Bacterial detection in apheresis platelets: blood systems experience with a two-bottle and one-bottle culture system

BACKGROUND: United Blood Services (UBS) began bacterial testing of platelets (PLTs) using one‐bottle cultures in September 2003. Collection of 7‐day PLTs using two‐bottle cultures began in April 2006. This study compares our experience using both systems. STUDY DESIGN AND METHODS: PLTs from 13 UBS regional centers cultured from September 1, 2003, to September 1, 2007, were included in the analysis. Positive‐signal bottles from a commercially available microbial detection system (BacT/ALERT, bioMérieux) were sent, with corresponding PLTs if available, for confirmatory testing using plate culture media. AABB definitions were used with modifications. RESULTS: A total of 51,265 7‐day PLT collections and 191,521 5‐day PLT collections were tested with bacterial cultures. The overall true‐positive (TP) rate for the two‐bottle system (1:8544) was comparable to the TP rate with the previous one‐bottle system (1:6344). In three of six yield cases, only the anaerobic bottle was positive (two cases of Group D Streptococci, one case of Corynebacterium spp.). The false‐positive (FP) and indeterminate (IND) rates in the anaerobic bottle (1:1767 and 1:1830, respectively) were significantly higher than those in the aerobic bottle (1:6408 and 1:17,088, respectively; p < 0.001). One confirmed transfusion‐related septic reaction, classified as a late TP after investigation, was reported out of 242,786 tested PLT donations. CONCLUSION: The rate of TP cases by the two‐bottle system was not increased over the one‐bottle system, although anaerobic‐bottle‐only positive cases were detected. FP and IND rates were increased in the two‐bottle system, attributable to the anaerobic bottle. Observation of only one documented transfusion‐related septic reaction in 4 years of bacterial screening at UBS is reassuring, although limitations in passive surveillance and higher rates of reactions reported by others indicate the need for continued vigilance.     

The residual risk of sepsis: modeling the effect of concentration on bacterial detection in two-bottle culture systems and an estimation of false-negative culture rates

BACKGROUND: Septic reactions continue to be reported with culture-tested platelet (PLT) products, probably due to false-negative results associated with inadequate sampling of low-concentration bacteria. The mechanism of test failure was modeled and false-negative rates were estimated utilizing published data. STUDY DESIGN AND METHODS: The effect of concentration on the probability of sampling one or more viable bacteria in an 8-mL sample of a 300-mL product was determined. The ratio of single- versus dual-bottle-positive tests in repeated cultures utilizing two-bottle systems was used to assess mean bacterial concentrations and to predict false-negative tests. RESULTS: Published reports reveal a mean residual risk of sepsis of 2.3 per 10(5) products tested. Modeling the effect of concentration predicts that 50 and 95 percent of samples are detected at 0.09 and 0.36 CFU per mL for organisms that grow under both aerobic and anaerobic conditions, and suggest a 50 and 5 percent false-negative culture rate at these concentrations. Reanalysis of published studies documenting single-bottle growth of nonfastidious organisms indicate that low bacterial concentrations are frequently encountered and predict false-negative cultures in products contaminated with common bacteria at rates that varied from 4 to more than 75 percent. The model highlights the weakness of the AABB definition of false-positive culture results at low bacterial concentrations, especially for organisms that grow poorly in aerobic storage conditions. CONCLUSION: Continuing reports of septic reactions after transfusion of culture-tested apheresis PLTs, and frequent single-bottle-positive results when testing with two-bottle systems, suggest appreciable levels of false-negative results with a commercially available bacterial detection system (BacT/ALERT, bioMéreiux) as implemented.     

Culture-based bacterial evaluation of the appendix lumen in patients with and without acute appendicitis

PurposeControversy exists over whether bacterial flora within the appendix differs between patients with and without appendicitis. To examine these potential differences, we cultured the appendiceal luminal microbiota of patients with and without acute appendicitis, and identified the bacterial species therein.MethodsFifty-seven patients with acute appendicitis and 37 patients without acute appendicitis who underwent curative resection of colorectal cancer and prophylactic appendectomies (control group) were included. Appendicitis patients were classified into the phlegmonous group or the gangrenous appendicitis group histopathologically. There was no patient with perforated appendicitis. Aerobic isolates were identified using standard identification schemata, and anaerobic isolates were identified according to the Japanese guidelines.ResultsThere were no significant differences among the three groups in the median number aerobe species present per patient. However, the median number anaerobe species in the gangrenous appendicitis group was significantly higher than that of the control group and the phlegmonous appendicitis group. In addition, the incidence of patients with Bacillus species, Fusobacterium nucleatum, and Bilophila wadsworthia increased as the disease progressed from phlegmonous to gangrenous appendicitis.ConclusionThe present results suggest that increased diversity of anaerobes and the translocation of Bacillus species, F. nucleatum, and B. wadsworthia are associated with the progression of acute appendicitis.     

测定抗-HBs的两种方法在不同温育时间的结果比较

目的探讨检测乙型肝炎（下称乙肝）病毒表面抗体（抗-HBs）两种方法在不同温育时间的结果差异。方法采用免疫酶法（EIA）一步法、时间分辨荧光（TRF）法检测抗-HBs,OD值在0．056-0．380之间标本温育时间变化而造成的结果值的差异。结果用30min EIA一步法检测抗-HBs,结果37例弱阳性和59例阴性,再用60min EIA一步法抗-HBs检测,结果41例呈弱阳性和55例阴性。用TRF两步法抗-HBs测定出42例弱阳性和54例阴性,发现有6例标本不同于温育30min的EIA一步法抗-HBs检测的结果。结论酶联免疫吸附试验作为一种固相免疫测定,抗原抗体的结合反应在固相上进行,要使液相中的抗原或抗体与固相上的特异抗体或抗原完全结合,必须在一定的温度、时间条件下反应。温育所需时间与温度成反比,尤其在冬天,标本从室温到恒温箱这一过程很容易造成实际测定温育时间不够,也是造成灰区误差的原因之一。     

Evaluation of the enhanced bacterial detection system for screening of contaminated platelets

BACKGROUND: The Pall third-generation enhanced bacterial detection system (eBDS) was recently approved for detection of bacterial contamination in leukoreduced platelets (PLTs). The method is based on the measurement of the oxygen content as a marker for bacteria. eBDS incorporates major modifications including removal of the sample-set filter, modification of the culture medium, and incubation with agitation of the sample pouch. STUDY DESIGN AND METHODS: Ten whole blood-derived random-donor PLT units collected on Day 1 after donation and 10 single-donor apheresis PLT units were spiked with low levels of bacteria in three different blood transfusion centers. Inoculation was performed at a final concentration of 5 to 50 colony-forming units per mL with reference strains of five organisms involved in severe transfusion-associated infections. PLT units were stored at 22 degrees C for 24 hours before sampling. Six sample sets were then sterile-connected to each unit and placed on a horizontal agitator at 35 degrees C for 18 or 24 hours of incubation. RESULTS: No false-positive results were obtained, indicating a 100 percent specificity of the assay. Of 126 spiked sample pouches tested, 61 of 63 (96.82%) and 63 of 63 (100%) were detected positive after 18 or 24 hours of incubation, respectively. In the two missed cases that failed to detect Bacillus cereus, the measured oxygen was slightly above the detection threshold but was markedly different from the negative samples. CONCLUSION: The eBDS method allows definitive testing of PLTs as soon as 42 hours after collection and offers an alternative culture method to the BacT/ALERT system.     

Reassessment of the routine anaerobic culture and incubation time in the BacT/Alert FAN blood culture bottles

A total of 9,130 blood cultures were collected from adult patients with suspected bloodstream infections. The recommended 20 mL sample of blood was divided equally between the aerobic and anaerobic FAN bottles and monitored in the BacT/Alert Microbial Detection System for a total of 5 days. There were 757 clinically significant positive culture pairs from 291 patients. Significant differences were found with greater recovery of Pseudomonas aeruginosa (p < 0.001), Acinetobacter spp. (p = 0.002), coagulase-negative staphylococci other than Staphylococcus epidermidis (p = 0.002), and Candida spp. (p < 0.001) from the aerobic bottle and greater recovery of anaerobic bacteria (p < 0.001) from the anaerobic bottle. Significantly more episodes of P. aeruginosa bacteremia (p < 0.003) and candidemia (p < 0.001) were detected by the aerobic FAN bottle and significantly more episodes of anaerobic bacteremia (p < 0.001) were detected by the anaerobic FAN bottle (Table 2). No other significant differences between systems in their detection of bacteremias were noted. Anaerobic bacteremias were encountered in diverse and often unpredictable clinical settings. All clinically significant episodes of bloodstream infection were detected within 4 days of incubation of their cultures. We conclude routine, rather than selective, use of the anaerobic FAN bottle in the blood culture set and a 4-day incubation of blood cultures in the BacT/Alert aerobic and anaerobic FAN bottles is an appropriate routine procedure.     

Use of simulated blood cultures for time to detection comparison between BacT/ALERT and BACTEC 9240 blood culture systems

In order to avoid the influence of pre-analytical steps, the following study was performed by using sterile blood spiked with defined loads of microorganisms as inoculum. Time-to-Detection (TTD) was evaluated for the most frequently encountered bacteria and fungi in septicemia, comparing two commercially available blood culture systems, BACTEC 9240 (Becton Dickinson) and BacT/ALERT (Organon Teknika). A specific medium, Bactec Mycosis IC/F (Becton Dickinson), was compared with the Bactec Plus Aerobic (Becton Dickinson) and FAN Aerobic (Organon Teknika) media for recovery of fungi in general and in case of mixed bacterial/fungal septicemia. The results show that the BACTEC system detects nearly all enrolled microorganisms significantly faster than the BacT/ALERT; the anaerobic vial contributes to the detection of anaerobes and facultative anaerobes and, in the case of BACTEC, shortens TTD; the Bactec Mycosis IC/F bottle shortens TTD of fungi.     

Evaluation of an automated culture system for detecting bacterial contamination of platelets: an analysis with 15 contaminating organisms

BACKGROUND: Approximately 1 in 2000 platelet components are bacterially contaminated. The time to detection of 15 seeded organisms in platelets recovered from an automated culture system was studied. STUDY DESIGN AND METHODS: Isolates of Bacillus cereus, Bacillus subtilis, Candida albicans, Clostridium perfringens, Corynebacterium species, Enterobacter cloacae, Escherichia coli, Klebsiella oxytoca, Propionibacterium acnes, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Serratia marcescens, Streptococcus pyogenes, and Streptococcus viridans were inoculated into Day 2 apheresis platelet components to obtain a final concentration of approximately 10 and 100 CFU per mL (2 units/organism). Each bag was sampled 10 times (20 mL/sample). Four mL of each sample was inoculated into standard aerobic and anaerobic bottles and into aerobic and anaerobic bottles containing charcoal; 2 mL was inoculated into pediatric aerobic bottles (so as to maintain a 1:10 ratio of sample to media) and 1 mL into thioglycollate broth. RESULTS: With the exception of P. acnes, all organisms were detected in a mean of 9.2 to 25.6 hours. A range of 10 serial dilutions in inoculating concentrations was associated with an overall 10.1-percent difference in detection time. A mean of 74.4 and 86.2 hours (100 and 10 CFU/mL inocula, respectively) was required for the detection of P. acnes in anaerobic bottles. CONCLUSION: Bacteria thought to be clinically significant platelet contaminants can be detected in 9.2 to 25.6 hours when the starting concentration is approximately 10 to 100 CFU per mL. P. acnes required considerably longer incubation times for detection (in either aerobic or anaerobic bottles). However, P. acnes is of questionable clinical significance. Such a detection system could be used in either a blood collection center or a transfusion service to screen platelet concentrates for bacterial contamination. Such testing (with sterile sampling performed so as to maintain a closed-bag system) would be expected to save lives and might allow an extension of platelet storage.     

Reassessment of the incubation time in a controlled clinical comparison of the BacT/Alert aerobic FAN bottle and standard anaerobic bottle used aerobically for the detection of bloodstream infections

This study assessed the minimum incubation time required to detect bloodstream infections during a controlled clinical comparison of the performance characteristics of the BacT/Alert aerobic FAN bottle and the standard anaerobic bottle used aerobically except on a selective basis. Blood was collected from adults with suspected bloodstream infections and inoculated into each bottle, which was monitored in the BacT/Alert Microbial Detection System. The anaerobic bottle was vented before incubation except when cultures were obtained from patients on the colorectal and gynecologic surgical and emergency services. Statistical analysis was limited to those culture sets in which each bottle was inoculated with ≥8 mL of blood and bacterial growth was considered to be clinically significant. A total of 682 positive cultures from 243 patients satisfied the inclusion criteria. Significantly more isolates of Staphylococcus aureus (p <0.001), S. epidermidis (p <0.001), other coagulase-negative staphylococci (p <0.001), Enterococcus spp. (p = 0.04), Escherichia coli (p = 0.03), all Enterobacteriaceae (p <0.001), Pseudomonas aeruginosa (p = 0.001), and Candida spp. (p <0.001) were detected by the aerobic FAN bottle. Significantly more septic episodes due to S. aureus, S. epidermidis, other coagulase-negative staphylococci, Enterobacteriaceae, P. aeruginosa, and Candida spp. were detected by the aerobic FAN bottle. Significantly more bacterial isolates were detected by the aerobic FAN whether or not antibiotics were being administered at the time of blood culture, whereas there were significantly fewer positive cultures in the vented standard anaerobic bottle when patients were receiving antimicrobial therapy than when they were not. All but 5% of positive cultures were detected within three days. Only six of the cultures requiring four or five days of incubation represented true misses, and only one of these six resulted in a change in therapy which, however, did not affect the patent’s outcome.     

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

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 < 0.05) and Acinetobacter baumannii (14/15 versus 7/15, P < 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 < 0.001).     

The effect of two different lancets and heel warming on duration of crying and procedure time during blood sampling: A randomized controlled study

ObjectiveThe study was performed to investigate the effect of two different lancets and heel warming during blood sampling from the heel on procedure duration and crying.MethodsThis was a randomized controlled trial study. The data were obtained from the Newborn Intensive Care Unit of a hospital in Istanbul between January 2015 and January 2016. One hundred twenty newborns were randomly assigned to four groups — automatic lancet with/without warming and manual lancet with/without warming. The newborns were administered heel puncture for routine blood bilirubin monitoring.ResultThere was no statistically significant difference between the four groups in terms of the characteristics that could affect the outcome of the study. The infants in the manual lancet group without warming were found to have longer duration of crying and longer procedure durations than the other groups. The procedure durations of infants in the manual lancet group with warming were significantly longer than those in the automatic lancet group with warming.ConclusionThis study show that both heel warming and using automatic lancet are effective in reducing the durations of the procedure and crying during blood sampling from the heel.     

Limiting and detecting bacterial contamination of apheresis platelets: inlet-line diversion and increased culture volume improve component safety

BACKGROUND: Septic transfusion reactions to apheresis platelets (PLTs) continue to occur despite preventive measures. This study evaluated the effect of two operational changes designed to reduce bacterial risk: 1) introducing inlet-line sample diversion on two-arm procedures and 2) increasing the sample volume cultured from 4 to 8 mL from all donations.
STUDY DESIGN AND METHODS: Aerobic culture results and septic transfusion reactions reported between December 1, 2006, and July 31, 2008 (Period 2), were compared to March 1, 2004, to May 31, 2006 (Period 1).
RESULTS: During Period 2, a total of 781,936 apheresis PLT collections were cultured, of which 130 donations (1:6015) were confirmed positive and 9 (1:86,882) had negative culture results but were associated with 11 septic reactions. Confirmed-positive cultures from two-arm procedures decreased (27.2 to 14.7 per 10 ⁵ collections; odds ratio [OR], 0.54; 95% confidence interval [CI], 0.41-0.70) in Period 2, owing to a lower rate of skin flora contamination. Detection of contamination of one-arm collections significantly increased by 54% in Period 2 (13.7 vs. 21.1 per 10 ⁵ collections; OR, 1.54; 95% CI, 1.05-2.27). Fewer septic transfusion reactions occurred in Period 2, but the difference did not reach significance (1.7 vs. 1.2 per 10 ⁵ donations; OR, 0.68; 95% CI, 0.30-1.53).
CONCLUSION: Inlet-line diversion decreased bacterial contamination during two-arm collections by more than 46%. Concurrently, doubling the sample volume was associated with a 54% relative increase in culture sensitivity. These interventions act cooperatively to decrease bacterial risk.     

Delayed insertion of blood culture bottles into automated continuously monitoring blood culture systems increases the time from blood sample collection to the detection of microorganisms in bacteremic patients

This study examined the effects on patients with bacteremia of delaying the insertion of a blood culture bottle into an automated, continuously monitoring blood culture system. We investigated the time taken from the collection of blood samples (collection) to the insertion of blood culture bottles inoculated with blood samples into the instrument (insertion), and compared the mean detection time from collection to a positive signal from the instrument with the time between collection and insertion. The study was conducted from January 2003 to December 2004 at Kyoto University Hospital. Insertion into the system on the day of blood sample collection was defined as same-day insertion, and insertion on a different day to collection was defined as delayed insertion. The 7394 aerobic and anaerobic blood culture bottle sets obtained during the study period included 4361 sets with same-day insertion and 3033 sets with delayed insertion. For same-day insertion, 458 microorganisms were isolated from 432 positive sets in which at least one blood culture bottle was positive. For delayed insertion, 405 microorganisms were isolated from 379 positive sets in which at least one blood culture bottle was positive. The mean detection time for all microorganisms was significantly earlier for same-day insertion than for delayed insertion (28.3 h vs. 45.0 h, respectively, P < 0.0001). Delays from collection to insertion affect the time from collection to the detection of microorganisms.     

Improving the performance of culture-based bacterial screening by increasing the sample volume from 4 mL to 8 mL in aerobic culture bottles

BACKGROUND: In the setting of bacterial detection of apheresis platelets (PLTs), the manufacturer recommended PLT inoculation volume for BacT/ALERT culture bottles (bioMérieux) ranges from 4 to 10 mL. This study compares the rate of capture of true‐positive (TP) contaminations between aerobic culture bottles inoculated with either 4 or 8 mL of sample and assesses if a larger sample volume reduces time to detection. STUDY DESIGN AND METHODS: Detection of TP samples and mean time to detection were compared for 4‐ and 8‐mL samples collected between September 1, 2003, and May 2, 2011. RESULTS: A total of 180,263 and 283,114 PLT collections were tested with an 8‐ and 4‐mL sample, respectively. Analysis of TP rates by volume sampled show an increase in the rate of detection of TP with the 8‐mL sample relative to the 4‐mL sample (139 vs. 106 per million events; odds ratio, 1.31; 95% confidence interval, 0.77‐2.23). Comparison of mean time to detection for TP shows a decrease in mean time to detection using 8 mL compared with 4 mL (12.36 ± 3.7 hr to 15.97 ± 6.3 hr, p = 0.012). CONCLUSION: Doubling the sample volume to 8 mL showed a trend in improvement for the rate of detection of TP and shortened the mean time of detection for TP by 23% when compared to a sample volume of 4 mL. The decrease in mean time to detection using a larger sample volume suggests that a shorter release time after inoculation could be achieved without significantly increasing patient risk.