Laboratory diagnosis - Melioidosis

Isolation and Identification

B. pseudomallei is not difficult to isolate, and standard procedures should detect the organism in samples from normally sterile sites. Microscopy may show bipolar or unevenly staining Gram-negative bacilli, but this is neither specific nor sensitive. Isolation rates for B. pseudomallei from sites with a normal flora (superficial wound swabs, throat swabs, stool or rectal swabs) may be increased by the use of selective media (e.g. Ashdown’s medium, which may be obtained from the Reference Laboratory). More recently, commercial selective medium for the isolation of B. cepacia has been found to perform as well as Ashdown’s medium, and is more likely to be available in most diagnostic laboratories.

Persons to contact for Advice on diagnosis.

Culture

Samples should be processed according to usual isolation procedures. If melioidosis is suspected, all samples from sites with a normal flora (see above) should be cultured on B. cepacia or Ashdown’s medium in addition to routine culture media. All media should be incubated for a minimum of 48 hours before being discarded.

In difficult cases, the yield of cultures from sites with a normal flora (e.g. throat swabs, sputum) may be further increased by a pre-enrichment in a specific selective broth incubated at 42°C (contact the Reference Laboratory for details).

The appearance of B. pseudomallei on non-selective media is quite variable. Often there is a metallic surface sheen and a sweet, earthy smell. After 48 hours incubation, some strains form the characteristic rugose colonies described in text books, but other strains are smooth and shiny. Colonies on Ashdown’s agar are usually rough and wrinkled and violet to purple after 48 hours, irrespective of the strain. Colonies on B. cepacia medium resemble B. cepacia.

See here for Photographs of cultures of B. pseudomallei (and B. mallei).

Confirmation and Additional tests

Most isolates will be identified by routine laboratory methods, including commercial kits, of which the API20NE is best validated. The commonest profiles for B. pseudomallei using this kit are 1156577 and 1556577. The API20E and Vitek 1 automated systems will also correctly identify 96-99% of isolates. The Vitek 2 automated system, which misidentified a high proportion of B. pseudomallei isolates, has reportedly had a software adjustment to address this problem. Other useful clues to the identity of B. pseudomallei include the characteristic antibiogram (resistance to gentamicin, ampicillin and colistin, susceptibility to chloramphenicol, tetracyclines and co-amoxiclav).

Suspect colonies of B. pseudomallei can be screened by simple tests:

• Oxidase (positive)
• Growth right up to 10 µg gentamicin and colistin discs on any susceptibility testing agar
• Susceptibility to co-amoxiclav
• Characteristic morphology on Ashdown’s medium

Confirm suspect isolates by biochemical test kits (e.g. API 20NE).

Send isolates to the Reference Laboratory (see below) for confirmation, having warned them in advance that isolates are being sent.

Serology

Acute and convalescent sera can be sent to the Reference Laboratory for testing. The laboratory should be warned in advance that sera are being submitted.

Reference Laboratory

Isolates for confirmation of identity and serum samples should be sent to:

Laboratory of Healthcare Associated Infection
Opportunistic Pathogens Unit
HPA Centre for Infections
61 Colindale Avenue
London NW9 5HT
TEL: 020 8327 7233

A Decade of experience of the UK Melioidosis Diagnostic Service (PDF, 945 KB) 

Transport arrangements

(see here for more information)

For the purposes of transport , cultures of B. pseudomallei fall into Category A - Infectious Substances Capable of Causing Disease in Humans or Animals - for a full list see Biological agents: Managing the risks (PDF, 379 KB) (ACDPs Appendix 1.2, Table A2).

Category A infectious substances are assigned to UN 2814 and must be packaged in accordance with UN Packaging Instructions PI620(road/ rail) or PI602 (air). Category A transfers should be individually requested through an approved courier. The service will be a next day tracked door-to-door delivery, which must be signed for at collection and receipt.

[Current HPA arrangements for transport of samples are detailed in the "Procurement Handbook, Ch 4 section 7: Mail, overnight specimen transfer and courier services".]

Clincial samples such as serum are generally classified as Category B and assigned to UN3373 (Biological Substance, Category B) and should be packaged in accordance with PI650. Clinical samples assigned to UN3373 may be posted.
 

Reference
Peacock SJ, Chieng G, Cheng AC, Dance DAB, Amornchai P, Wongsuvan G et al. Comparison of Ashdown's medium, Burkholderia cepacia medium, and Burkholderia pseudomallei selective agar for clinical isolation of Burkholderia pseudomallei. J Clin Microbiol 2005; 430:5359-61.

Last reviewed: 15 March 2012