Melioidosis: Problems with Current Identification Methods (API, Vitek and other methods) Rob Baird Royal Darwin Hospital.

Melioidosis: Problems with Current Identification

Methods (API, Vitek and other methods)

Rob BairdRoyal Darwin Hospital

or

J Med Microbiol. 2012 Oct;61(Pt 10):1483-4.Epub 2012 Jul 19.

Christoph Weissert, et al Emerging Infec DisVolume 15, Number 11—November 2009

Common phenotypic mis-identifications of B. pseudomallei are primarily due to:

Regional biochemical differences in B. pseudomallei strains

Lack of the regional biochemical profiles in the phenotypic databases

A proliferation of taxonomic change in the genus, occurring well ahead of phenotypic database updates

and

Ashdown’s media: growth not limited to B. pseudomallei. Coliforms, yeast, S. maltophilia, Pseud sp. and B. cepacia can all grow.

Can survive the decontamination process of TB and grows quite well on the media used making isolation of TB difficult.

Mass spectrometry, while an excellent aid in diagnosis, is dependent on the quality of the protein spectra database of the instrument. Misidentification has been reported in the literature.

Currently 85 Burkholderia are taxonomically recognised

Burkholderia acidipaludis Burkholderia hospita Burkholderia soliBurkholderia ambifaria Burkholderia kururiensis Burkholderia sordidicolaBurkholderia andropogonis Burkholderia lata Burkholderia stabilisBurkholderia anthina Burkholderia latens Burkholderia symbiotica

Burkholderia arboris Burkholderia mallei Burkholderia terraeBurkholderia bannensis Burkholderia megapolitana Burkholderia terricola

Burkholderia brasilensis Burkholderia metallica Burkholderia thailandensisBurkholderia bryophila Burkholderia mimosarum Burkholderia tropicaBurkholderia caledonica Burkholderia multivorans Burkholderia tuberumBurkholderia caribensis Burkholderia nodosa Burkholderia ubonensisBurkholderia caryophylli Burkholderia norimbergensis Burkholderia unamaeBurkholderia cenocepacia Burkholderia oklahomensis Burkholderia vandiiBurkholderia cepacia Burkholderia oxyphila Burkholderia vietnamiensisBurkholderia cepacia complex Burkholderia phenazinium Burkholderia xenovoransBurkholderia cocovenenans Burkholderia phenoliruptrix Burkholderia zhejiangensisBurkholderia contaminans Burkholderia phymatum "Candidatus Burkholderia calva"Burkholderia denitrificans Burkholderia phytofirmans "Candidatus Burkholderia crenata"Burkholderia diazotrophica Burkholderia pickettii "Candidatus Burkholderia hispidae"Burkholderia diffusa Burkholderia plantarii "Candidatus Burkholderia kirkii"

Burkholderia dolosa Burkholderia pseudomallei "Candidatus Burkholderia mamillataBurkholderia endofungorum Burkholderia pyrrocinia "Candidatus Burkholderia nigropunctata"Burkholderia ferrariae Burkholderia rhizoxinica "Candidatus Burkholderia rigidae"Burkholderia fungorum Burkholderia sabiae "Candidatus Burkholderia schumannianae"Burkholderia ginsengisoli Burkholderia sacchari "Candidatus Burkholderia verschuerenii"Burkholderia gladioli Burkholderia sartisoli "Candidatus Burkholderia virens" Burkholderia glathei Burkholderia sediminicolaBurkholderia glumae Burkholderia seminalisBurkholderia graminis Burkholderia silvatlanticaBurkholderia grimmiae Burkholderia singaporensisBurkholderia heleia Burkholderia solanacearum

Comparison of the VITEK 2 system biochemical profiles of 68 confirmed B. pseudomallei clinical strains from hospitals in Sabah and Sarawak, Malaysian Borneo with 149 B. pseudomallei and 18 B. cepacia isolates from the Royal Darwin Hospital (RDH) in the Northern Territory, Australia. One isolate per patient was analyzed. All isolates were collected between September 2010 and June 2012 except for 17 isolates collected in 1994 from Sabah.

Regional differences

Regional differences

Typical/usual B. pseudomallei profile:88% of isolates with BNAG identified correctly, only 13% without BNAG identified correctly

Figure 1. Nonmetric multidimensional scaling (nMDS) ordination on the Euclidean distance resemblance matrix of the Vitek-2 biochemical profile of 235 B. pseudomallei and B. cepacia isolates from Australia and Malaysian Borneo. (A) Samples were identified as either B. pseudomallei, B. cepacia, B. pseudomallei misidentified as B. cepacia or isolates with low discrimination. (B) The bubble size reflects the presence (large) or absence (small) of BNAG substrate in an isolate. (C) Analysis based on isolates from both countries, Australia and Malaysia. (D) The bubble size reflects the presence (large) or absence (small) of NAGA substrate in an isolate.

Abbreviations: Bps, B. pseudomallei; Bcep, B. cepacia; Bcep misID, B. pseudomallei misidentified as B. cepacia; BNAG, β-N-acetyl-glucosaminidase; NAGA, N-acetylgalactosamine.

B

DC

A

Unfamiliarity with B. pseudomallei and problems with inaccurate speciation using some automated commercial biochemical identification systems have resulted in laboratories misidentifying the bacterium as a Pseudomonas or other Burkholderia species.

Confirmation of B. pseudomallei identity by real-time polymerase chain reaction (PCR) of DNA extracted from cultured bacterial colonies is increasingly the standard for many laboratories. Various genetic targets have been published for PCR identification of B. pseudomallei from bacterial cultures and also for direct detection from clinical samples, with a recent review showing the TTS1-orf2 assay to be superior in detecting B. pseudomallei directly from clinical specimens.

Apart from molecular methods, B. pseudomallei from cultures can also be confirmed by antigen detection assays such as latex agglutination. More recently matrix-assisted laser desorption ionization-time of flight Mass Spectrometry (Maldi-TOF MS) has been adapted to identify cultured bacteria based on protein fingerprint profiles.

Common phenotypic mis-identifications of B. pseudomallei are primarily due to:

Regional biochemical differences in B. pseudomallei strains

Lack of the regional biochemical profiles in the phenotypic databases

A proliferation of taxonomic change in the genus, occurring well ahead of phenotypic database updates

The move towards genotypic and molecular identification, as the gold standard, will reduce this problem for reference labs, but

local labs still require a high index of clinical and microbiological suspicion, when identifying Burkholderia isolates from patients with compatible clinical illnesses

and

Ashdown’s media: growth not limited to B. pseudomallei. Coliforms, yeast, S. maltophilia, Pseud sp. and B. cepacia can all grow.

Can survive the decontamination process of TB and grows quite well on the media used making isolation of TB difficult.

Mass spectrometry, while an excellent aid in diagnosis, is dependent on the quality of the protein spectra database of the instrument. Misidentification has been reported in the literature.