Comparison of microscopic, phenotypic and molecular techniques for the rapid identification and susceptibility testing of Staphylococci from positive blood culture bottles

Introduction

The ability to rapidly identify and determine the antimicrobial susceptibility of bacterial pathogens is an undisputed requirement for strategies aimed at improving the management of sepsis. Staphylococci are amongst the most common organisms isolated from blood cultures but it is difficult to rapidly distinguish between those representing contamination of the blood culture with harmless skin commensals (Staphylococcus epidermidis) and those that contain pathogenic species (Staphylococcus aureus).

Objective

A number of phenotypic and genotypic tests with a vast range of complexity, speed and cost have been proposed to help distinguish these organisms. We evaluated the sensitivity, specificity and speed of a range of these tests compared with the standard laboratory identification protocol which takes up to 48 hours.

Methods

Positive blood culture sets (BACT/Alert 3D) (n = 113) in which Gram-positive cocci in clusters were seen on the initial film were included. Further identification as S. epidermidis versus S. aureus was attempted using the following techniques directly from the positive bottles: (1) morphology and organization of the cocci on Gram stain (10 minutes), (2) rapid tube coagulase (2 hours) (3) direct DNAse test using toluedine blue agar (4 hours), (4) species-specific multiplex PCR (4 hours), (5) direct inoculation of selective commercial chromogenic media (18 hours), and (6) direct inoculation of mannitol-supplemented Mueller-Hinton agar (MMHA) combined with disc diffusion susceptibility testing (18 hours). Identifications were compared with the standard laboratory identification at 36 hours.

Results

Definitive laboratory identification revealed S. epidermidis 87% (n = 98), S. aureus 11% (methicillin-sensitive S. aureus (MSSA) n = 9, methicillin-resistant S. aureus (MRSA) n = 3), Micrococcus spp. 2% (n = 2); one culture contained a mixture of S. aureus and S. epidermidis. The sensitivity and specificity of each of the direct techniques was calculated as follows: (1) microscopy 53% and 98%, (2) rapid coagulase 92% and 100%, (3) direct DNAse 69% and 98%, (4) multiplex PCR 88% (no amplification in 12 samples) and 99%, (5) chromogenic S. aureus media 90% and 100%, and (6) MMHA 100% and 78%.

Conclusion

All the methods had specificity >95%, except for MMHA, although this has the added advantage of providing susceptibility results. Chromogenic agar had the highest sensitivity and specificity, although it provided a result only 12 hours quicker than the standard protocol. Rapid tube coagulase was highly specific and one of the most rapid tests. This may be the most useful, inexpensive technique providing preliminary results to guide empirical therapy, especially in resource-poor settings.