Rapid Evaluation Procedures

RAPID EVALUATION PROCEDURES

In most of the tests mentioned in the other articles results are not available, until visible microbial growth occurs, at least in the controls. This usually takes 24 hours or more. The potential benefits of rapid antimicrobial susceptibility screening procedures are obvious, particularly in aggressive infections or rapidly progressing nosocomial infections of immunocompromised patients where appropriate antimicrobial selection is critical. To date only a few ‘rapid’ methods for detecting microbial viability or growth are presently employed in assessing the efficacy of antimicrobials. These include epifluorescent and bioluminescence techniques. The former relies on the fact that when exposed to the vital stain acridine orange and viewed under UV light, viable cells fluoresce green or greenish yellow, while dead cells appear orange. Live/dead staining of sessile bacterial populations has the potential to yield important data with respect to antimicrobial susceptibility, but requires skilled personnel and specialized microscopy equipment.

With tests involving liquid systems the early growth of viable cells can be assessed by some light scattering processes, blood culture techniques have classically used the production of CO₂ as an indicator of bacterial metabolism and growth. In addition, the availability of molecular techniques, such as quantitative PCR, may be useful in demonstrating the presence or growth of microorganisms that are slow or difficult to culture under usual laboratory conditions, e.g. viruses. This may obviate the need to neutralize residual disinfectant with some assays.

Recently, rapid colorimetric assays for antimicrobial susceptibility have been developed including the commercially available Vitek and Vitek2 systems (Biomerieux) and colourimetric tests based on the extracellular reduction of tetrazolium salts 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2 H-tetrazolium monosodium (WST-8) and 2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2 H-tetrazolium-5-carboxanilide (XTT). These latter studies have demonstrated the potential for the tetrazolium salts WST-8 and XTT to be used in the rapid, accurate and facile screening of antimicrobial susceptibility and MIC determination in a range of bacteria, including staphylococci, extended β-lactamase producing clinical isolates (E. coli, Ent. faecalis) and Ps. aeruginosa (see Tunney et al., 2004). Using this method, MIC values in agreement with those obtained using standard methods were obtained after 5 hours.