- Email: [email protected]
Alert FAN aerobic and anaerobic blood culture bottles for the detection of bacteremia in ICU patients
Diagnostic Microbiology and Infectious Disease 73 (2012) 239–242
Contents lists available at SciVerse ScienceDirect
Diagnostic Microbiology and Infectious Disease j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / d i a g m i c r o b i o
Evaluation of BACTEC Plus aerobic and anaerobic blood culture bottles and BacT/Alert FAN aerobic and anaerobic blood culture bottles for the detection of bacteremia in ICU patients☆ Kyoung Ho Roh a, Ju Yeon Kim a, Ha Nui Kim a, Hye Jin Lee a, Jang Wook Sohn b, Min Ja Kim b, Yunjung Cho a, Young Kee Kim a, Chang Kyu Lee a,⁎ a b
Department of Laboratory Medicine, Korea University College of Medicine, Seoul, Korea Department of Internal Medicine, Korea University College of Medicine, Seoul, Korea
a r t i c l e
i n f o
Article history: Received 31 August 2011 Received in revised form 8 March 2012 Accepted 19 March 2012 Available online 25 April 2012 Keywords: Blood culture Evaluation BACTEC BacT/Alert Culture bottle Time to detection
a b s t r a c t Blood culture is the most valuable laboratory test for the diagnosis of bacteremia and sepsis. The BACTEC FX and BacT/Alert 3D automated blood culture systems are commonly used in Korean health care facilities. A controlled clinical evaluation of the resin-containing BACTEC Plus aerobic (BA) and anaerobic (BN), and the charcoal-containing FAN aerobic (FA) and anaerobic (FN) bottles using blood from intensive care unit (ICU) patients was designed. The performances of these 2 systems with media containing particle absorbing antimicrobial agents were evaluated using the culture positivity rate and time to detection (TTD). TTD was collected using data management systems, either the Epicenter (BD Diagnostic Systems) or the hospital laboratory information system. A total of 1539 four-bottle sets were collected from 270 patients in medical and surgical ICUs. Blood culture samples included 1539 bottles each of BA, BN, FA, and FN, and yielded 113 (7.3%), 90 (5.8%), 104 (6.8%), and 80 (5.2%) positive bacterial or fungal isolates, respectively. There were significant differences between the resin-containing BA and BN samples in culture positivity and also between the charcoal-containing FA and FN samples, especially for Escherichia coli (25/27 versus 17/27, P b 0.05) and Acinetobacter baumannii (14/15 versus 7/15, P b 0.05). Significantly shorter recovery time was observed in BACTEC Plus aerobic bottles than in FAN aerobic bottles (17.2 and 24.7 h, respectively) (P b 0.001). © 2012 Elsevier Inc. All rights reserved.
1. Introduction Blood culture is the most valuable laboratory test for the diagnosis of bacteremia and sepsis (Smith-Elekes and Weinstein, 1993). The recovery of pathogenic microorganisms from blood culture is essential for appropriate clinical management of infection. The timely reporting of identified organisms and antimicrobial agent susceptibility is helpful for the proper selection of antimicrobial agents and treatment. The automated blood culture system is the most sensitive method introduced in blood culture and is currently in widespread use (Mirrett et al., 2003). The BACTEC FX (BD Diagnostic Systems, Sparks, MD, USA) (FX) and BacT/Alert 3D (bioMérieux, Durham, NC, USA) (3D) automated blood culture systems are commonly used in Korea. The BACTEC FX system is a newly introduced automated blood culture system. The FX and 9240 systems detect the growth of microorganisms by a fluorescent sensor for CO2, whereas the 3D uses a colorimetric detection sensor for CO2. A 5-day incubation protocol is generally used in clinical microbiology laboratories (Huang et al., ☆ This work was supported by a grant from BD Korea. ⁎ Corresponding author. Tel.: +82-2-920-5381; fax: +82-2-920-5538. E-mail address: [email protected] (C.K. Lee). 0732-8893/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2012.03.022
1998). In addition to the culture positivity rate, the time to detection (TTD) of a positive culture is an important determinant for the rapid reporting of results. Various types of blood culture bottles have been introduced to increase the culture positivity rate and shorten the TTD. Higher culture positivity rates and shorter TTD give clinicians more opportunities for providing appropriate patient management. By the time the blood cultures are collected, a significant proportion of patients are usually already receiving antibiotic therapy. This can result in the inhibition of growth of the causative organism, especially in intensive care unit (ICU) patients. Blood culture bottles with antimicrobial agent–removing resin or charcoal-containing media demonstrate higher recovery rates compared to standard bottles (Weinstein et al., 1995; Wilson et al., 2001). The BACTEC Plus aerobic (BA) and anaerobic (BN) bottles contain resin, and the bioMérieux FAN aerobic (FA) and anaerobic (FN) bottles include charcoal to minimize the effects of antimicrobial agents in the patients' blood. A few investigators have tested the in vitro performance of blood culture bottles using blood culture bottles containing various antimicrobial agents and microorganisms (Flayhart et al., 2007; Vigano et al., 2002). Some studies evaluated only aerobic culture bottles (Jorgensen et al., 1997; Pohlman et al., 1995). Few clinical investigations have concurrently evaluated aerobic and anaerobic
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K.H. Roh et al. / Diagnostic Microbiology and Infectious Disease 73 (2012) 239–242
blood culture bottles with the BACTEC FX or 9240 automated blood culture system and the BACTEC Plus blood culture bottles with 3D FAN blood culture bottles (Ziegler et al., 1998). In this study, we designed a controlled clinical evaluation of both aerobic and anaerobic bottles using blood from ICU patients only. The positivity rates of both blood culture bottles and the TTD of positive bottles detected by FX or 3D were also investigated.
2. Materials and methods 2.1. Subjects and samples Between January 2010 and May 2010, 1539 four-bottle sets were collected from 270 medical and surgical ICU inpatients at a university hospital. The study began after approval had been received from the institutional review board (IRB) (IRB approval number AN09217-002). Blood cultures were performed in all patients with clinically suspected sepsis or bacteremia. Peripheral blood was drawn using an aseptic technique with alcohol and povidone iodine. ICU physicians and phlebotomists were educated regarding the technique for drawing blood with a 20-mL syringe and injecting each 5-mL blood aliquot into 4 different blood culture bottles: BA, BN, FA, and FN. Inoculated blood culture bottles were transferred to a clinical microbiology laboratory by hand or by an automated carrier system within 1 h of sampling. When the laboratory technicians received the blood culture bottles, they checked blood volumes by comparing samples against a premarked blood culture bottle in order to ensure that the sample volumes were the same.
2.2. Cultures and identification of microorganisms Each pair of bottles was loaded into the FX and 3D blood culture systems and incubated for 5 days. Growth status was continuously monitored by 2 different automated blood culture systems. The culture results were categorized as either culture positive or culture negative. In culture-positive cases, the culture bottle was mixed well. Gram staining and culture with blood agar and chocolate agar plates at 35 °C in a 5% CO2 incubator were subsequently performed. On the following day, the inoculated plates were observed and the presence of visible colonies was noted. Identification of the grown organism was performed using the Vitek2 system (bioMérieux) and/or API identification kits (bioMérieux). Correct organism identifications were also recorded for further data analysis.
2.3. Time to detection In all positive cultures, the TTD was checked from the time of loading into the blood culture system to the time of receiving a positive result. TTD data were collected using data managing systems, either the Epicenter (BD Diagnostic Systems) or the laboratory information system of Korea University Anam Hospital (KUMC). TTD was expressed as hours and minutes. TTD values were calculated according to bottle type and microorganisms and compared.
3. Results 3.1. Subjects and samples A total of 1539 four-bottle sets were collected from 270 patients in medical and surgical ICUs. Repeated blood culture sets from the same patient were included because blood cultures were performed whenever bacteremia was suspected, especially for long-term patients. The average and median number of sets collected per patient were 5.7 and 3 (range 1–37), respectively. The male-to-female ratio was 60:40. The age of the patients (mean ± SD) was 61.5 ± 16.2 years, and 63.2% of the patients were over 60 years of age. Among the study sample, 87.7% of the patients concurrently received 1 or more antimicrobial agents in the ICU. 3.2. Cultures and identification of microorganisms A total of 387 isolates from 74 patients were grown and evaluated. The 1539 bottles of BA, BN, FA, and FN each yielded a growth of 113 (7.3%), 90 (5.8%), 104 (6.8%), and 80 (5.2%) bacteria or fungi isolates (positive rate), respectively. Nineteen more positive culture bottles were detected in the FX Plus media compared to the 3D FAN media (Table 1).The detection rates for all organisms were higher in the FX BA and BN (80.6%) than in the 3D FA and FN (73.0%) culture bottles. The BA and BN culture bottles were able to detect significantly more isolates of Escherichia coli (P b 0.05) and Acinetobacter baumannii (P b 0.05) than the FA and FN bottles (Table 1). There were 27 isolates of E. coli from 6 patients, of which 10 isolates were recovered in FX only from 4 of those patients. There were 15 isolates of A. baumannii from 6 patients, of which 8 were recovered in FX only from 4 of those patients. Fourteen isolates of A. baumannii were detected by the FX culture bottles, whereas only 7 isolates were detected by 3D culture bottles. Twice of A. baumannii isolates were detected by FX compared with the 3D culture bottles. Pseudomonas aeruginosa and Enterococcus Table 1 Comparison of recovered microorganisms in the BACTEC FX and BacT/Alert 3D blood culture bottles. Microorganism(s)
Statistical analysis was performed using SPSS 12.0 (SPSS, Chicago, IL, USA). The McNemar χ 2 test and Wilcoxon signed rank test were used to determine the significances of differences in culture positivity and TTD, respectively (Mirrett et al., 2003; Riedel et al., 2009). The level of statistical significance was defined as P b 0.05.
FX Plus/F bottle only
P value
3D FAN bottle only
Gram-negative Escherichia coli Other Enterobacteriaceaea Pseudomonas aeruginosa Acinetobacter baumannii
15 22 1 6
10 5 3 8
2 7 0 1
0.039 1.0 0.250 0.039
Gram-positive cocci Staphylococcus aureus Coagulase-negative staphylococcib Enterococcus spp.c Streptococcus spp.d Gram-positive bacilli Anaerobe Candida spp.
34 33 5 11 1 0 6
9 22 7 2 1 1 1
7 24 3 1 3 1 1
0.607 0.766 0.344 1.0 0.625 1.0 1.0
110 24 134
66 3 69
45 5 50
0.055 0.727 0.096
80.6%
73.0%
With antibiotics Without antibiotics All Recovery rate
2.4. Statistical analysis
No. of isolates detected in Both bottles
The P values were calculated using McNemar chi-squared test. a Other Enterobacteriaceae included Citrobacter freundii, n = 1; Enterobacter aerogens, n = 4; Enterobacter cloacae, n = 11; Klebsiella oxytoca, n = 1; Klebsiella pneumoniae, n = 5; Serratia marcescens, n = 12. b Coagulase-negative staphylococci included S. capitis, n = 8; S. epidermidis, n = 58; S. haemolyticus, n = 1; S. hominis, n = 11; S. vitulinus, n = 1. c Enterococcus spp. included Enterococcus faecalis, n = 2; Enterococcus faecium, n = 13. d Streptococcus spp. included S. pneumoniae, n = 2; S. mitis/S. oralis, n = 5; S. salivarius, n = 4; S. vestibularis, n = 3.
K.H. Roh et al. / Diagnostic Microbiology and Infectious Disease 73 (2012) 239–242 Table 2 Comparison of the median TTD and mean TTD in BACTEC FX and BacT/Alert 3D blood culture bottles. BACTEC FX Plus (h)
BacT/Alert 3D FAN (h)
Median
Mean (95% CI)
Median
Mean (95% CI)
P value
Aerobic culture bottle Anaerobic culture bottle
17.2
21.7 (6.6–63.2)
24.7
30.9 (9.2–80.0)
b0.001
21.9
27.5 (8.9–81.7)
22.5
29.4 (2.2–95.6)
0.124
Total
18.3
24.2 (7.2–77.8)
23.8
30.3 (5.1–80.0)
b0.001
CI = Confidence interval. The P values were calculated using Wilcoxon signed rank test.
spp. were also isolated more frequently using FX culture bottles. However, similar detection rates were found between the FX and the 3D bottles for Staphylococcus aureus and coagulase-negative staphylococci. In the antibiotic-use subgroup, increased statistical significance was found (P = 0.055) compared with the without-antibiotics subgroup (Table 1). Detection rates according to microorganism subset are summarized in Table 1. 3.3. Time to detection The median and mean TTD for all the clinical isolates according to the brand of bottles are shown in Table 2. The median TTD of aerobic cultures in BA and FA bottles was 17.2 and 24.7 h, respectively (P b 0.001). The median TTD of anaerobic cultures in BN and FN bottles was 21.9 and 22.5 h, respectively. Overall, shorter TTD was observed for the BACTEC Plus blood culture bottles than for the 3D FAN culture bottles. The median TTD was significantly lower in the aerobic cultures of the BA bottles than in those of the FA bottles. The same difference was also noticed in each organism group: Gram-positive cocci, Gram-negative bacilli, and coagulasenegative staphylococci (data not shown). 4. Discussion A prospective clinical study was performed using BD Plus and 3D FAN bottles in a sample of ICU patients. Most (87.7%) of the patients received antimicrobial treatment before blood culture. Significant differences in culture positivity were noted for the Gram-negative bacilli. In particular, E. coli and A. baumannii showed a significantly higher recovery rates in BD Plus bottles than in 3D FAN bottles (all P = 0.039). As E. coli and Gram-negative bacilli bacteremias are common among inpatients (Weinstein et al., 1994), a higher recovery rate of those isolates in BD Plus bottles can be helpful in managing sepsis patients. Carbapenem-resistant A. baumannii, which is frequently resistant to aminoglycosides, fluoroquinolones, and third-generation cephalosporins, is increasing in prevalence in Korea (Lee et al., 2011). Therefore, antimicrobial susceptibility tests that isolate A. baumannii by blood culture may be helpful for choosing antibiotics. Inappropriate antimicrobial treatment is an important independent risk factor for mortality among hospitalized patients with serious infections, including bloodstream infections (Ibrahim et al., 2000). Multiple sample collections might influence the results of FX culture testing; however, in our sample, a total of 27 E. coli–positive bottles came from 6 patients. In 4 of 6 total patients, more than 1 positive bottle was detected. FX demonstrated better performance in more than half of the E. coli– or A. baumannii–positive patients. FX detected more isolates of S. aureus, Enterococcus spp., and Streptococcus spp. than did 3D. However, the differences were not significant. We divided the patients into 2 subgroups—patients treated with and without antibiotics—and analyzed the corresponding results. In the antibiotic-use subgroup, increased statistical signifi-
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cance was found (from P = 0.096 to P = 0.055), which may indicate that FX provides superior performance in antibiotic-treated patients. Adsorption of antimicrobial agents with charcoal or resin within media may enhance the viability of pathogens and increase the microorganism detection rate. Some studies have shown that the use of charcoal-containing aerobic and anaerobic culture bottles increases blood culture positivity rates, especially for S. aureus, coagulase-negative staphylococci (CNS), and Escherichia coli, compared with standard bottles without charcoal (Weinstein et al., 1995; Wilson et al., 1995). Another study has shown that resincontaining culture bottles also result in increased overall blood culture positivity rates, particularly for S. aureus, CNS, streptococci, E. coli, and Klebsiella pneumoniae (Wilson et al., 2001). Some investigators previously performed clinical comparisons between different automated blood culture systems and culture bottles (Flayhart et al., 2007; Horvath et al., 2004; Jorgensen et al., 1997; Mirrett et al., 2003; Pohlman et al., 1995; Riedel et al., 2009). Two clinical evaluations showed no significant differences in clinical performance between the BACTEC Plus Aerobic/F and BacT/Alert FAN media (Jorgensen et al., 1997; Pohlman et al., 1995). These 2 studies used only aerobic blood culture bottles, while anaerobic blood culture bottles were included in the present study. Ziegler et al. (1998) evaluated both aerobic and anaerobic bottles in the BACTEC 9240 and BacT/Alert 3D systems, and reported a shorter TTD for the BACTEC system and a higher detection rate of Enterobacteriaceae with 3D. In the Ziegler et al. study, 41.9% of patients received antibiotics, whereas, in our study, 87.7% patients received antibiotics. The difference in the detection rate of Enterobacteriaceae may reflect overall antibiotic use. Another in vitro study demonstrated that FAN media detected more fungal isolates than did BACTEC Plus media (Horvath et al., 2004). A total of only 8 fungal isolates were isolated in the present study, and there is no difference of culture positivity rate between the BACTEC Plus and 3D FAN culture bottles. During our investigation, the overall blood culture positivity rate in ICU patients was 6.8%, which is higher than the mean positivity rate of inpatients (5.9%) housed in the general ward of KUMC. The use of resin-containing medium may reduce the concentrations of antimicrobial agents in blood culture media. The concentrations of various antibiotics have been observed to decrease by 30–80% within 30 min after inoculation to resin-containing medium in an in vitro experiment (Spaargaren et al., 1998). In the study by Flayhart et al. (2007), a 50% increase in recovery rate was observed in the BD Plus bottles compared to the 3D FAN bottles in those cases containing antibiotics. However, that previous study was performed in vitro. ICU patients are often injected with many different antibiotics, such as cephalosporins, quinolones, or glycopeptides. In vivo concentrations of various antibiotics and different formulations of media within blood culture bottles could cause differences in the blood culture positive rates. In a clinical microbiology laboratory, TTD is considered as important as the culture positivity rate. A shorter TTD and rapid reporting could guide clinicians and enable them to choose more appropriate antibiotics for sepsis patients, which could lead to better prognosis. However, the TTD results were different among research studies. Pohlman et al. (1995) reported that FAN was more rapid than BACTEC Plus Aerobic/F in the detection of isolates of S. aureus. Whereas Jorgensen et al. (1997) insisted that BACTEC Plus Aerobic/F had a shorter TTD than FAN media in the case of Gram-positive cocci, including S. aureus. Simulated blood cultures were utilized for comparing Plus and FAN bottles in 13 groups of organisms (Vigano et al., 2002) and demonstrated that facultative anaerobes and fungi were detected earlier in BACTEC. A comparison of aerobic and anaerobic TTD from detection of E. coli and K. pneumoniae revealed shorter TTD when anaerobic bottles were used (Park et al., 2010). This present study suggested that both aerobic and anaerobic TTD
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performance should be evaluated in order to identify superior laboratory methods. In aerobic cultures, BD Plus had a remarkably shorter TTD than did 3D FAN media. The median TTD for BA and FA culture bottles was 17.2 and 24.7 h, respectively. This result was similar to that of a previous study by Jorgensen et al. (1997), although the difference in the median TTD between the BA and FA culture bottles was greater in our study. In the Jorgensen et al. study, the difference in the median TTD between BA and FA culture bottles was only 1.9 h. This difference between these 2 studies was possibly due to the different study samples included. ICU patients receive more substantial antibiotic treatment than do general ward patients. Spaargaren et al. (1998) reported that differences in TTD are due to different concentrations of antibiotics. TTD is significantly different in patients with S. aureus undergoing treatment by penicillin or glycopeptides (3–13 h). We suggest that using resin-containing media may make the rapid detection of bacteremia in blood cultures from critically ill patients possible. In present anaerobic cultures, faster results were also shown for BN bottles, but significant differences were not noticed between the 2 brands. When the TTD is shortened by 7 h, organism identification time and antimicrobial agent susceptibility testing are also reduced by as much as 1 day in a 24-h shift laboratory. Shortened TTD combined with a rapid bacterial identification system such as matrix-assisted laser desorption/ ionization–time of flight mass spectrometry (Stevenson et al., 2010) could result in immediate treatment with proper antibiotics and save the lives of patients with bloodstream infections. The shortened TTD (11 h) in patients with CNS diseases could allow earlier clinical decisions, enabling discontinuation of antimicrobial treatment in cases of contamination. One of our study limitations is that about 25% to 30% of the recovered isolates were in CNS, possible contaminants, in FX (55/ 203 isolates) and 3D (57/184). More careful skin decontamination is necessary to improve our results. Due to the limitations of available resources, we were unable to increase the size of our study sample. It would be possible to get more significant data by increasing the sample size and reducing the influences of possible contaminants. In conclusion, BA bottles showed better performance for isolating E. coli and A. baumannii from ICU patients than did FA bottles. In particular, significantly higher positive rate and shorter recovery time were observed for the BA bottles than for the FA bottles. Acknowledgments The authors would like to thank BD Korea for their technical contributions regarding the BACTEC FX system and for supplying Plus Aerobic and Anaerobic bottles. The authors also acknowledge the assistance of the laboratory staff of the Korea University Anam Hospital.
References Flayhart D, Borek AP, Wakefield T, Dick J, Carroll KC. Comparison of BACTEC PLUS blood culture media to BacT/Alert FA blood culture media for detection of bacterial pathogens in samples containing therapeutic levels of antibiotics. J Clin Microbiol 2007;45:816–21. Horvath LL, George BJ, Murray CK, Harrison LS, Hospenthal DR. Direct comparison of the BACTEC 9240 and BacT/ALERT 3D automated blood culture systems for candida growth detection. J Clin Microbiol 2004;42:115–8. Huang AH, Yan JJ, Wu JJ. Comparison of five days versus seven days of incubation for detection of positive blood cultures by the Bactec 9240 system. Eur J Clin Microbiol Infect Dis 1998;17:637–41. Ibrahim EH, Sherman G, Ward S, Fraser VJ, Kollef MH. The influence of inadequate antimicrobial treatment of bloodstream infections on patient outcomes in the ICU setting. Chest 2000;118:146–55. Jorgensen JH, Mirrett S, McDonald LC, Murray PR, Weinstein MP, Fune J, et al. Controlled clinical laboratory comparison of BACTEC plus aerobic/F resin medium with BacT/Alert aerobic FAN medium for detection of bacteremia and fungemia. J Clin Microbiol 1997;35:53–8. Lee K, Kim MN, Kim JS, Hong HL, Kang JO, Shin JH, et al. Further increases in carbapenem-, amikacin-, and fluoroquinolone-resistant isolates of Acinetobacter spp. and P. aeruginosa in Korea: KONSAR Study 2009. Yonsei Med J 2011;52: 793–802. Mirrett S, Reller LB, Petti CA, Woods CW, Vazirani B, Sivadas R, et al. Controlled clinical comparison of BacT/ALERT standard aerobic medium with BACTEC standard aerobic medium for culturing blood. J Clin Microbiol 2003;41:2391–4. Park SH, Shim H, Yoon NS, Kim MN. Clinical relevance of time-to-positivity in BACTEC9240 blood culture system. Korean J Lab Med 2010;30:276–83. Pohlman JK, Kirkley BA, Easley KA, Basille BA, Washington JA. Controlled clinical evaluation of BACTEC Plus Aerobic/F and BacT/Alert Aerobic FAN bottles for detection of bloodstream infections. J Clin Microbiol 1995;33:2856–8. Riedel S, Junkins A, Stamper PD, Cress G, Widness JA, Doern GV. Comparison of the Bactec 9240 and BacT/Alert blood culture systems for evaluation of placental cord blood for transfusion in neonates. J Clin Microbiol 2009;47:1645–9. Smith-Elekes S, Weinstein MP. Blood cultures. Infect Dis Clin North Am 1993;7:221–34. Spaargaren J, van Boven CP, Voorn GP. Effectiveness of resins in neutralizing antibiotic activities in BACTEC plus Aerobic/F culture medium. J Clin Microbiol 1998;36: 3731–3. Stevenson LG, Drake SK, Murray PR. Rapid identification of bacteria in positive blood culture broths by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol 2010;48:444–7. Vigano EF, Vasconi E, Agrappi C, Clerici P. Use of simulated blood cultures for time to detection comparison between BacT/ALERT and BACTEC 9240 blood culture systems. Diagn Microbiol Infect Dis 2002;44:235–40. Weinstein MP, Mirrett S, Reimer LG, Wilson ML, Smith-Elekes S, Chuard CR, et al. Controlled evaluation of BacT/Alert standard aerobic and FAN aerobic blood culture bottles for detection of bacteremia and fungemia. J Clin Microbiol 1995;33: 978–81. Weinstein MP, Mirrett S, Wilson ML, Reimer LG, Reller LB. Controlled evaluation of 5 versus 10 milliliters of blood cultured in aerobic BacT/Alert blood culture bottles. J Clin Microbiol 1994;32:2103–6. Wilson ML, Mirrett S, Meredith FT, Weinstein MP, Scotto V, Reller LB. Controlled clinical comparison of BACTEC plus anaerobic/F to standard anaerobic/F as the anaerobic companion bottle to plus aerobic/F medium for culturing blood from adults. J Clin Microbiol 2001;39:983–9. Wilson ML, Weinstein MP, Mirrett S, Reimer LG, Feldman RJ, Chuard CR, et al. Controlled evaluation of BacT/alert standard anaerobic and FAN anaerobic blood culture bottles for the detection of bacteremia and fungemia. J Clin Microbiol 1995;33: 2265–70. Ziegler R, Johnscher I, Martus P, Lenhardt D, Just HM. Controlled clinical laboratory comparison of two supplemented aerobic and anaerobic media used in automated blood culture systems to detect bloodstream infections. J Clin Microbiol 1998;36: 657–61.
Contents lists available at SciVerse ScienceDirect
Diagnostic Microbiology and Infectious Disease j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / d i a g m i c r o b i o
Evaluation of BACTEC Plus aerobic and anaerobic blood culture bottles and BacT/Alert FAN aerobic and anaerobic blood culture bottles for the detection of bacteremia in ICU patients☆ Kyoung Ho Roh a, Ju Yeon Kim a, Ha Nui Kim a, Hye Jin Lee a, Jang Wook Sohn b, Min Ja Kim b, Yunjung Cho a, Young Kee Kim a, Chang Kyu Lee a,⁎ a b
Department of Laboratory Medicine, Korea University College of Medicine, Seoul, Korea Department of Internal Medicine, Korea University College of Medicine, Seoul, Korea
a r t i c l e
i n f o
Article history: Received 31 August 2011 Received in revised form 8 March 2012 Accepted 19 March 2012 Available online 25 April 2012 Keywords: Blood culture Evaluation BACTEC BacT/Alert Culture bottle Time to detection
a b s t r a c t Blood culture is the most valuable laboratory test for the diagnosis of bacteremia and sepsis. The BACTEC FX and BacT/Alert 3D automated blood culture systems are commonly used in Korean health care facilities. A controlled clinical evaluation of the resin-containing BACTEC Plus aerobic (BA) and anaerobic (BN), and the charcoal-containing FAN aerobic (FA) and anaerobic (FN) bottles using blood from intensive care unit (ICU) patients was designed. The performances of these 2 systems with media containing particle absorbing antimicrobial agents were evaluated using the culture positivity rate and time to detection (TTD). TTD was collected using data management systems, either the Epicenter (BD Diagnostic Systems) or the hospital laboratory information system. A total of 1539 four-bottle sets were collected from 270 patients in medical and surgical ICUs. Blood culture samples included 1539 bottles each of BA, BN, FA, and FN, and yielded 113 (7.3%), 90 (5.8%), 104 (6.8%), and 80 (5.2%) positive bacterial or fungal isolates, respectively. There were significant differences between the resin-containing BA and BN samples in culture positivity and also between the charcoal-containing FA and FN samples, especially for Escherichia coli (25/27 versus 17/27, P b 0.05) and Acinetobacter baumannii (14/15 versus 7/15, P b 0.05). Significantly shorter recovery time was observed in BACTEC Plus aerobic bottles than in FAN aerobic bottles (17.2 and 24.7 h, respectively) (P b 0.001). © 2012 Elsevier Inc. All rights reserved.
1. Introduction Blood culture is the most valuable laboratory test for the diagnosis of bacteremia and sepsis (Smith-Elekes and Weinstein, 1993). The recovery of pathogenic microorganisms from blood culture is essential for appropriate clinical management of infection. The timely reporting of identified organisms and antimicrobial agent susceptibility is helpful for the proper selection of antimicrobial agents and treatment. The automated blood culture system is the most sensitive method introduced in blood culture and is currently in widespread use (Mirrett et al., 2003). The BACTEC FX (BD Diagnostic Systems, Sparks, MD, USA) (FX) and BacT/Alert 3D (bioMérieux, Durham, NC, USA) (3D) automated blood culture systems are commonly used in Korea. The BACTEC FX system is a newly introduced automated blood culture system. The FX and 9240 systems detect the growth of microorganisms by a fluorescent sensor for CO2, whereas the 3D uses a colorimetric detection sensor for CO2. A 5-day incubation protocol is generally used in clinical microbiology laboratories (Huang et al., ☆ This work was supported by a grant from BD Korea. ⁎ Corresponding author. Tel.: +82-2-920-5381; fax: +82-2-920-5538. E-mail address: [email protected] (C.K. Lee). 0732-8893/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2012.03.022
1998). In addition to the culture positivity rate, the time to detection (TTD) of a positive culture is an important determinant for the rapid reporting of results. Various types of blood culture bottles have been introduced to increase the culture positivity rate and shorten the TTD. Higher culture positivity rates and shorter TTD give clinicians more opportunities for providing appropriate patient management. By the time the blood cultures are collected, a significant proportion of patients are usually already receiving antibiotic therapy. This can result in the inhibition of growth of the causative organism, especially in intensive care unit (ICU) patients. Blood culture bottles with antimicrobial agent–removing resin or charcoal-containing media demonstrate higher recovery rates compared to standard bottles (Weinstein et al., 1995; Wilson et al., 2001). The BACTEC Plus aerobic (BA) and anaerobic (BN) bottles contain resin, and the bioMérieux FAN aerobic (FA) and anaerobic (FN) bottles include charcoal to minimize the effects of antimicrobial agents in the patients' blood. A few investigators have tested the in vitro performance of blood culture bottles using blood culture bottles containing various antimicrobial agents and microorganisms (Flayhart et al., 2007; Vigano et al., 2002). Some studies evaluated only aerobic culture bottles (Jorgensen et al., 1997; Pohlman et al., 1995). Few clinical investigations have concurrently evaluated aerobic and anaerobic
240
K.H. Roh et al. / Diagnostic Microbiology and Infectious Disease 73 (2012) 239–242
blood culture bottles with the BACTEC FX or 9240 automated blood culture system and the BACTEC Plus blood culture bottles with 3D FAN blood culture bottles (Ziegler et al., 1998). In this study, we designed a controlled clinical evaluation of both aerobic and anaerobic bottles using blood from ICU patients only. The positivity rates of both blood culture bottles and the TTD of positive bottles detected by FX or 3D were also investigated.
2. Materials and methods 2.1. Subjects and samples Between January 2010 and May 2010, 1539 four-bottle sets were collected from 270 medical and surgical ICU inpatients at a university hospital. The study began after approval had been received from the institutional review board (IRB) (IRB approval number AN09217-002). Blood cultures were performed in all patients with clinically suspected sepsis or bacteremia. Peripheral blood was drawn using an aseptic technique with alcohol and povidone iodine. ICU physicians and phlebotomists were educated regarding the technique for drawing blood with a 20-mL syringe and injecting each 5-mL blood aliquot into 4 different blood culture bottles: BA, BN, FA, and FN. Inoculated blood culture bottles were transferred to a clinical microbiology laboratory by hand or by an automated carrier system within 1 h of sampling. When the laboratory technicians received the blood culture bottles, they checked blood volumes by comparing samples against a premarked blood culture bottle in order to ensure that the sample volumes were the same.
2.2. Cultures and identification of microorganisms Each pair of bottles was loaded into the FX and 3D blood culture systems and incubated for 5 days. Growth status was continuously monitored by 2 different automated blood culture systems. The culture results were categorized as either culture positive or culture negative. In culture-positive cases, the culture bottle was mixed well. Gram staining and culture with blood agar and chocolate agar plates at 35 °C in a 5% CO2 incubator were subsequently performed. On the following day, the inoculated plates were observed and the presence of visible colonies was noted. Identification of the grown organism was performed using the Vitek2 system (bioMérieux) and/or API identification kits (bioMérieux). Correct organism identifications were also recorded for further data analysis.
2.3. Time to detection In all positive cultures, the TTD was checked from the time of loading into the blood culture system to the time of receiving a positive result. TTD data were collected using data managing systems, either the Epicenter (BD Diagnostic Systems) or the laboratory information system of Korea University Anam Hospital (KUMC). TTD was expressed as hours and minutes. TTD values were calculated according to bottle type and microorganisms and compared.
3. Results 3.1. Subjects and samples A total of 1539 four-bottle sets were collected from 270 patients in medical and surgical ICUs. Repeated blood culture sets from the same patient were included because blood cultures were performed whenever bacteremia was suspected, especially for long-term patients. The average and median number of sets collected per patient were 5.7 and 3 (range 1–37), respectively. The male-to-female ratio was 60:40. The age of the patients (mean ± SD) was 61.5 ± 16.2 years, and 63.2% of the patients were over 60 years of age. Among the study sample, 87.7% of the patients concurrently received 1 or more antimicrobial agents in the ICU. 3.2. Cultures and identification of microorganisms A total of 387 isolates from 74 patients were grown and evaluated. The 1539 bottles of BA, BN, FA, and FN each yielded a growth of 113 (7.3%), 90 (5.8%), 104 (6.8%), and 80 (5.2%) bacteria or fungi isolates (positive rate), respectively. Nineteen more positive culture bottles were detected in the FX Plus media compared to the 3D FAN media (Table 1).The detection rates for all organisms were higher in the FX BA and BN (80.6%) than in the 3D FA and FN (73.0%) culture bottles. The BA and BN culture bottles were able to detect significantly more isolates of Escherichia coli (P b 0.05) and Acinetobacter baumannii (P b 0.05) than the FA and FN bottles (Table 1). There were 27 isolates of E. coli from 6 patients, of which 10 isolates were recovered in FX only from 4 of those patients. There were 15 isolates of A. baumannii from 6 patients, of which 8 were recovered in FX only from 4 of those patients. Fourteen isolates of A. baumannii were detected by the FX culture bottles, whereas only 7 isolates were detected by 3D culture bottles. Twice of A. baumannii isolates were detected by FX compared with the 3D culture bottles. Pseudomonas aeruginosa and Enterococcus Table 1 Comparison of recovered microorganisms in the BACTEC FX and BacT/Alert 3D blood culture bottles. Microorganism(s)
Statistical analysis was performed using SPSS 12.0 (SPSS, Chicago, IL, USA). The McNemar χ 2 test and Wilcoxon signed rank test were used to determine the significances of differences in culture positivity and TTD, respectively (Mirrett et al., 2003; Riedel et al., 2009). The level of statistical significance was defined as P b 0.05.
FX Plus/F bottle only
P value
3D FAN bottle only
Gram-negative Escherichia coli Other Enterobacteriaceaea Pseudomonas aeruginosa Acinetobacter baumannii
15 22 1 6
10 5 3 8
2 7 0 1
0.039 1.0 0.250 0.039
Gram-positive cocci Staphylococcus aureus Coagulase-negative staphylococcib Enterococcus spp.c Streptococcus spp.d Gram-positive bacilli Anaerobe Candida spp.
34 33 5 11 1 0 6
9 22 7 2 1 1 1
7 24 3 1 3 1 1
0.607 0.766 0.344 1.0 0.625 1.0 1.0
110 24 134
66 3 69
45 5 50
0.055 0.727 0.096
80.6%
73.0%
With antibiotics Without antibiotics All Recovery rate
2.4. Statistical analysis
No. of isolates detected in Both bottles
The P values were calculated using McNemar chi-squared test. a Other Enterobacteriaceae included Citrobacter freundii, n = 1; Enterobacter aerogens, n = 4; Enterobacter cloacae, n = 11; Klebsiella oxytoca, n = 1; Klebsiella pneumoniae, n = 5; Serratia marcescens, n = 12. b Coagulase-negative staphylococci included S. capitis, n = 8; S. epidermidis, n = 58; S. haemolyticus, n = 1; S. hominis, n = 11; S. vitulinus, n = 1. c Enterococcus spp. included Enterococcus faecalis, n = 2; Enterococcus faecium, n = 13. d Streptococcus spp. included S. pneumoniae, n = 2; S. mitis/S. oralis, n = 5; S. salivarius, n = 4; S. vestibularis, n = 3.
K.H. Roh et al. / Diagnostic Microbiology and Infectious Disease 73 (2012) 239–242 Table 2 Comparison of the median TTD and mean TTD in BACTEC FX and BacT/Alert 3D blood culture bottles. BACTEC FX Plus (h)
BacT/Alert 3D FAN (h)
Median
Mean (95% CI)
Median
Mean (95% CI)
P value
Aerobic culture bottle Anaerobic culture bottle
17.2
21.7 (6.6–63.2)
24.7
30.9 (9.2–80.0)
b0.001
21.9
27.5 (8.9–81.7)
22.5
29.4 (2.2–95.6)
0.124
Total
18.3
24.2 (7.2–77.8)
23.8
30.3 (5.1–80.0)
b0.001
CI = Confidence interval. The P values were calculated using Wilcoxon signed rank test.
spp. were also isolated more frequently using FX culture bottles. However, similar detection rates were found between the FX and the 3D bottles for Staphylococcus aureus and coagulase-negative staphylococci. In the antibiotic-use subgroup, increased statistical significance was found (P = 0.055) compared with the without-antibiotics subgroup (Table 1). Detection rates according to microorganism subset are summarized in Table 1. 3.3. Time to detection The median and mean TTD for all the clinical isolates according to the brand of bottles are shown in Table 2. The median TTD of aerobic cultures in BA and FA bottles was 17.2 and 24.7 h, respectively (P b 0.001). The median TTD of anaerobic cultures in BN and FN bottles was 21.9 and 22.5 h, respectively. Overall, shorter TTD was observed for the BACTEC Plus blood culture bottles than for the 3D FAN culture bottles. The median TTD was significantly lower in the aerobic cultures of the BA bottles than in those of the FA bottles. The same difference was also noticed in each organism group: Gram-positive cocci, Gram-negative bacilli, and coagulasenegative staphylococci (data not shown). 4. Discussion A prospective clinical study was performed using BD Plus and 3D FAN bottles in a sample of ICU patients. Most (87.7%) of the patients received antimicrobial treatment before blood culture. Significant differences in culture positivity were noted for the Gram-negative bacilli. In particular, E. coli and A. baumannii showed a significantly higher recovery rates in BD Plus bottles than in 3D FAN bottles (all P = 0.039). As E. coli and Gram-negative bacilli bacteremias are common among inpatients (Weinstein et al., 1994), a higher recovery rate of those isolates in BD Plus bottles can be helpful in managing sepsis patients. Carbapenem-resistant A. baumannii, which is frequently resistant to aminoglycosides, fluoroquinolones, and third-generation cephalosporins, is increasing in prevalence in Korea (Lee et al., 2011). Therefore, antimicrobial susceptibility tests that isolate A. baumannii by blood culture may be helpful for choosing antibiotics. Inappropriate antimicrobial treatment is an important independent risk factor for mortality among hospitalized patients with serious infections, including bloodstream infections (Ibrahim et al., 2000). Multiple sample collections might influence the results of FX culture testing; however, in our sample, a total of 27 E. coli–positive bottles came from 6 patients. In 4 of 6 total patients, more than 1 positive bottle was detected. FX demonstrated better performance in more than half of the E. coli– or A. baumannii–positive patients. FX detected more isolates of S. aureus, Enterococcus spp., and Streptococcus spp. than did 3D. However, the differences were not significant. We divided the patients into 2 subgroups—patients treated with and without antibiotics—and analyzed the corresponding results. In the antibiotic-use subgroup, increased statistical signifi-
241
cance was found (from P = 0.096 to P = 0.055), which may indicate that FX provides superior performance in antibiotic-treated patients. Adsorption of antimicrobial agents with charcoal or resin within media may enhance the viability of pathogens and increase the microorganism detection rate. Some studies have shown that the use of charcoal-containing aerobic and anaerobic culture bottles increases blood culture positivity rates, especially for S. aureus, coagulase-negative staphylococci (CNS), and Escherichia coli, compared with standard bottles without charcoal (Weinstein et al., 1995; Wilson et al., 1995). Another study has shown that resincontaining culture bottles also result in increased overall blood culture positivity rates, particularly for S. aureus, CNS, streptococci, E. coli, and Klebsiella pneumoniae (Wilson et al., 2001). Some investigators previously performed clinical comparisons between different automated blood culture systems and culture bottles (Flayhart et al., 2007; Horvath et al., 2004; Jorgensen et al., 1997; Mirrett et al., 2003; Pohlman et al., 1995; Riedel et al., 2009). Two clinical evaluations showed no significant differences in clinical performance between the BACTEC Plus Aerobic/F and BacT/Alert FAN media (Jorgensen et al., 1997; Pohlman et al., 1995). These 2 studies used only aerobic blood culture bottles, while anaerobic blood culture bottles were included in the present study. Ziegler et al. (1998) evaluated both aerobic and anaerobic bottles in the BACTEC 9240 and BacT/Alert 3D systems, and reported a shorter TTD for the BACTEC system and a higher detection rate of Enterobacteriaceae with 3D. In the Ziegler et al. study, 41.9% of patients received antibiotics, whereas, in our study, 87.7% patients received antibiotics. The difference in the detection rate of Enterobacteriaceae may reflect overall antibiotic use. Another in vitro study demonstrated that FAN media detected more fungal isolates than did BACTEC Plus media (Horvath et al., 2004). A total of only 8 fungal isolates were isolated in the present study, and there is no difference of culture positivity rate between the BACTEC Plus and 3D FAN culture bottles. During our investigation, the overall blood culture positivity rate in ICU patients was 6.8%, which is higher than the mean positivity rate of inpatients (5.9%) housed in the general ward of KUMC. The use of resin-containing medium may reduce the concentrations of antimicrobial agents in blood culture media. The concentrations of various antibiotics have been observed to decrease by 30–80% within 30 min after inoculation to resin-containing medium in an in vitro experiment (Spaargaren et al., 1998). In the study by Flayhart et al. (2007), a 50% increase in recovery rate was observed in the BD Plus bottles compared to the 3D FAN bottles in those cases containing antibiotics. However, that previous study was performed in vitro. ICU patients are often injected with many different antibiotics, such as cephalosporins, quinolones, or glycopeptides. In vivo concentrations of various antibiotics and different formulations of media within blood culture bottles could cause differences in the blood culture positive rates. In a clinical microbiology laboratory, TTD is considered as important as the culture positivity rate. A shorter TTD and rapid reporting could guide clinicians and enable them to choose more appropriate antibiotics for sepsis patients, which could lead to better prognosis. However, the TTD results were different among research studies. Pohlman et al. (1995) reported that FAN was more rapid than BACTEC Plus Aerobic/F in the detection of isolates of S. aureus. Whereas Jorgensen et al. (1997) insisted that BACTEC Plus Aerobic/F had a shorter TTD than FAN media in the case of Gram-positive cocci, including S. aureus. Simulated blood cultures were utilized for comparing Plus and FAN bottles in 13 groups of organisms (Vigano et al., 2002) and demonstrated that facultative anaerobes and fungi were detected earlier in BACTEC. A comparison of aerobic and anaerobic TTD from detection of E. coli and K. pneumoniae revealed shorter TTD when anaerobic bottles were used (Park et al., 2010). This present study suggested that both aerobic and anaerobic TTD
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performance should be evaluated in order to identify superior laboratory methods. In aerobic cultures, BD Plus had a remarkably shorter TTD than did 3D FAN media. The median TTD for BA and FA culture bottles was 17.2 and 24.7 h, respectively. This result was similar to that of a previous study by Jorgensen et al. (1997), although the difference in the median TTD between the BA and FA culture bottles was greater in our study. In the Jorgensen et al. study, the difference in the median TTD between BA and FA culture bottles was only 1.9 h. This difference between these 2 studies was possibly due to the different study samples included. ICU patients receive more substantial antibiotic treatment than do general ward patients. Spaargaren et al. (1998) reported that differences in TTD are due to different concentrations of antibiotics. TTD is significantly different in patients with S. aureus undergoing treatment by penicillin or glycopeptides (3–13 h). We suggest that using resin-containing media may make the rapid detection of bacteremia in blood cultures from critically ill patients possible. In present anaerobic cultures, faster results were also shown for BN bottles, but significant differences were not noticed between the 2 brands. When the TTD is shortened by 7 h, organism identification time and antimicrobial agent susceptibility testing are also reduced by as much as 1 day in a 24-h shift laboratory. Shortened TTD combined with a rapid bacterial identification system such as matrix-assisted laser desorption/ ionization–time of flight mass spectrometry (Stevenson et al., 2010) could result in immediate treatment with proper antibiotics and save the lives of patients with bloodstream infections. The shortened TTD (11 h) in patients with CNS diseases could allow earlier clinical decisions, enabling discontinuation of antimicrobial treatment in cases of contamination. One of our study limitations is that about 25% to 30% of the recovered isolates were in CNS, possible contaminants, in FX (55/ 203 isolates) and 3D (57/184). More careful skin decontamination is necessary to improve our results. Due to the limitations of available resources, we were unable to increase the size of our study sample. It would be possible to get more significant data by increasing the sample size and reducing the influences of possible contaminants. In conclusion, BA bottles showed better performance for isolating E. coli and A. baumannii from ICU patients than did FA bottles. In particular, significantly higher positive rate and shorter recovery time were observed for the BA bottles than for the FA bottles. Acknowledgments The authors would like to thank BD Korea for their technical contributions regarding the BACTEC FX system and for supplying Plus Aerobic and Anaerobic bottles. The authors also acknowledge the assistance of the laboratory staff of the Korea University Anam Hospital.
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