Samples for method validation
Clinical strains, primarily analyzed according to conventional identification strategies such as culture on selective agar (esculin, DNAse), biochemical testing (optochin, PYR, bacitracin, CAMP) including analytical profile index (API) and complementary agglutination tests were used. For evaluation experiments, these strains were inoculated at the approximate amount of 100 CFU in former culture-negative blood culture bottles. The bottles used were aerobic Plus/F for the BACTEC system and BacT/Alert aerobic bottle (FA) for the BacT/Alert system.
Material from inoculated, previously negative blood cultures bottles and henceforth called blood culture material.
Setting and patient samples
The major part of the rapid method testing was performed, at the Division for Clinical Microbiology, Linköping University Hospital, on material from inoculated negative BACTEC blood culture bottles and agar plates. When stated that BacT/Alert was used, experiments were performed at the division for Clinical Microbiology in Kalmar.
The consecutive samples evaluated in this study were 166 patient blood cultures containing GPC from Kalmar County Hospital (catchment population 233 000) where 4 300 pairs of blood culture bottles are collected per year, of which 18% are positive. GPC are found in roughly half of the positive blood cultures. The species distribution was for the year of 2009: S. aureus 30%, CoNS 20%, Pneumococci 14%, Enterococci, 14%, group A streptococci 4%, group B streptococci 2%, other streptococci 8%, other GPC 8% (CoNS singlets excluded as probable contaminations).
Samples were taken as part of standard patient care. Ethical approval was obtained from the Regional Ethical Review Board in Linköping, number M17-09.
Tube coagulase test (TCT)
TCT was performed with 25 μL, 75 μL, 200 μL and 300 μL blood culture material and 0.5 mL of plasma; from rabbit (BBL Coagulase plasma with EDTA, Becton Dickinson, USA), horse (Swedish National Veterinary Institute, Uppsala, Sweden) and human (Division of Transfusion Medicine, Linköping University Hospital, Linköping, Sweden) to find the optimal blood volume and plasma type. The samples were incubated at 36°C and examined for coagulation every 15 minutes during 4 hours and finally at 24 h. Staphylococcus aureus ATCC 25923 and Staphylococcus saprophyticus CCUG 3706 isolates were used as positive and negative controls.
Staphylococcal agglutination
Three Staphylococcal agglutination kits; Prolex™ Staph latex kit (Pro-lab Diagnostics, Richmond Hill, ON, Canada), Slidex Staph kit (bioMérieux, Marcy l’Etoile, France), Prolex™ Staph Xtra Latex kit (Pro-lab Diagnostics, Richmond Hill, ON, Canada) were evaluated at the microbiology laboratory of Linköping University Hospital. The tests were performed on material from blood culture bottles inoculated with S. aureus (2 drops were used, which corresponds to the volume used for colony testing according to the manufacturers recommendation) and colonies grown 4–8 hours on blood agar plates from the same blood cultures according to the manufacturer’s instructions.
Pneumococcal agglutination
The Oxoid Dryspot Pneumo Test (Oxoid, Hampshire, UK) was performed with inoculated samples incubated in the BACTEC system the microbiology laboratory of Linköping University Hospital both on blood culture material and colonies grown 4–8 hours on blood agar plates according to manufacturer’s instructions except that the centrifugation step was left out.
Streptococcal agglutination
Prolex Streptococcal grouping test (Pro-lab Diagnostics, Richmond Hill, ON, Canada) was evaluated both at the microbiology laboratory of Linköping University Hospital and Kalmar County Hospital and compared to the streptococcal grouping test used in the routine at each laboratory, Phadebact Streptococcus test (Bactus AB, Huddinge, Sweden) was evaluated in Linköping and Streptex Test kit (Remel Inc. Lenexa, KN, USA) in Kalmar. The kits were tested on bacterial colonies according to manufacturers’ instructions and on blood culture material by using two drops of blood material to one drop of test reagent without prior pre-treatment. Two drops were chosen because it corresponds to the volume used to mix colonies in when tested as described in the manufacturer’s instruction.
The agglutinations from colonies were complemented with PYR-test (L-pyrrolidonyl-ß-naphthylamide, O.B.I.S.-PYR, Oxoid LTD, Basingstoke, England) for discriminating GAS and enterococci from other streptococci [15].
Time until signalled growth
Time to signalled growth (the time evolved from the start of the incubation until the blood culture system signalled growth), was analysed retrospectively for 360 patient samples positive for GPC incubated in BACTEC, in the Linköping laboratory. Analyses involved retrieving the sensitivity, specificity and positive and negative predictive values of the cut-off time points suggested by Martínez et al. [16].
Consecutive samples
Our strategy for GPC in clusters was to immediately culture the blood bottle material from positive blood culture bottles onto solid blood agar plates and to initiate a TCT. During the time it takes for S. aureus to produce a coagulate in the TCT, colonies will shortly appear on the solid agar, which can be used for a latex agglutination test. The process is illustrated in Figure 1.
For streptococci the strategy was to identify blood cultures containing beta-haemolytic streptococci, enterococci and pneumococci by using agglutination kits directly on material from positive blood culture bottles; with repeated testing on colonies after 6 hour incubation on solid blood agar. Prolex Streptococcal grouping kit, Oxoid Dryspot Pneumo Test and subcultures on blood, chocolate and esculin agar was performed on all GPC in pairs or chains. The process is illustrated in Figure 2.
This strategy was applied at the microbiology laboratory of Kalmar County Hospital, Sweden during March 1st 2009 to January 31st 2010. Blood cultures, positive weekdays before 8 am, showing GPC, were included. Blood cultures from 691 patients were positive for bacterial growth during this time period. Out of these, 164 contained GPC and were subjected to the processes and reported as shown in Figures 1 and 2 in addition to the conventional procedure used in the laboratory. Two independent species-specific tests were always used for species determination.
Statistics
Sensitivity and specificity are presented as percentage of results obtained by golden standard methods. Chi-square tests were carried out for non-metrical results. Significance levels are shown as stars (p < 0.001 = ***, p < 0.01 = **, p < 0.05 = *, n.s. = non-significant).