Comparison of the BACTEC 9240 and BacT/Alert Blood Culture Systems for the Evaluation of. Placental Cord Blood for Transfusion in Neonates

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Comparison of the BACTEC 9240 and BacT/Alert Blood Culture Systems for the Evaluation of

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Placental Cord Blood for Transfusion in Neonates

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Stefan Riedel1*, Alan Junkins2, Paul D. Stamper1, Gretchen Cress3, John A. Widness3, and

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Gary V. Doern2

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The Johns Hopkins University, School of Medicine, Department of Pathology, Division of Microbiology,

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Baltimore, Maryland1, and

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University of Iowa Roy J. and Lucille A. Carver College of Medicine,

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Department of Pathology, Division of Microbiology, Iowa City, Iowa2_{, and}

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University of Iowa Roy J. and Lucille A. Carver College of Medicine,

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Department of Pediatrics, Division of Neonatology, Iowa City, Iowa3

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*Corresponding author: Stefan Riedel, M.D., Ph.D.

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The Johns Hopkins University, School of Medicine

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Department of Pathology – Division of Microbiology

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Johns Hopkins Bayview Medical Center

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4940 Eastern Avenue; A Building, Room 102-B

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Baltimore, MD 21224

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Phone: 410-550-6618

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Fax: 410-550-2109

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E-mail: sriedel2@jhmi.edu

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Key words: Umbilical/Placental Cord Blood, Sterility testing, BACTEC, BacT/Alert

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Abstract

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The BACTEC 9240 and the BacT/Alert blood culture systems were compared as a means for detection of

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bacterial contaminants in whole blood, concentrated red cells, and plasma preparations prepared from

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umbilical cord blood samples (UCB). Ninety-two UCB units seeded with low levels of various bacteria were

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evaluated. In more than 50% of cases, growth was not detected in plasma using either system (p<0.001).

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When concentrated red cells and whole blood were compared, the BACTEC system detected bacterial

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growth consistently sooner than the BacT/Alert system in all seeded bacteria except Staphylococcus

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species in whole blood. The median length of time to detection (LTD) for whole blood and concentrated

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cells in BacT/Alert was 18.7hrs and 18.5 hrs, respectively. The median LTD for the same blood fractions

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using the BACTEC system were 16.05hrs and 15.64hrs. These differences in LTD by blood culture system

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and sample type were statistically significant (whole blood: p-value=0.0449; concentrated cells: p-value=

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0.0037). Based on the results of our study, we recommend the use of either concentrated red cells or

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whole blood for sterility testing in umbilical cord blood samples. In our laboratory, the BACTEC system

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compared to the BacT/Alert system was the superior method for rapid detection of bacterial contaminants

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in cord blood.

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Introduction

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While all neonates experience a decline in their circulating red blood cells (RBC) immediately after birth,

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anemia is a more common complication for premature neonates (1, 2). Annually in the United States, an

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estimated 130,000 anemic, critically ill infants receive approximately one million RBC transfusions (3).

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Autologous blood transfusions have been shown to be safe in both adult and pediatric patients (4-6).

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Umbilical/placental cord blood is autologous blood from a neonate (7), and the use of autologous umbilical

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cord blood (UCB) has long been discussed among neonatologists (8-12). Owing to the increasing

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utilization of umbilical cord blood for the transplantation of hematopoietic stem cells, significant progress

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has been made in developing safer and more efficient collection techniques for UCB (13, 14). In neonates,

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bacterial contamination has been described as the third most common cause of transfusion-related

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fatality, with most fatalities occurring in gram-negative sepsis (15). Unfortunately, many cases of

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transfusion-transmitted bacterial infection remain unrecognized and underreported (16-18). While much

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experience exists now regarding the efficacy, recovery, and safety of umbilical cord blood, only few

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studies investigated the prevalence of bacterial contamination of cord blood. These studies report variable

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bacterial contamination rates between 1.85 and 12 percent (11, 14, 19-21). Bacterial contamination

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predominantly consists of organisms known as typical skin contaminants similar to those described in

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adult blood culture collections. Organisms of the vaginal flora have been described as an additional and

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important component of contaminants in UCB. The American Association of Blood Banks (AABB)

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standards require that a small volume of collected umbilical cord blood is used for sterility testing. While

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general regulations exist for the evaluation of safety including bacterial and viral pathogens in adult blood

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and platelet collections as well as human cell therapy products, to our knowledge no specific method

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requirements for the evaluation of bacterial contamination of umbilical cord blood for autologous

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transfusions have been published to date (22, 23). The two most frequently used FDA-approved

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automated, continuous monitoring blood culture systems in the United States are the BacT/Alert system

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(bioMérieux, Durham, NC) and the BACTEC system (BD Microbiology, Sparks, MD). In the current study,

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we investigated the performance of the BACTEC 9240 and BacT/Alert continuous monitoring blood culture

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systems for the detection of “seeded” bacterial contaminants in umbilical cord blood samples compared to

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adult blood collections, with an additional focus on detection of “seeded” bacteria in various fractions of

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cord blood components.

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Materials and Methods

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Between 2007 and 2008, 97 umbilical cord blood samples and 61 adult blood samples were collected at

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the University of Iowa Hospitals and Clinics (Children’s Hospitals and the DeGowin Blood Center), Iowa

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City, IA. Additional cord blood collections from neonates were obtained at the Genesis Medical Center,

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Davenport, IA. The study protocol was approved by the Institutional Review Boards at all participating

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hospitals. The study was conducted in three phases, first evaluating the contamination rate for UCB

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collection, using a specific collection method, and second evaluating the performance of the BACTEC and

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BacT/Alert continuous monitoring blood culture systems for detection of organisms in seeded UCB

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samples. During the final third phase we evaluated both BC systems for detection of organisms in adult

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blood collections as a comparison to UCB samples.

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For placental cord blood collections written informed consent was obtained from a parent prior to

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delivery of the infant. Eligible patients were those born to mothers older than 18 years with delivery

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between 23 and 41 weeks of gestation. Exclusion criteria were clinically suspected and/or laboratory

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confirmed fetal anemia, major congenital malformations, or chorioamnionitis. Because a large number of

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neonates requiring transfusions of various blood products are preterm infants, an equal number of

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term and term infants were enrolled in this study.

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For adult patients older than 18 years, written informed consent was obtained prior to phlebotomy. Eligible

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patients were those with the diagnosis of hemochromatosis (prior confirmation by genetic analysis), who

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were undergoing therapeutic phlebotomy during maintenance phase of their disease. The maintenance

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phase was defined by normal iron status (ferritin < 50g/dl, transferring < 35%) and a greater than 4

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femtoliter decrease in mean erythrocyte corpuscular volume. Patients with diabetes mellitus and/or clinical

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or laboratory evidence of liver disease during the previous 2 years were excluded from the study.

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Collection of umbilical cord blood and adult blood:

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After delivery of the newborn and immediately after delivery of the placenta, the umbilical cord was

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cleansed using a povidone-iodine scrub followed by isopropyl alcohol swab and allowed to dry for 10-15

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seconds before needle puncture. Using a gravity-based method system, placental/umbilical cord blood

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was then collected into 250ml blood collection bags (13). Each collection bag contained 33ml of citrate

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phosphate dextrose (CPD) anticoagulant storage media (Fenwal Single BLOOD-PACK Unit, Lake Zurich,

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IL; product code 4R0837MC). On average, 47ml of cord blood were collected, accounting for total volume

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of 80ml per collection bag. Procedures followed manufacturer’s and published guidelines for cord blood

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banking. Adult blood collections from phlebotomies for hemochromatosis maintenance phase therapeutic

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interventions were collected into CPD containing blood collection bags (63ml CPD per bag), achieving a

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final volume of 420-450 ml of whole blood/CPD per bag. All procedures followed blood donation and

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collection guidelines by the American Association of Blood Banks (24).

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Study Design for Phases 1-3:

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During phase 1, 10 ml of cord blood was collected into Wampole™ Isolator Blood Tube Lysis

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Centrifugation System (Wampole Laboratories, Cranbury, NJ) for 68 consecutive cord blood collections at

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UIHC. Isolator blood tubes were processed according to manufacturer’s guidelines, and the samples were

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plated onto sheep blood agar (SBA), chocolate agar (ChA), and eosin methylene blue agar (EMB). All

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agar plates were examined for bacterial growth at 24, 48, and 72 hours of incubation (35°C, 5% CO2

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atmosphere).

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During Phase 2, 97 UBC collections were collected and subsequently inoculated with 97 recent clinical

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laboratory isolates of various bacteria (Table 1). The bacterial isolates were selected as being

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representative of those bacteria most frequently isolated from clinical cord blood samples (14, 15, 18).

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Suspensions of test organisms approximately equivalent to 102 CFU/ml were prepared in Trypticase soy

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broth. Using a sterile coupling device, an aliquot from the final stock solution was aseptically transferred

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into the blood collection bag, achieving a final organism concentration of less than 10 CFU/ml per blood

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collection bag. This target concentration was verified by culture quantitation of an aliquot from each final

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stock solution. Seeded cord blood preparations were gently agitated for approximately 5 minutes. Using a

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single 20ml syringe, 16ml of cord blood were aseptically withdrawn using a 21-gauge needle.

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milliliter aliquots of this sample were then immediately transferred aseptically into BacT/Alert FA, FAN®

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Aerobic and BACTEC™ Plus Aerobic/F bottles. The remainder of the seeded cord blood sample was then

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centrifuged (1000 x g for 10 minutes) for separation of concentrated erythrocytes and plasma. The

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procedures followed AABB recommendations for processing blood and stem cell component donations.

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The plasma fraction (PF) was aseptically removed using a single 20ml syringe, leaving the concentrated

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erythrocytes fraction (EF) in the original blood collection bag. Using aseptic technique, 8 ml aliquots of

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plasma were transferred into BacT/Alert FA, FAN®_{Aerobic and BACTEC™ Plus Aerobic/F bottles. Finally,}

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corresponding aliquots of concentrated erythrocytes were aseptically removed from the blood bags and

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transferred into the corresponding blood culture bottles. In some cases (n=34), less than 16ml (range 2-14

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ml) of concentrated erythrocytes had remained in the blood collection bag. In these cases, the remaining

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volume was equally split for inoculation into BacT/Alert FA, FAN®_{Aerobic and BACTEC™ Plus Aerobic/F}

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bottles (mean volume: 4ml per BC bottle). All inoculated bottles were immediately placed on their

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respective continuous monitoring instruments and incubated for a period of up to 5 days (120 hours). For

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all samples and specimens, the order of bottle inoculation was random to ensure that each bottle was

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inoculated first approximately the same number of times.

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The time that bottles first registered as being positive was recorded. Subcultures of positive bottles were

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performed to ensure that the organisms that grew were the same as the organisms used to seed the

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respective cord blood samples. The lengths of time in hours to detection (LTD) for each system for all

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tested organisms were compared.

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During Phase 3, a total of 10 adult blood samples were collected and divided into equal aliquots similar

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to the volumes of cord blood samples. This was done to ensure that a corresponding number of different

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bacterial organisms was inoculated matching the corresponding UCB samples. Each adult blood collection

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bag contained 63ml of citrate phosphate dextrose (CPD) anticoagulant storage media to account for an

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equal concentration of citrated when compared to cord blood samples. As described previously for UCB,

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each aliquot of adult blood was inoculated with a microbial suspension of the same selected organisms for

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UCB, achieving a final organism concentration of less than 10 CFU/ml per blood collection bag. After

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gentle agitation, eight milliliters of adult blood were aseptically transferred into BacT/Alert FA, FAN®

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Aerobic and BACTEC™ Plus Aerobic/F bottles and immediately placed on the corresponding continuous

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monitoring system. Bottles were incubated for a period of up to 5 days. LTD was determined as previously

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described for UCB. Plasma and concentrated red cell components were not prepared and tested for adult

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blood samples.

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Statistical Analysis:

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Blood cultures were defined as negative after 120 hours incubation without growth, and with positive

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cultures, length of time to detection (in hours) was used for the analysis. Recovery of the organism

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(growth / no growth) was evaluated using Fisher exact or Chi squared tests. Length of time to detection

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was analyzed using Wilcoxon rank-sum (Mann-Whitney) test. Measures of association and descriptive

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statistics were performed using Stata 9 (Stata Corporation, Texas, USA).

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Results

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During phase 1, 68 isolator blood tubes were collected. 63 did not yield any growth of bacterial organisms

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after 72 hours incubation. Four of five samples with growth had coagulase negative Staphylococci (CNS)

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at day 3 of inoculation, one other sample was positive for CNS at 48 hours. All positive samples

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demonstrated growth of a single colony on a single type media, only. We postulate that these organisms

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represent contaminants in the laboratory rather than being true pathogens present in the cord blood (e.g.

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contamination at the time of collection), since only a one of three media (SBA, ChA, EMB) per sample

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demonstrated growth in each case after at least 48 hours of incubation.

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During phase 2, an analysis for bacterial growth in either blood culture system compared results for the

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three tested compartments (whole blood, concentrated erythrocytes, and plasma) in 92 of 97 cord blood

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samples. In 5 instances comparisons were not possible, due to absence of at least one compartment

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being used for inoculation and testing. These 5 samples were excluded from further analysis. For the

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remaining 92 samples, bacterial growth was detected more often in whole blood and concentrated

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erythrocytes when compared to plasma (Table 1). This was statistically significant using Pearson's

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squared (p value of <0.001 for comparison of whole blood vs. plasma). When compared similarly to whole

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blood, bacterial growth was more often detected in concentrated erythrocytes (p value of <0.05); however,

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this comparison was not as strong as the comparison to plasma.

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Based on these findings, the results for plasma were excluded from further comparison of the BACTEC

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and BacT/Alert systems for evaluation of mean length of time in hours to detection (LTD).

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The difference in LTD in the two blood culture systems for individual organisms is presented in Table 2.

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Overall, a shorter LTD was observed for the BACTEC system compared to the BacT/Alert except with

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Staphylococcus species in Whole Blood; statistical significance was observed. The median and mean LTD

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was more pronounced for detection of growth in concentrated red cells (p value of 0.0037). Although in

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whole blood the mean of the LTD was shorter, this overall mean was heavily influenced by the greater

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mean LTD for Staphylococcus species. By comparing the medians, whole blood LTD was shorter for the

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BACTEC system compared to the BacT/Alert (p value of 0.0449). When stratified by organisms, the

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BACTEC system detected growth of E. coli, enterococci, K. pneumoniae, and P. aeruginosa sooner in

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whole blood and concentrated red cells when compared to the BacT/Alert. Comparison of median LTD

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between the two systems did not show significant differences for C. albicans, coagulase negative

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staphylococci, Group B streptococci, and S. aureus in either whole blood or concentrated red cells. For

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detection of H. influenza, the BACTEC system detected growth in concentrated red cells on average 7.58

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hours sooner than the BacT/Alert system (p value 0.02). Although BACTEC detected growth sooner

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(average of 0.5 hours) in whole blood, the mean difference in LTD for both systems for H. influenzae was

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not statistically significant.

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A total of 61 adult blood and corresponding cord blood samples were analyzed as bacterial growth was

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detected in both blood culture systems for these samples. Growth was consistently detected sooner

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(average 2.18 hours) in cord blood when compared to adult blood. While this observation was made

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independent of the type of blood culture system, the observation was not statistically significant (p=0.68).

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When cord blood and adult blood were compared for each system growth was detected faster in cord

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blood than adult blood (BACTEC 2.72 hrs. and BacT/Alert 1.64 hrs.). These differences, however, were

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not statistically significant (p=0.32 for BACTEC and p=0.74 for BacT/Alert). Therefore, detailed data on

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adult and cord blood comparison are not described further.

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Discussion

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The use of umbilical cord blood as the means of autologous blood transfusions is a novel and emerging

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form of treatment for anemia in critically ill and preterm neonates. To date the use of cord blood is still

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subject to clinical investigations, and universal guidelines for harvesting, processing and utilization have

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not been developed. Current guidelines within the 21 CFR 1271 address sterility testing for cell therapy

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products. UCB is listed in this section; however, the regulations in 21 CFR 1271 do not explicitly require a

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specific method to be used for sterility testing in cell therapy products, incl. UCB (22, 23). The results of

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our study clearly indicate that either whole blood or concentrated erythrocytes should be the preferred

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specimen for detection of bacterial organisms in umbilical cord blood. The plasma fraction appears not to

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be a suitable surrogate medium for sterility testing. These findings are contrary to those described by

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Honohan et.al., who stated that cord blood did not show a preferential location of bacteria after

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centrifugation and prior to processing for transfusion (25). The higher centrifugation speed (1000 x g) used

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in our study compared to the study by Honohan et.al. (50 x g) as expected resulted in organisms to be

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more likely to be concentrated within the red cell fraction rather than within the plasma fraction. Additional

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studies are necessary to further evaluate the effects of centrifugation speed on the concentration of

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bacteria in various fractions of blood. The differences for organism recovery in plasma fraction were most

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striking for coagulase negative Staphylococci, Group B Streptococci, and S. aureus. These organisms

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represent important UCB contaminants and also are significant causes of neonatal bacteremia (15, 16,

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26). In further support of our findings, we identified other studies investigating the utility of umbilical cord

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blood for autologous transfusions in neonates that have shown successful use of either whole blood or

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concentrated red cells for detection of bacterial contamination with adequate organism recovery (8, 17,

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27). The majority of laboratories in the United States use either the BacT/Alert system (bioMérieux,

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Durham, NC) or the BACTEC system (BD Microbiology, Sparks, MD) as their automated continuous

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monitoring blood culture system. Khuu et.al. found that the BACTEC and BacT/Alert automated blood

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culture systems are at least equivalent if not superior to the CFR based culture methods for detection of

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bacterial contaminants in UCB and other human cell therapy products (27). The results of our study

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indicate that when using the LTD as a basis of comparison from seeded samples, the BACTEC system

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was statistically superior to the BacT/Alert system for detection of bacteria in umbilical cord blood. These

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observations are consistent with the findings by other authors who compared the performance of the

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BACTEC and BacT/Alert systems (28, 29). However, it is important to mention that both blood culture

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systems had a less than optimal recovery of GBS and S. aureus in whole blood, 20% and 50%

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respectively; whereas, recovery was much better for these bacteria in concentrated red cells, 30% and

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100% respectively. The initial inoculum concentrations for seeded neonatal cord blood samples were

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rigorously verified by colony counts and had a mean of 1.7 CFU/10µl (range 0 to 7 CFU). We postulate

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that in the effort to seed cord blood samples with a low concentration of organisms, some bottles by

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chance may not have received a large enough viable inoculum size, initially. This finding could be

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attributable to the overall small sample size for GBS and S. aureus in this study. Furthermore, the

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presence of citrate from the CPD preservative used for cord blood banking in our study may have had a

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negative effect on the ability of certain organisms to grow. A low recovery rate for GBS and S. aureus was

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also observed in a study by Smith demonstrating the possible effects of different additives present in the

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blood culture bottles on bacterial growth (29). The bactericidal activity of citrate and other organic acids

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has been previously described by Lee and Richards in two independent studies (30, 31). Additional

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studies with larger sample size examining the effects of such additives are necessary to detect possible

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differences in overall recovery rates and LTD between different blood culture systems for these particular

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organisms in UCB.

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Although not statistically significant, both blood culture systems detected growth faster in cord blood

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than adult blood, and the BACTEC system registered bacterial growth consistently faster in adult blood

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compared to the BacT/Alert system. The lack of statistical significance in observations for adult vs. cord

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blood may be due to the small sample size and the use of a specific adult population (hemochromatosis

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patients) in this study. Additional studies comparing the use of different methods for detection of bacterial

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contaminants in adult and cord blood may be necessary to further evaluate the differences in LTD by

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system and blood component fractions.

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In summary, the most important conclusion from our work is that plasma appears to be a substandard

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specimen for the detection of bacterial contamination of umbilical cord blood intended for transfusion in

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neonates. Either whole blood or concentrated red cells post centrifugation should be the preferred

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specimen type. We believe that centers utilizing umbilical cord blood for autologous transfusion in

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neonates may select and implement a continuously monitoring, automated blood culture system for

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sterility testing, after appropriate on-site validation has been performed. In addition we conclude that in our

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laboratory, the BACTEC system is a better method when compared to the BacT/Alert system for the

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screening of umbilical cord blood units because of more rapid detection of bacteria.

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Acknowledgements

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This work was supported by the United States Public Health Service National Institutes of Health (NIH)

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Grants P01 HL46925 and 5UL1RR024979 (PI: John A. Widness, M.D.) from the National Center for

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Research Resources (NCRR).

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The authors would like to thank Dr. Yasuko Erikson, Dr. Zahi Zeidan, and the CRU nurses Karen Johnson,

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Nancy Krutzfield, Ruthann Schrock, Sara Scott, and Laura Knosp for their assistance in the collection and

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management of the adult blood as well the cord blood samples.

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25. Honohan, A., H. Olthuis, A.T. Bernards, J.M. van Beckhoven, and A. Brand. Microbial

441

contamination of cord blood stem cells. Vox Sanguinis 2002; 82: 32-38

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26. Schelonka, R., L., M.K. Chai, B.A. Yoder, D. Hensley, R.M. Brockett, and D.P. Ascher.

443

Volume of blood required to detect common neonatal pathogens. J Pediatrics 1996;129: 275-278

444

27. Khuu, H.M., N. Patel, C.S. Carter, P.R. Murray, and E.J.Read. Sterility testing of cell therapy

445

products: parallel comparison of automated methods with a CFR-compliant method.

446

Transfusion 2006; 46: 2071-2082

447

28. Riedel, S., G. Siwek, S.E. Beekmann, S.S. Richter, T. Raife, and G.V. Doern. Comparison of the

448

BACTEC 9240 and BacT/Alert blood culture systems for detection of bacterial contamination in

449

platelet concentrates. J Clin Microbiol 2006; 44: 2262-2264

450

29. Smith, J., A., E.A. Bryce, J.H. Ngui-Yen, and F.J. Roberts. Comparison of BACTEC 9240 and

451

BacT/Alert blood culture systems in an adult hospital. J Clin Microbiol 1995; 33: 1905-1908

452

30. Lee, Y.-L., L. Thrupp, J. Owens, T. Cesario, and E. Shanbrom. 2001. Bactericidal activity of

453

citrate against Gram-positive cocci. Letters in Applied Microbiol; 33: 349-351

454

31. Richards, R., M., E., D. K. L. Xing, and T. P. King. 1995. Activity of p-aminobenzoic acid

455

compared with other organic acids against selected bacteria.

456

J Applied Microbiol; 78: 209-215

457

458

459

460

461

462

463

464

(18)

TABLE 1. Number of positive cultures (5 days incubation): Comparing BacT/Alert and BACTEC in

465 Whole Blood, Plasma, in Concentrated Cells.

466

467

468

469 Whole Blood

Plasma

Concentrated Cells

Organism (n)

BacT/Alert

BACTEC

BacT/Alert

BACTEC

BacT/Alert

BACTEC

C. albicans

(10)

10

3

4

10

10 CoNS (11)

8

9

1

4

7

9 E. coli

(10)

10

8

9

10

10 Enterococcus (12)

12

11

12

12 GBS (10)

2

5

1

3

8 H. influenzae

(10)

8

10

4

3

9

8 K. pneumoniae

(9)

9

8

9

9 P. aeruginosa

(10)

10

10 S. aureus

(10)

3

5

2

9

10 Total (92)

72

80

49

53

79

86 Grand Total (184)

152

102

b

165

c

470

a

CoNS, Coagulase Negative

Staphylococcus

species; GBS, Group B Beta-hemolytic

Streptococcus

.

471

b

Total Microorganism Recovery in Whole Blood

vs.

Plasma p-value<0.001 (Chi-squared,

α=0.05 level.)

.

472

c

Total Microorganism Recovery in Whole Blood

vs

. Concentrated Cells p-value

≤

0.05 (Chi-squared,

α=0.05

473

level

).

474

475

476

477

478

479

480

481

482

483

484

485

486

487

488

(19)

TABLE 2. Median and Mean Length of Time to Detection [in hours]: Comparing BacT/Alert and

489 BACTEC in Whole Blood and Concentrated Cells

a

490

491 Median and Mean LTD [h (95% confidence interval)] in:

Whole Blood

Concentrated Cells

BacT/Alert

BACTEC

BacT/Alert

BACTEC

Organism

Median Mean Median Mean

P

value

b

Median Mean Median Mean

P

value

b C. albicans 29.65 30.23 (27.69-32.77) 27.90 28.14 (25.87-30.40) 0.3258 27.35 27.89 (25.79-29.99) 25.83 25.63 (22.04-29.21) 0.1735 CoNS 31.25 32.99 (25.88-40.1) 28.87 36.13 (19.1-53.17) 0.7728 27.30 28.84 (22.29-35.4) 19.20 27.23 (12.29-42.17) 0.1530 E. coli 13.10 13.08 (12.41-13.75) 10.49 10.57 (10.12-11.01) 0.0002 12.00 11.93 (11.64-12.22) 9.87 10.12 (9.61-10.64) 0.0006 Enterococcus 17.90 17.91 (16.05-19.76) 14.96 14.65 (13.84-15.45) 0.0018 16.25 17.67 (14.17-21.16) 14.05 14.25 (13.48-15.03) 0.0039 GBS 14.00 14.0 (0-33.06) 13.52 13.44 (11.25-15.63) 1.0000 14.10 21.13 (0-52.48) 15.01 15.60 (12.58-18.62) 0.6831 H. influenzae 22.55 24.09 (18.21-29.97) 17.53 23.59 (13.19-33.99) 0.1551 22.10 25.0 (18.0-32.0) 16.18 17.42 (14.77-20.07) 0.0208 K. pneumoniae 13.30 13.29 (12.51-14.07) 11.50 11.48 (11.13-11.83) 0.0007 12.60 12.47 (11.96-12.97) 10.90 10.91 (10.57-11.26) 0.0007 P. aeruginosa 18.55 22.07 (16.73-27.41) 16.76 20.21 (15.16-25.26) 0.0493 18.35 21.51 (16.47-26.55) 16.26 20.00 (14.41-25.58) 0.0587 S. aureus 35.30 38.6 (21.07-56.13) 63.16 54.29 (30.45-78.14) 0.1797 23.70 22.51 (19.35-25.68) 20.35 19.55 (16.95-22.16) 0.1331 Total 18.70 22.07 (19.91-24.22) 16.05 22.10 (18.68-25.51) 0.0449 18.50 20.64 (18.85-22.42) 15.64 17.79 (15.85-19.72) 0.0037 a