CHAPTER 2 Michael L. Wilson1, Melvin P. Weinstein2, and L. Barth Reller3 1 University of Colorado School of Medicine, Aurora, CO, USA 2 College of Medicine and Dentistry of New Jersey, New Brunswick, NJ, USA 3 Duke University School of Medicine, Durham, NC, USA Techniques for detecting pathogenic microorganisms in blood have evolved from manual methods using broth media, once prepared in individual clinical laboratories, to widely available commercial systems and media prepared and sold by manufacturers to hospitals and clinical laboratories. Currently, both manual and automated systems are available, with the market now dominated by instrument-based automated systems and methods. This chapter will emphasize those manual and automated systems and media cleared for diagnostic use in the United States, as well as developments in the use of molecular methods for detecting pathogenic microorganisms directly from blood or in conjunction with traditional blood cultures. Over the past 15 years it has become increasing difficult to perform controlled clinical evaluations or comparisons of blood culture products. Good studies are expensive, require a substantial investment of time and effort (studies may run for a year or more at several institutions), institutional review boards have become increasingly demanding in terms of the requirements for clinical trials of this nature, and relatively few hospitals have the numbers and types of blood culture isolates needed to generate sufficient statistical power for comparisons between products. In addition, there are few investigators with the experience and expertise required to perform these studies. Because of these factors, relatively few such studies have been published since the first edition of this book. The principle of blood cultures is straightforward: a defined volume of blood, obtained by sterile venipuncture, is inoculated into a broth culture medium that will support the growth of most pathogenic bacteria and fungi. Manual methods employ this concept in its most basic form. Although there formerly were many commercial manual blood culture media, there now are relatively few. This is due, in part, to consolidations and mergers among manufacturers, elimination of some systems because of declining demand, and the availability of better evidence demonstrating which systems are acceptable for clinical use. However, broth-based manual blood culture systems, and a manual lysis-centrifugation method, still are marketed and used in the United States and other countries. The SEPTI-CHEK blood culture system (BD Biosciences, Sparks, MD) was developed as an alternative to older, more labor-intensive conventional blood cultures, which, in order to maximize recovery of pathogenic microorganisms, must be subcultured manually after the first overnight incubation and at the end of the incubation period. SEPTI-CHEK consists of a conventional blood culture bottle containing broth medium to which is attached an agar-coated plastic paddle, creating a biphasic system similar to that of the classic Castaneda bottle. Blood is inoculated into the culture bottle through a threaded plastic cap with a rubber diaphragm, which is then removed and replaced with an agar-coated paddle after the blood specimen is received by the laboratory. After the paddle is attached, the bottle is inverted so that the blood–broth mixture floods the paddle, thereby inoculating the agar with microorganisms that may be present. Aerobic and anaerobic bottles, the latter without the agar paddle device (which is permeable to atmospheric oxygen), are then incubated with or without agitation and inspected for evidence of microbial growth once or twice daily. The agar-coated paddle can be removed for better inspection by unscrewing it from the top of its plastic container. Following each examination of the agar paddle, the bottle is inverted, in effect repeating the subculture of the blood–broth mixture to the agar. If growth occurs on the paddles first or, on paddles and in broth at the same time, colonies are available immediately for identification and antimicrobial susceptibility testing. Moreover, preliminary microbiologic information may be available (e.g., lactose-positive or lactose-negative colonies on the MacConkey agar). SEPTI-CHEK bottles contain a broth medium with 0.05% sodium polyanethol sulfonate (SPS) as the anticoagulant. Bottles contain a CO2 atmosphere. Two bottle sizes are available: larger bottles containing 70 mL of liquid medium that accommodate blood specimen volumes of up to 10 mL, and smaller bottles containing 20 mL of liquid medium that accommodate blood specimen volumes of up to 3 mL (the latter being considered pediatric bottles). Several sizes and medium formulations are currently available, including four adult (70 mL) bottles containing soybean-casein digest broth with or without resins, soybean-casein digest/Columbia broth, soybean-casein digest thioglycollate broth, and two pediatric bottles containing either soybean-casein digest broth or brain-heart infusion broth. The paddles contain both nonselective and differential selective agars (chocolate agar, MacConkey agar, and malt agar) in conjunction with traditional blood cultures [19,28]. In a number of controlled clinical evaluations, the SEPTI-CHEK system performed well [2,25,41–43]. As with any other manual blood culture system, the system is adequate for smaller laboratories processing relatively low numbers of blood cultures, as a back up for automated systems, or in circumstances where a number of broth options are desirable. As per the manufacturer, this product has been discontinued but may be available through some distributors. Please check with your local vendors to determine availability. The Oxoid Signal blood culture system (Remel, Lenexa KS), like SEPTI-CHEK, also was developed as an alternative to older conventional blood culture systems. Unlike SEPTI-CHEK, it was developed and marketed as a one-bottle blood culture system. Similar to SEPTI-CHEK, Oxoid bottles are inoculated with blood through a conventional rubber diaphragm. Once in the laboratory, the diaphragm is removed and the plastic signal device is attached to the top of the bottle where it is anchored by a plastic outer sleeve. The device consists of a long needle that goes below the level of the blood–broth mixture and extends up into the clear plastic cylinder of the signal device. When microbial growth occurs in the bottle, gases are released into the bottle headspace, increasing the atmospheric pressure within the bottle and forcing some of the blood–broth mixture up through the needle into the signal device. Thus, in addition to the conventional macroscopic examinations of blood culture bottles (looking for hemolysis, gas formation, colonies growing in liquid media), the Oxoid Signal system offers the potential advantage of easy detection of positive cultures. This is because fluid present in the signal cylinder can be seen without difficulty when blood culture bottles are inspected each day. Positive cultures can then be worked up in the traditional manner. Only one medium formulation of the Oxoid Signal system has been marketed. Published controlled clinical evaluations performed in the United States comparing both manual and automated blood culture systems have been reported previously [23,36,37,39]. An additional manual system currently available in the United States is the Isolator blood culture system (Wampole Laboratories, Cranbury, NJ), sometimes referred to as the “DORN Procedure.” Unlike SEPTI-CHEK and Oxoid Signal, Isolator is based on the principle of lysis-centrifugation, making it the only commercially available blood culture system that does not utilize broth culture medium. Both adult as well as pediatric tubes are available. Blood is inoculated to Isolator tubes (Figure 2.1) that contain a lysing solution consisting of saponin, an anticoagulant, and a fluorocarbon that acts as a cushion during the centrifugation step of blood processing. Once the lysis and centrifugation steps have taken place, the rubber stopper is removed from the Isolator tube and the supernatant is removed by aspiration with a disposable pipette. The pellet is then resuspended and can be inoculated directly to the culture media that will support the various potential pathogens in which detection is desired. The system can be used for detection of routine bacterial pathogens, occasionally for quantitation of bacteria and yeasts, but has been shown, in some studies, to yield decreased recovery of anaerobes, Haemophilus species, and pneumococci if specimens are not processed within 8 h of receipt in the laboratory [12,13,16,35]. Isolator has been shown to be an excellent system for detecting yeasts and dimorphic fungi, mycobacteria, and Bartonella spp. [1]. The system is labor-intensive, however, particularly in the initial processing of specimens. Figure 2.1 ISOSTAT and ISOLATOR microbial tubes. (Courtesy of Alere, Inc.) Commercial automated blood culture systems were first introduced in the early 1970s. The first clinically and commercially successful system was the BACTEC 460 radiometric system (Becton Dickinson, Sparks, MD). This system was succeeded in turn by the BACTEC 660, 730, and 860 nonradiometric systems during the subsequent 20 years; systems that were all based on a similar technology and instrumentation, with each generation representing an incremental improvement over the preceding one. Because they were based on the same method for detection growth of microorganisms, when the continuous-monitoring blood culture systems were introduced, the BACTEC 460, 660, 730, and 860 systems were phased out of use and are no longer available. In these systems, production of CO2 by microorganisms was detected in the bottle headspace by detection of radioactive isotopes of CO2 or by infrared spectrophotometry. Although they are no longer in use, the fundamental principle on which these instruments were based, detection of CO2 production, was the basis for the development of continuous-monitoring blood culture systems. In the early 1990s, the first continuous-monitoring blood culture system, BacT/Alert (bioMérieux, Durham, NC) was introduced [33]; it was followed shortly thereafter by the BACTEC 9000 (BD Biosciences, Sparks, MD) and ESP/VersaTREK (Thermo Scientific, Cleveland, OH) blood culture systems. The continuous-monitoring systems have a number of characteristics in common [49]. The systems are modular, allowing a single computer to coordinate the function of one to as many as 50 incubator units. Blood culture bottles are placed in individual cells of an incubator unit, and testing is performed without further manipulation of the bottle (until a bottle is flagged as positive and removed for Gram stain and subcultures), thereby reducing technologist hands-on time. In each system, blood culture bottles are individually monitored at intervals of 10–24 min for evidence of microbial growth. Individual bottle readings are transmitted to the system’s computer for storage and analysis, and growth curves are calculated according to sophisticated instrument algorithms. Because of the frequent testing that takes place round-the-clock, it is possible to detect microbial growth approximately 1 to 1.5 days earlier than was the case for the BACTEC radiometric and nonradiometric systems [20,24,48]. The more frequent measurements and greater number of data points also seem to be associated with fewer instrument false-positive signals than was the case with the earlier automated systems [48]. Table 2.1 summarizes some of the relevant information on the current generation of commercially available continuous-monitoring systems. Table 2.1 Commercially available continuous monitoring blood-culture systems *O2 detection is for Myco/F Lytic medium only. †Maximum number of bottles accommodated depends on data management system selected. In 1991, the BacT/Alert blood culture system was introduced as the first commercial continuous-monitoring system (Figure 2.2). In 1999, the next generation system, the BacT/Alert 3D, was introduced. Compared with the earlier BacT/Alert systems, the 3D system is more compact and incorporates a touch screen for the instrument’s computer, enabling the technologist to more easily load and unload bottles as well as to perform quality control functions on the instrument. Figure 2.2 BacT/ALERT™ 3D instrument. (Courtesy of bioMérieux.) In the BacT/Alert system, a carbon dioxide sensor is incorporated at the base of each culture bottle, separated from the blood–broth mixture by a CO2 semipermeable membrane that monitors the amount of CO2 in the bottle. The sensor changes color as the concentration of CO2 increases in the bottle [33]. At the base of the cell in which the bottle resides in the incubating unit are light-emitting and light-sensing diodes. When microbial growth occurs, the sensor changes color, and the amount of light reflected from the sensor increases; the increased reflectance is measured by the instrument as increased voltage and the data are transmitted to the central computer. The computer’s detection algorithms have the ability to flag a culture as positive in several ways: (i) by recognition that the arbitrary threshold has been exceeded; (ii) by recognition of a linear increase in CO2; or (iii) by recognition of a change in the rate of CO2 production. Several medium formulations are available for use in the BacT/Alert system. These include standard aerobic and anaerobic media that contain 40 mL of soybean casein digest broth and accept up to 10 mL of blood per culture vial; aerobic and anaerobic FAN media, which contain 40 mL of brain heart infusion broth, activated charcoal, and Fuller’s earth (Ecosorb) designed to inactivate or bind antimicrobial agents in the blood and accept up to 10 mL of blood per culture vial; and the PF Pediatric bottle, which contains 20 mL of soybean casein digest broth supplemented with brain heart infusions solids and activated charcoal, which accepts up to 4 mL of blood. The Pediatric bottle has been promoted for use with pediatric patients and those elderly patients from whom it is difficult to obtain larger volumes of blood. The latter use is to be discouraged, as it fosters inadequate volumes of blood cultures from adult patients. The MB blood bottle for detection of mycobacteria in blood has been introduced. Refer to Chapter 14 (Mycobacteria) for additional information.
Commercial Blood Culture Systems and Methods
2.1 Manual blood cultures
2.1.1 SEPTI-CHEK
2.1.2 Oxoid Signal
2.1.3 Isolator
2.2 Automated blood culture systems
System (manufacturer)
Method(s) for detecting growth
Bottle capacity per module
Maximum number of modules
(number of bottles†) per system
Test cycle (min)
Agitation type (speed [speed/min])
Dimensions (cm)
BacT/Alert 3D (bioMérieux)
CO2 detection, colorimetric
240
6 (2880)
10
Rocking (48)
90 × 49 × 61
BacT/Alert 3D 60
CO2 detection, colorimetric
120
6 (720)
10
Rocking (48)
87 × 87 × 55
BACTEC 9240 (BD Biosciences)
CO2 detection, O2 detection,* fluorescence
240
5 (1200) (core)
20 (4800) (Vision)
50 (12,000) (EpiCenter)
10
Rocking (48)
93 × 128 × 55
BACTEC 9120
CO2 detection, O2 detection,* fluorescence
120
5 (600) (core)
20 (2400) (Vision)
50 (6000) (EpiCenter)
10
Rocking (48)
61 × 129 × 56
BACTEC 9050
CO2 detection, O2 detection,* fluorescence
50
1 (50)
10
Continuous rotation
61 × 72 × 65
VersaTREK 240
(ThermoFisher Scientific)
Manometric, pressure change
240
5 (640)
12 (aerobic) or 24 (anaerobic)
Rotary (aerobic only) (160)
90 × 86 × 65
VersaTREK 528
Manometric, pressure change
256
12 (aerobic) or 24 (anaerobic)
Rotary (aerobic only) (160)
199 × 86 × 65
2.2.1 BacT/Alert
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
