Conventional and Molecular Methods for Verification of Results Obtained with BacT/Alert Nonvent Blood Culture Bottles

ABSTRACT A strategy comparing molecular and conventional methods for verification of the BacT/Alert nonvent blood culture bottles (Organon Teknika, Durham, N.C.) was performed with seeded isolates. The bottles were evaluated with 12 common organisms from bloodstream infections. Overall, the bottles were equivalent as determined by conventional and molecular methods.

Blood culture is currently the only routine method for detecting bacterial bloodstream infections. Over the past 2 decades, advances in blood culture technology have resulted in significant decreases in the time to detection and identification of these etiological agents (8,12). Consequently, one of the most challenging tasks facing the clinical microbiology laboratory is the meaningful verification of a new blood culture system. Suggested guidelines available for verification of new blood culture bottles include parallel and seeded blood culture studies (5).
In conjunction with advances in conventional blood culture methodologies, there have been several studies examining the use of nucleic acid tests for the rapid detection of positive blood cultures (4, 6, 7, 10, 11, 13, 14; D. Bruckner, L. Gibson, J. Hindler, J. Hogan, I. Andruszkiewicz, K. Clark-Dickey, and W. Weisburg, Abstr. 101st Gen. Meet. Am. Soc. Microbiol., abstr. C- 1, p. 147, 2001). Specifically, the Gen-Probe Incorporated hybridization protection assay (HPA), which uses a DNA probe to detect rRNA, has been utilized to evaluate positive blood cultures. The chemiluminescent signal generated by the HPA has been found elsewhere to directly correlate with CFU ( Organon Teknika (Durham, N.C.) has replaced its standard vented BacT/Alert bottle with a redesigned nonvent (NV) blood culture bottle. As a result, laboratories have been confronted with performing a verification study of these new blood culture bottles. The objective of this study was to perform a seeded study to (i) evaluate the NV bottle for the ability to support growth in a timely manner by conventional methods and (ii) compare conventional methods with molecular probe detection as a possible rapid alternative method for blood culture bottle verification.
In this seeded study performed at UCLA Medical Center, Corp.) according to the manufacturer's recommendation. Bottles remained incubated until they flagged positive or until 7 days had elapsed. The time from loading to detection was recorded for each bottle. If isolates failed to grow, 5 to 10 ml of blood was added to bottles and retested in duplicate. Furthermore, isolates still failing to grow were replaced with a patient strain and evaluated with and without blood. When bottles were triggered as positive, cell mass was measured by viable plate counts and probe detection was performed with the Gen-Probe Incorporated all-bacterial and all-fungal DNA probes directed toward rRNA targets. Bottles not triggered as positive were terminally subcultured and probed after 7 days. The all-bacterial and all-fungal probes were derived from the 23S and 18S regions of the rRNA genes, respectively. For probing a 1-ml sample was centrifuged to pellet the organism, washed twice with a saponin-based cocktail, lysed at 100°C in a succinate-buffered detergent cocktail, and frozen for further analysis. Frozen pellets were diluted when necessary to obtain relative light unit (RLU) levels within the linear range of the luminometer when run in an HPA (1,9).
Overall, the BacT/Alert NV bottles were equivalent to the standard BacT/Alert bottles as determined by both conventional and molecular methods. These results agree with others with clinical samples (2,12). The comparative mean times to detection are summarized in Table 1. In the bottles that did not require blood, the standard and NV bottles were equivalent in their mean times to positivity. Isolates failing to grow without blood were equivalent in their mean times to positivity for the standard and NV bottles after blood was added to the bottles (Table 1), excluding C. jeikeium.
The equivalent mean times for detection of the isolates are comparable to those reported by Snyder et al. (12) and within the range of performance characteristics reported in the package insert. The manufacturer states in the package insert that organisms may require added blood or other supplements to support growth. Consequently, S. pneumoniae, S. viridans, and C. jeikeium required blood in some of the bottles to support growth. This was necessary for S. pneumoniae and S. viridans only in the anaerobe bottles. It is important to keep in mind that any nonblood clinical specimen, such as joint fluids and peritoneal dialysates, cultured in these bottles may require supplements for adequate growth. Those isolates that required blood because they failed to grow well were also retested with a patient strain, with and without blood. The original isolates and the patient strains showed no difference in growth performance.
The comparative mean CFU per milliliter in the positive blood culture bottles for the various representative organisms and bottles are summarized in Table 2. For bacteria, the positive bottles were detected in the late log phase of growth, within a range of 1.53 ϫ 10 7 to 7.41 ϫ 10 9 CFU/ml, excluding H. aphrophilus. For yeast, positive bottles were detected with approximately 4.5 ϫ 10 6 CFU/ml. Interestingly, molecular probe detection was proportionally equivalent to conventional culture methods for verification of the positive bottles. Results of the mean comparative RLU/ml are summarized in Table 3. The CFU-per-milliliter numbers for H. aphrophilus may be underrepresented due to the organism's granular growth in broth. Despite clumping of cells, the RLU-per-milliliter signals for H. aphrophilus had substantially lower standard deviations than did the CFU per milliliter from culture. This phenomenon demonstrates that measurement of total amount of rRNA is not dependent on the organisms being monodispersed. This suggests that a nucleic acid detection system is a better measure of organisms when used for quality control of growth media. When bottles failed to flag as positive after 7 days, they were terminally subcultured and probed.
Both molecular and conventional methods were able to detect organisms below the limit of detection of the BacT/ Alert3D instrument (1.53 ϫ 10 7 to 7.41 ϫ 10 9 CFU/ml). The limit of detection was approximately 10 4 CFU/ml for the HPA probe system, while culture displayed a sensitivity of 300 to 3,000 CFU/ml. For example, CFU and RLU probe signals were detected in the 7-day subcultures of P. aeruginosa in all anaerobic bottles even though the Bact/Alert3D instrument did not detect them. This reflects a 10,000-fold-greater sensitivity of the probe assay than of the Bact/Alert3D for detecting positive blood cultures.
The 7-day subcultures of C. albicans from the standard aerobic bottles had a mean of 35 CFU/ml, while the 7-day subcultures of the standard and new NV anaerobic bottles had means of Ͻ30 and 2.3 ϫ 10 3 CFU/ml, respectively. Either no probe or low probe signals were detected for the 7-day subcultures of C. albicans.
The ratio of the RLU to CFU for a given organism is dependent upon a number of variables. The growth state, plating