media, including a nutrient agar such as eosin-methylene blue. The organism may be obscured in sputum culture by faster growing contaminants. Ashdown's selective medium has proven reliable in dealing with this problem, especially where melioidosis is endemic. Blood culture has proven to be an important adjunct in diagnosing melioidosis. More dilute (1:10) cultures, those subcultured earlier and more often, and those incubated in a resin-containing medium improve the possibility of isolating the organism in patients receiving antibiotics. Isolation rates have been as high as 86% in patients not receiving antibiotics at the time of culture, with as little as 5 ml of blood incubated at a high (1:4) blood-to-broth ratio in an unsupplemented broth bottle.

Because seropositivity reflects evidence of infection rather than actual disease, complement fixation, hemagglutination, and fluorescent antibody tests are primarily used for confirmation of the clinical diagnosis. The indirect hemagglutination (IHA) test is considered to be more specific than the complement fixation tests. Titers of 1:40 or higher are significant for chronic or subclinical infection; however, these levels are not diagnostic of acute infection but are most significant when rising to 1:80 or more. Rapid screening of the patient's serum can be performed with an indirect or direct immunofluorescent antibody technique, which detects both IgM and IgG. More recently, enzyme-linked immunosorbent assays, measuring specific IgM and IgG, have been developed (Ashdown et al, 1989; Kunakorn et al, 1990). Using a combination of IHA and IgM ELISA, a sensitivity of 100% and a specificity of 95% can be achieved.

Once a positive diagnosis is made, either by isolation of the organism or by one or more of the above serological procedures, subsequent complement fixation and hemagglutination tests or serial assays for C-reactive protein can be used to monitor the patient's response to treatment. Other laboratory data are within normal limits, except for an occasional increased sedimentation rate, elevated white blood cell count, or normochromic, normocytic anemia.

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