Fig 1.
Routine diagnostic workflow for B. pseudomallei bloodstream infection: From sample collection to susceptibility testing.
Created in BioRender. Gassiep, I. (2025) https://BioRender.com/5209mfb. Blood cultures are collected aseptically at the patient’s bedside. The inoculated bottles are transported promptly to the microbiology laboratory under appropriate conditions and loaded into an automated blood culture incubator system for continuous monitoring and incubation (where automated systems are not available visual inspection and blind subculture are required, delaying identification by days). Upon detection of a positive signal, an aliquot of the blood culture broth is removed for Gram staining, which provides a preliminary report of the organism’s morphology and Gram reaction. Simultaneously, subcultures are made onto suitable solid media for isolation of pure colonies. While not routinely performed, organism identification can be achieved directly from positive blood culture broth using antigen detection, proteomic, or molecular techniques. Following incubation, discrete colonies obtained from subcultures are subjected to organism identification using a biochemical identification systems, latex agglutination, or mass spectrometry. Antimicrobial susceptibility testing is then performed on the identified isolate using disk diffusion. Notably, on receipt in the laboratory, nonblood specimen are directly inoculated onto solid media and therefore, organism identification may occur 24–48 hours sooner.
Table 1.
Differentiation of B. pseudomallei from its closely related clinically relevant species (other Burkholderia spp., Ralstonia spp., Stenotrophomonas maltophilia, and Pseudomonas spp.) by biochemical characteristics.
Fig 2.
Created in BioRender. Gassiep, I. (2025) https://BioRender.com/xbq39lt. A B. pseudomallei colony is picked from an agar plate with a toothpick and placed on a target slide. α-Cyano-4-hydroxycinnamic acid is added and once dry creates a crystallized matrix which both inactivates B. pseudomallei and allows for even ionisation. The target slide is placed into the instrument where laser ionisation results in an organism-specific mass-to-charge ratio and relative abundance of ions.
Fig 3.
Created in BioRender. Gassiep, I. (2025) https://BioRender.com/o3h78f4. Clinical specimens including pus, sputum, and urine are potential samples for direct analysis. A liquid sample containing B. pseudomallei antigens is placed onto the sample pad. The sample is wicked across the nitrocellulose test pad (left to right). The antigen in the sample is bound by mobile antigen-specific gold-conjugated-antibodies which travel across the membrane and are subsequently bound to the immobilised B. pseudomallei-specific antibodies on the test strip. The aggregation of this gold-conjugated antibody-antigen-antibody complex is seem as a visible line (colour dependant on conjugate).
Fig 4.
Molecular detection by real-time Polymerase Chain Reaction.
Created in BioRender. Gassiep, I. (2025) https://BioRender.com/r9l042c. Direct molecular detection of B. pseudomallei from clinical samples involves several steps. After collecting a sample of blood, sputum, or urine, bacterial DNA is extracted using a commercial kit which isolates the DNA and removes inhibiting substances. Next, a real-time PCR assay targeting a specific gene, such as the Type 3 Secretion System 1 (TTS1), is performed. The assay contains nucleotides, DNA polymerase, primers, and probes, and occurs under optimised thermal cycling conditions including denaturation, annealing, and extension. A fluorescent signal, which is inhibited by proximity to a quencher, is released when the probe is hydrolysed. The amount of fluorescence is measured in real-time, and a result is generated once a threshold is overcome.
Fig 5.
Molecular detection by loop-mediated isothermal amplification and CRISPR-CasCreated in BioRender.
Gassiep, I. (2025) https://BioRender.com/r9l042c. Simplified schematic of Loop-mediated isothermal amplification (LAMP) paired with CRISPR-Cas system. LAMP achieves high specificity by using 4–6 primers that recognise multiple distinct regions of the B. pseudomallei genome. A strand-displacing DNA polymerase initiates synthesis without thermal denaturation, displacing the complementary strand as it extends. The displaced DNA forms stem–loop structures due to the complementary sequences introduced by the primers, enabling rapid, continuous amplification at a constant temperature. In CRISPR-based diagnostics, a guide RNA (gRNA) is designed to match a specific DNA or RNA target sequence, which in this context, corresponds to the LAMP amplification product. The gRNA directs a Cas nuclease—such as Cas12a for DNA targets or Cas13a for RNA—toward the complementary sequence. Upon binding, the nuclease is activated and exhibits collateral activity, indiscriminately cleaving nearby single-stranded DNA (Cas12a) or RNA (Cas13a). This property is harnessed by adding synthetic reporter molecules that release a detectable signal when cleaved. Detection can be performed using methods such as fluorescence measurement or lateral flow assays.
Fig 6.
Indirect hemagglutination assay.
Created in BioRender. Gassiep, I. (2025) https://BioRender.com/xbq39lt. B. pseudomallei isolates are combined to form a crude, nonstandardised, antigen rich lysate. This lysate is used to sensitise sheep red blood cells (RBCs) with B. pseudomallei antigens. Patient serum is incubated with nonsensitised sheep RBCs to remove nonspecific agglutinins. The incubated patient sera are diluted by 2-fold serial dilutions in a 96-microtiter plate and antigen-sensitised red cells are then added to the wells. B. pseudomallei antibodies present in a patient’s serum will create an antigen-antibody complex, resulting in haemagglutination and the appearance of a hazy or lattice-like structure.
Table 2.
Performance assessment of laboratory techniques in identifying B. pseudomallei.