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Department of Medicine, Brooke Army Medical Center, Fort Sam Houston, Texas
ABSTRACT
Simulated candidemia was produced with 20 Candida isolates at three inoculum sizes (100, 10, and 1 CFU/ml of blood). Growth detection was better with larger inocula. The time to growth detection was shorter with larger inocula. Inoculum size does effect Candida growth detection and time to detection in BACTEC 9240 automated systems.
TEXT
Candida species rank fourth among organisms isolated from the blood of hospitalized patients, with non-albicans Candida species accounting for about 50% of these Candida isolates (1, 2, 5, 9, 11, 14). Most hospitals rely on automated blood culture systems for the detection of blood pathogens, including Candida spp. The College of American Pathologists recommends that blood cultures be incubated for 5 days and then discarded without terminal subculture if growth is not detected by the automated system. In two previous studies of simulated candidemia with the BACTEC 9240 automated blood culture system (Becton Dickinson and Co., Sparks, Md)., we noted poor detection in anaerobic medium, false-negative results on terminal subculture, and prolonged times to growth detection for C. glabrata in aerobic medium despite the use of a large inoculum (100 CFU/ml of blood) (7, 8).
It has been suggested that patients with candidemia have <10 CFU/ml of blood. Simulation of candidemia is not standardized throughout the literature, with inoculum sizes ranging from <1 to >100 CFU/ml of blood (6, 7, 8, 16, 18). Furthermore, the inoculum recommended by the manufacturer for quality control testing of the BACTEC 9240 system, and similar automated systems, is exponentially larger than what would be expected to exist in clinical candidemia (approximately 1,000 organisms/bottle). This study was therefore conducted to determine if inoculum size affected Candida growth detection or time to detection in the BACTEC 9240 system.
(This work was presented in part previously [B. J. George, L. L. Horvath, and D. R. Hospenthal, 103rd Gen. Meet. Am. Soc. Microbiol., abstr. C-267, 2003].)
Twenty Candida isolates, including four each of C. albicans, C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis, were evaluated. Yeast suspensions of 106 CFU/ml were produced as described in NCCLS antifungal susceptibility testing guidelines and our previous publications (7, 8, 12). Serial dilutions of the suspensions were performed, and dilution accuracy was confirmed as previously described (7, 8).
Sixty aerobic (BACTEC Plus Aerobic/F) and 60 anaerobic (BACTEC Plus Anaerobic/F) blood culture bottles (Becton Dickinson and Co.) were inoculated with 10 ml of fresh, unprocessed blood from healthy volunteers and then with a 0.1-ml aliquot of yeast suspension, resulting in final inoculum sizes of approximately 1, 10, and 100 CFU/ml of blood and 10, 100, and 1000 CFU/bottle, respectively. Prepared bottles were placed in the BACTEC 9240 system and incubated at 35°C.
Once growth was detected by the BACTEC 9240 system, each bottle was removed from the system and a terminal subculture was performed as previously described (7, 8). Terminal subculture was performed for all bottles without growth detection after 17 days of incubation. Data analysis was performed using SPSS software version 11.5 (SPSS Inc., Chicago, Ill.).
The BACTEC 9240 system detected Candida growth in 68 of 120 bottles (57%) (Table 1). Growth detection was better in aerobic medium (59 of 60 bottles [98%]) than in anaerobic medium (9 of 60 bottles [15%]) (P < 0.001). The BACTEC 9240 system detected all C. albicans, C. krusei, C. parapsilosis, and C. tropicalis isolates in aerobic medium, regardless of inoculum size. C. glabrata and C. tropicalis were the only species with growth detected in anaerobic medium. Overall, there was a trend towards better Candida growth detection with increasing inoculum size that did not reach statistical significance: 25 of 40 (63%) bottles with growth detected at 1,000 CFU/bottle, 22 of 40 (55%) bottles at 100 CFU/bottle, and 21 of 40 (53%) bottles at 10 CFU/bottle.
One hundred percent of the 68 bottles with growth detected in the BACTEC 9240 system grew yeast cells on terminal subculture, representing true-positive results. Of the 52 bottles without growth detection by the BACTEC 9240 system, 42 (81%) grew yeast cells on terminal subculture. Terminal subculture detected more Candida growth (110 of 120 bottles or 92%) than the BACTEC 9240 system did (68 of 120 bottles or 57%) (P < 0.05). Overall, 42 of 120 bottles had yeast growth upon terminal subculture that was not detected by the BACTEC 9240 system (35% false negatives). Ten bottles had no growth detected by either the BACTEC 9240 system or terminal subculture. All 10 of these bottles had been inoculated with the smallest yeast inoculum (10 CFU/bottle). Colony counts confirmed the inoculum size, with a mean of 9.6 CFU/bottle for all Candida isolates. Eight of the 10 bottles with no growth by either method contained less than the average 9.6 CFU/bottle. However, each of these 10 isolates grew in their corresponding bottles with the same small inoculum.
Time to growth detection by the BACTEC 9240 system varied depending on inoculum size, species, and medium (Table 2). Time to growth detection was shorter with larger inoculum (P < 0.001). For C. glabrata isolates, growth was detected earlier in anaerobic than aerobic medium with similar inoculum sizes (P < 0.001). The mean time to growth detection of C. glabrata in aerobic medium was also longer than that for any other Candida species at similar inoculum sizes (P < 0.05).
Candida spp. are generally regarded as obligate aerobes (19); therefore, it is not surprising that the majority of bottles with undetected growth contained anaerobic medium. However, it is troubling that C. glabrata growth went undetected even in a single aerobic bottle. Clinical laboratories must be concerned about time to growth detection, as most limit the duration of blood culture incubation to 5 days due to space and monetary constraints. There are data indicating that 3 days may be sufficient for recovery of 97.5% of clinically significant pathogens in the BacT/ALERT automated blood culture system, including Candida spp. (4). Most of the Candida spp. evaluated in this study were detected within either a 3- or 5-day incubation period regardless of inoculum size, except for C. glabrata, which required incubation for longer than 5 days at the two smallest inoculum sizes in aerobic medium.
There are limited data on the usefulness of terminal subculture for detection of candidemia in conjunction with automated systems (3, 13, 17). It is notable that 81% of the bottles with Candida spp. not detected by the BACTEC 9240 system had yeast growth upon terminal subculture. Missed detection of growth is particularly concerning, since all of these bottles contained inocula of sizes similar to the levels encountered in human candidemia (16). While inoculum size did affect growth detection and time to growth detection, inoculum size alone does not explain the inability of this system to detect growth of viable yeasts.
The BACTEC 9240 system was able to detect the majority of simulated candidemia episodes at all inoculum sizes. However, this study illustrates several potential problems, including difficulty detecting Candida growth in anaerobic medium at all inoculum sizes, longer time to detection with small inoculum sizes, and the unusual growth characteristics of C. glabrata. This is a particular problem since C. glabrata is the second most common Candida species isolated in U.S. hospitals, accounting for 24 to 42% of all cases of candidemia (5, 14). These problems raise the question of whether clinical laboratories should prolong the incubation time, perform terminal subcultures, or utilize specific mycology media for the detection of Candida when utilizing the BACTEC 9240 system (3, 7, 8, 10, 13, 15, 17). With an increasing incidence of candidemia from non-albicans Candida spp., automated systems need to detect all clinically relevant candidal pathogens or laboratories will have to consider adjusting their protocols accordingly.
ACKNOWLEDGMENTS
The views expressed are those of the authors and do not reflect the official policy or position of the Department of the Army, Department of Defense, or the U.S. Government.
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