prepared blood culture bottles is usually at a low oxidation reduction potential, permitting the growth of most facultative and some anaerobic organisms. To encourage the growth of obligate (strict) aerobes, such as yeast and Pseudomonas aeruginosa, transient venting of the bottles with a sterile, cotton-plugged needle may be necessary. Constant agitation of the bottles during the first 24 hours of incubation also enhances the growth of most aerobic bacteria.

Self-Contained Subculture System

A modification of the biphasic blood culture medium is the BD Septi-Chek system (Becton Dickinson Microbiology Systems, Sparks, Maryland) (Figure 1) consisting of a conventional blood culture broth bottle with an attached chamber containing a slide coated with agar or several types of agars. Special media for isolation of fungi and mycobacteria are also available. To subculture, the entire broth contents are allowed to contact the agar surface by inverting the bottle, a simple procedure that does not require opening the bottle or using needles. The large volume of broth sub cultured and the enclosed method provide faster detection for many organisms than is possible with conventional systems. The Septi Chek system appears to enhance the recovery of Streptococcus pneumoniae, but such biphasic systems do not efficiently recover anaerobic isolates.

Fig1. Becton Dickinson Septi-Chek pediatric-size biphasic blood culture bottle. The medium-containing base bottle is inoculated with blood, and the top piece containing agar paddles is added in the laboratory. The agar is inoculated by tipping the bottle to allow the blood-containing medium to flow over the agar.

Lysis Centrifugation

The Isolator (Alere, Waltham, MA) is a lysis centrifugation system commercially available. The Isolator consists of a stoppered tube containing saponin to lyse blood cells and SPS as an anticoagulant (Figure 2). After centrifugation, the supernatant is discarded, the sediment containing the pathogen is vigorously vortexed, and the entire sediment is plated to solid agar. Benefits of this system include rapid and improved recovery of filamentous fungi, the presence of actual colonies for direct identification and susceptibility testing after initial incubation, the ability to quantify the colony-forming units present in the blood, rapid detection of polymicrobial bacteremia, dispensing with the need for a separate antibiotic-removal step, the ability to choose special media for initial culture setup based on clinical impression (e.g., direct plating onto media supportive of Legionella spp. or Mycobacterium spp.), and potential enhanced recovery of intracellular microorganisms caused by lysis of host cells. Possible limitations of the system seem to be a relatively high rate of plate contamination and a decreased ability to detect certain bacteria, such as Streptococcus pneumoniae, Listeria monocytogenes, Haemophilus influenzae, and anaerobic bacteria, compared with conventional systems. If a mixed infection is suspected, an additional blood culture collection tube should be inoculated simultaneously.

Fig2. Lysis centrifugation blood culture (Isolator System, Alere, Waltham, MA) uses vacuum-draw collection tubes with a lysing agent and special apparatus (Isostat Press) to facilitate removal of the supernatant without use of needles.

Instrument-Based Systems

 Conventional blood culture techniques are labor intensive and time consuming. During these times of cost constraints in health care and a corresponding requirement for clinically relevant care, the development of improved instrumentation for blood cultures was needed. Instruments are capable of rapid and accurate detection of organisms in blood specimens. By using newer instrumentation, laboratories processing a large volume of blood cultures can also provide results cost effectively.

BACTEC Systems. Many laboratories use the BACTEC system (Becton Dickinson Microbiology Systems, Sparks, Maryland), which measures the production of carbon dioxide (CO2) by metabolizing organisms. Blood or sterile body fluid for routine culture is inoculated into bottles containing appropriate substrates.

The first BACTEC systems were semiautomated. Vials, containing 14C-labeled substrates (glucose, amino acids, and alcohols) were incubated and often agitated on a rotary shaker. At predetermined time intervals thereafter, the bottles were placed into the monitoring module, where they were automatically moved to a detector. The detector inserted two needles through a rubber septum seal at the top of each bottle and withdrew the accumulated gas above the liquid medium and replaced it with fresh gas of the same mixture (aerobic or anaerobic). Any amount of radiolabeled CO2, the final end product of metabolism of the 14C-labeled substrates (above a preset baseline level), was considered to be suspicious for microbial growth. Microbiologists retrieved suspicious bottles and worked them up (performed sub cultured and identification procedures) for possible microbial growth.

Subsequent modifications further automated the incubation and measuring device, and detection was accomplished by nonradioactive means. The BACTEC blood culture systems are fully automated with the incubator, shaker, and detector all in one instrument. These fully automated blood culture systems use fluorescence to measure CO2 released by organisms; a gas-permeable fluorescent sensor is on the bottom of each vial (Figure 3). As CO2 diffuses into the sensor and dissolves in water present in the sensor matrix, hydrogen (H+) ions are generated. These H+ ions cause a decrease in pH, which, in turn, increases the fluorescent output of the sensor. There is continuous monitoring of each bottle and detection is external to the bottle. Of importance, the noninvasion of the blood culture bottle eliminates the potential for cross-contamination of cultures.

Fig3. A, Blood culture bottles for the BACTEC 9240, 9120, and 9050 continuous monitoring instruments. B, The BD BACTEC FX continuous monitoring blood culture system.

Fig3. cont’d C, Blood culture bottles for the BacT/Alert continuous monitoring blood culture instruments. D, The BacT/Alert continuous monitoring blood culture system. E, Blood culture bottles for Trek Diagnostic Systems, Inc., ESP Culture System II continuous monitoring instrument. (A courtesy Becton Dickinson Microbiology Systems, Sparks, Md; BACTEC is a trademark of Becton Dickinson Microbiology Systems. B courtesy steven D. Dallas, PhD, UT Health Science Center San Antonio, San Antonio, Texas. C courtesy bioMérieux, Durham, NC. D courtesy of Stacie Lansink, Sioux Falls, SD. E courtesy Trek Diagnostic Systems, Inc., Cleveland, Ohio.)

BacT/ALERT Microbial Detection System. Other laboratories use the BacT/Alert System (bioMérieux, Durham, North Carolina), which measures CO2-derived pH changes with a colorimetric sensor in the bottom of each bottle (see Figure 3). The sensor is separated from the broth medium by a membrane permeable to CO2. As organisms grow, they release CO2, which diffuses across the membrane and is dissolved in water present in the matrix of the sensor. As CO2 is dissolved, free hydrogen ions are generated. These free hydrogen ions cause a color change in the sensor (blue to light green to yellow as the pH decreases); a sensor in the instrument reads this color change.

Versa TREK System. The Versa TREK system (Thermo Scientific, TREK Diagnostics, Cleveland, Ohio) utilizes a unique agitation system during blood culture inoculation. The aerobic media bottles each contain a small magnetic stir bar enhancing oxygenation during incubation. Like the other systems, this is also a continuously monitoring instrument. Table 1 summarizes characteristics of some blood culture instruments that are available at the time of printing of the text.

Table1. Summary Characteristics of the More Commonly Used Continuous-Monitoring Blood Culture Systems

Techniques to Detect IV Catheter–Associated Infections

The insertion of an IV catheter during hospitalization is common practice. Infection, either locally at the catheter insertion site or sepsis, caused by bacteremia, is one of the most common complications of catheter placement. Because the skin of all patients is colonized with microorganisms that are also common pathogens in catheters, techniques used to diagnose catheter-related infections attempt to quantitate bacterial growth. Diagnosis of an IV catheter–related bacteremia (or fungemia) is difficult, because there are often no signs of infection at the catheter insertion site and the typical signs and symptoms of sepsis can overlap with other clinical manifestations; even the finding of a positive blood culture does not identify the catheter as the source. To date, various methods, such as semiquantitative cultures, Gram stains of the skin entry site, and culture of IV catheter tips following catheter removal. The terminal end of the IV catheter is removed and rolled several times across a blood agar plate. The tip is then removed from the agar plate and placed in enrichment broth. Both the plate and enrichment broth are incubated at 37° C for 18 to 24 hours. Following inoculation, the blood agar plates are examined, and any isolates are identified according to the laboratory protocol. The enrichment broth may be sub cultured to blood agar and anaerobic media for further analysis and potential detection of intraluminal colonization. Many methods involve some type of quantitation in an attempt to differentiate colonization of the catheter from probable infection. Two major approaches to the diagnosis of catheter-related infection (CRI) in which the catheter remains in place are based on the premise that a greater number of organisms will be present in the intravascular catheter compared to the number found in blood specimens obtained from distant peripheral veins. The first approach, differential quantitative cultures, involves drawing two blood cultures—one from a peripheral site and the other from the suspected infected line. Quantitative cultures are processed for each specimen by inoculating the same volume of blood to standard micro biology media and colonies counted the following day. A colony count ratio greater than 4 to 10 : 1 between the central venous blood and a peripheral blood specimen indicates a probable CRI with a sensitivity of 78% to 94% and a specificity of 99% to 100%. The second approach involves the comparison of the differential time to positivity of blood specimens obtained from a peripheral and intravascular site; a differential time to positivity greater than 2 hours between bottles inoculated with blood from the catheter and those from a peripheral vein indicates a probable CRI. Unfortunately, no single method has demonstrated a clear clinical benefit in diagnosing CRI, and the debate remains unsettled.

Handling Positive Blood Cultures

 Most laboratories use a broth-based automated blood culture method. When a positive culture is indicated according to the automated detection system, a Gram stained smear of an air-dried drop of medium should be performed. Methanol fixation of the smear preserves bacterial and cellular morphology, which may be especially valuable for detecting gram-negative bacteria among red cell debris. Designed to maximize sensitivity, detection algorithms of automated blood culture instruments lead to a certain percentage of false-positive results. Thus, in addition to performance of a Gram stain using methanol fixation, acridine orange (AO) staining is also useful for those blood culture bottles flagged by the instrument as positive but Gram stain-negative for organisms. Adler and colleagues found that AO staining proved particularly helpful in the early detection of candidemia—one third of all microorganisms missed by Gram stain of instrument-positive bottles were yeasts detected by AO staining. As soon as a morphologic description can be tentatively assigned to an organism detected in blood, the physician should be contacted and given all available information. Determining the clinical significance of an isolate is the physician’s responsibility. If no organisms are seen on microscopic examination of a bottle that appears positive, subcultures should be per formed anyway.

Subcultures from blood cultures suspected of being positive, whether proved by microscopic visualization or not, should be made to various media that would support the growth of most bacteria, including anaerobes. Initial subculture may include chocolate agar, 5% sheep blood agar, MacConkey agar (if gram-negative bacteria are seen), and supplemented anaerobic blood agar. In addition, some laboratories are subculturing to specialized chromogenic agar for the isolation of specific pathogenic organisms such as MNSA, yeast (Candida spp.). The incidence of polymicrobial bacteremia or fungemia ranges from 3% to 20% of all positive blood cultures. For this reason, samples must be resubcultured for isolated colonies.

Numerous rapid tests for identification and presump tive antimicrobial susceptibilities can be performed from the broth blood culture if a monomicrobic infection is suspected (based on microscopic evaluation). A suspension of the organism that approximates the turbidity of a 0.5 McFarland standard, obtained directly from the broth or by centrifuging the broth and resuspending the pelleted bacteria, can be used to perform either disk diffusion (qualitative) or broth dilution (quantitative) antimicrobial susceptibility tests. These suspensions may also be used to perform preliminary tests such as coagulase, thermostable nuclease, esculin hydrolysis, bile solubility, antigen detection by fluorescent-antibody stain or agglutination procedures for gram-positive bacteria, oxidase, and commercially available rapid identification kits for gram-negative bacteria. Presumptive results must be verified with conventional procedures using pure cultures. In addition to these approaches, the introduction of a number of molecular methods, including conventional and peptide nucleic acid hybridization assays using specific probes, conventional and real-time polymerase chain reaction assays and microarrays have been used to directly identify microorganisms in blood culture bottles.

In the event of possible future studies (e.g., additional susceptibility testing), all isolates from blood cultures should be stored for a minimum of 6 months by freezing at –70° C in 10% skim milk. A commercial preservation system, Microbank beads, is available for the preservation and storage or bacterial and fungal isolates (Pro-Lab Diagnostics, Austin, Texas). The vials contain pretreated beads and a cryopreservative solution that improves storage of microorganisms. Storing an agar slant of the isolate under sterile mineral oil at room temperature is a good alternative to freezing. It is often necessary to compare separate isolates from the same patient or iso lates of the same species from different patients months after the bacteria were isolated.

Interpretation of Blood Culture Results

 Because of the increasing incidence of blood/vascular infection caused by bacteria normally considered avirulent, indigenous microflora of a healthy human host, interpretation of the significance of growth of such bacteria in blood cultures has become increasingly difficult. On one hand, contaminants may lead to unnecessary antibiotic therapy, additional testing and consultation, and increased length of hospital stay. Costs related to false-positive blood culture results (i.e., contaminants) are associated with 40% higher charges for IV antibiotics and microbiology testing. On the other hand, failure to recognize and appropriately treat indigenous microflora can have dire consequences. Guidelines that can assist in distinguishing probable pathogens from contaminants are as follows:

 • Probable contaminant

• Growth of Bacillus spp., Corynebacterium spp., Propionibacterium acnes, or coagulase-negative staphylococci in one of several cultures

Note: Bacillus anthracis must be ruled out before dismissing Bacillus species as a probable contaminant.

• Growth of multiple organisms from one of several cultures (polymicrobial bacteremia is uncommon)

 • The clinical presentation or course is not consistent with sepsis (physician-based, not laboratory-based criteria)

• The organism causing the infection at a primary site of infection is not the same as that isolated from the blood culture

 • Probable pathogen

• Growth of the same organism in repeated cultures obtained either at different times or from different anatomic sites

• Growth of certain organisms in cultures obtained from patients suspected of endocarditis, such as enterococci, or gram-negative rods in patients with clinical gram-negative sepsis

• Growth of certain organisms such as members of Enterobacteriaceae, Streptococcus pneumoniae, gram negative anaerobes, and Streptococcus pyogenes

• Isolation of commensal microbial flora from blood cultures obtained from patients suspected to be bacteremic (e.g., immunosuppressed patients or those having prosthetic devices)

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مواضيع ذات صلة

Detection of Bacteremia: Types of Blood Culture Bottle

Detection of Bacteremia: Specimen Collection

The Study of the Intestinal Microbiota

Chlamydia psittaci and Psittacosis

Chlamydia Pneumonia and Respiratory Infections

Chlamydia trachomatis Genital Infections and Inclusion Conjunctivitis

Trachoma

Coxiella burnetii

Ehrlichia and Anaplasma

Rickettsia and Orientia

Cultivation of Mycoplasma and Ureaplasma

Laboratory Diagnosis of Mycoplasma and Ureaplasma