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Chapter 36 - Bacteremia and Sepsis.ppt.pptx
- 1.
- 2. BACTEREMIA: DEFINITIONS ▸ BACTEREMIAis the presence of viable bacteria in the blood, as determined by their growth in a blood culture ▸ PSEUDOBACTEREMIA explains the false positive result that occurs when blood cultures become positive due to contamination during phlebotomy ▸ OCCULT/UNSUSPECTED BACTEREMIA refers to a true-positive blood culture result in the absence of physical signs or symptoms of severe infection ▸ SEPTICEMIA is a term used clinically to indicate bacteremia PLUS the physical signs 2
- 3. Classification of bacteremia ▸Classification by site of origin allows clinicians to determine appropriate therapy and prognosis ○ Primary bacteremia is when bacteria are present in an endovascular source ■ Infected cardiac valve ■ Infected IV catheter ○ Secondary bacteremia is when bacteria come from an infected extravascular source ■ Lungs in patient with pneumonia ○ Bacteremia of unknown origin is when the source of bacteria remains undefined 3
- 4. Classification of bacteremia (con’t) ▸Classification by causative agent offers clues to the underlying source of infection ○ Gram positive bacteria ■ S.pneumoniae ■ S.aureus ■ E.faecium ○ Gram negative bacteria ■ E.coli ■ P.aeruginosa ○ Anaerobic bacteria ■ B.fragilis 4
- 5. Classification of bacteremia (con’t) ▸Classification by place of acquisition may be significant in guiding initial therapy ○ Community acquired bacteremia ■ Occurs in individuals living in the general community ○ Healthcare- associated bacteremia ■ Occurs in patient who are hospitalized or living in a nursing home ■ Defined as bacteremia occurring >72 hours after hospital admission 5
- 6. Classification of bacteremia (con’t) ▸Classification by duration ○ Transient ■ Occurs after a procedural manipulation of a body site colonized by indigenous flora ● Mouth; following dental procedure ● GI tract; following colonoscopy ● Urogenital tract; following cytoscopic procedure ○ Intermittent ■ Can occur due to abscesses somewhere in the body or certain types of infections ● Meningococcemia, gonococcemia or pneumonia ○ Continuous ■ Organisms consistently present in the bloodstream ■ Organisms coming from an intravascular source ● Infective endocarditis is the most common 6
- 7. BACTEREMIA: RISK FACTORS ▸Decreased immune competency ○ Especially in persons with cancers ○ Persons receiving immunosuppressive therapy ▸ Increased use of invasive procedures/indwelling devices ○ Widespread use of semi-permanent vascular catheters ○ Surgery of urinary/GI/biliary tracts disrupts mucosal barriers that block spread of normal flora ▸ Age of the patient ○ Increased incidence of bacteremia in the very young and very old ▸ Antimicrobial resistance ○ Broad-spectrum antimicrobials reduce normal flora and favor colonization/invasion by resistant bacteria 7
- 8. BACTEREMIA: SOURCES ▸ Catheter-relatedbloodstream infections ○ Semi-permanent catheters remain for weeks- months ▸ UTIs ▸ Pneumonias ▸ Intraabdominal infections ▸ Skin infections (bed sores, decubitus ulcers) ▸ Infective endocarditis ▸ Musculoskeletal infections ▸ Central nervous system infections 8
- 9. BACTEREMIA: Pathogenesis ▸ Thepathogenesis of bacteremia depends in part on the ○ Infecting pathogen ○ Portal of entry ○ Immune status of the patient ▸ Bacteremia often occurs because of disruption of normal skin or mucosal barriers, leading to bacterial invasion of the bloodstream. ▸ Patient’s immune system attempts to control infection via ○ Antibodies that opsonize organisms and activate complement-mediated killing by phagocytosis ○ Filtering mechanisms in the body that sequester organisms 9
- 10. BACTEREMIA: CLINICAL SIGNSAND SYMPTOMS ▸ SIGNS AND SYMPTOMS ○ Abrupt onset of chills, fever, or hypothermia, and hypotension ○ Prostration (exhaustion/weakness) and diaphoresis (profuse sweating) ○ Tachypnea (abnormal rapid breathing) ○ Delirium, agitation ○ Nausea, vomiting ▸ LABORATORY VALUES ○ Thrombocytopenia ○ Leukocytosis OR leukopenia ○ Lactic acidosis ○ hypo/hyperglycemia ○ Elevated CRP, haptoglobin, and fibrinogen ○ DIC 10
- 11. Specimen Collection ▸ Positiveblood culture is a critical value ▸ SPECIMEN COLLECTION ○ Aseptic collection is CRITICAL ■ Cleanse skin FIRST with isopropyl alcohol and THEN with chlorhexidine or tincture of iodine ■ Antiseptic should stay on skin for at least 30 seconds ■ Scrub in a concentric fashion ○ 1-3% of blood cultures become contaminated with normal flora ■ Called pseudobacteremia ■ Most common contaminant is CNS, which is also the most common skin flora ○ Ideally collected from venipuncture ■ More risk for contamination when drawing from IV lines ■ If collected from IV line, collect a 2nd set 11
- 12. Specimen Collection (con’t) ▸VOLUME, FREQUENCY AND NUMBER OF BLOOD CULTURES ○ Best practice for timing and volume that yields highest percentage of positive cultures ■ At least 60 mL of blood collected in a 24 hour period, with 10 mL in each bottle ■ 3 sets of 2 bottles each (aerobic, anaerobic) ○ VOLUME: ideal ratio of blood: broth is 1:5 to 1:10 ■ Bottles commonly underfilled ■ Collecting too much blood can induce anemia ○ FREQUENCY: 2-3 SETS within a 24 hour period ■ Multiple sets help discern contamination from true pathogen ○ TIMING: when experiencing a temperature spike ■ If transient/intermittent bacteremia suspected ■ Otherwise, organisms in bloodstream continuously and will be recovered whenever cultures are collected 12
- 13. Specimen Collection (con’t) ▸BLOOD CULTURE BOTTLES ○ Traditional set ■ Aerobic bottle will recover aerobes, facultative aerobes and aerotolerant anaerobes ● Contain media enriched with broth ■ Anaerobic bottle will recover anaerobes and facultative anaerobes ■ Historically, antibiotic removal device (ARD) neutralizes antimicrobials for patients currently on antibiotics 13
- 14. Specimen Collection (con’t) ▸ANTICOAGULANTS ○ It is critical that blood NOT be allowed to clot ■ Sodium polyanetholsulfonate (SPS) is a common additive ● Acts as an anticoagulant ● Neutralizes bactericidal activity of human serum ● Prevents phagocytosis ● Inactivates certain antimicrobials ■ Sodium amylosulfate (SAS) similar to SPS ● Less effective at neutralizing human serum ● Inhibitory to K.pneumoniae ■ Sodium citrate ● Can be inhibitory to some GPC 14
- 15. Manual blood cultureMethods ▸ MANUAL SYSTEMS ○ Septi-Chek, broth-slide system ■ Slide paddle with agar ■ Bottles are inoculated and incubated ■ Bottles tipped daily ■ Growth on paddle agar is indicator ○ Oxoid Signal system ■ Blood bottle with liquid medium and a clear device attached to the top ■ As organisms grow, CO2 builds up in headspace ■ Increased pressure pushes liquid medium and blood up through a needle into the clear signal device ■ Presence of fluid during daily inspection is indicator ■ Fluid then removed for gram stain and culture 15
- 16. Manual blood cultureMethods (con’t) ▸ Isolator, lysis-centrifugation method ○ Isolator tube contains additives that cause the lysis of WBCs and RBCs, prevents clotting, and neutralizes C’ ○ Organisms concentrated through high-speed centrifugation ○ Sediment then inoculated onto media 16
- 17. CONTINUOUS MONITORING BLOODCULTURE SYSTEMS ▸ BACTEC ○ Fluorescence to detect CO2 17
- 18. CONTINUOUS MONITORING BLOODCULTURE SYSTEMS (con’t) ▸ VersaTREK ○ Detects consumption/production of gases (CO2, H2, O2) by monitoring the change in headspace pressure 18
- 19. CONTINUOUS MONITORING BLOODCULTURE SYSTEMS (con’t) ▸ BacT Alert ○ pH sensitive membranes in bottom of bottle ○ Microbial growth releases CO2 and changes the color of the membrane ○ CO2 production measured colorimetrically 19
- 20. BLOOD CULTURE WORKUP ▸BLOOD CULTURE WORKUP ○ INCUBATION TIMES ■ Routine 5-7 days ■ Endocarditis 2 weeks ■ Brucella/Fungi/HACEK 3-4 weeks ○ REPORTING ■ Initial report at 24 hours ● “No growth Day 1” or “No growth at 24 hours” ■ Blood culture updated daily ● “No growth Day 2, etc.” ■ Final report sent out at 5-7 days ● “FINAL REPORT: No growth Day 5” 20 ○ POSITIVE CULTURES ■ Gram stain bottle, call results ■ Subculture to media ■ ID/AST ○ POTENTIAL PATHOGENS ■ S.aureus ■ S.pneumoniae ■ H.influenzae ■ Pseudomonas spp. ■ Group B strep ■ CNS ■ GNRs ■ Yeast ○ CONTAMINANTS ■ CNS ■ Bacillus spp. ■ diphtheroids
- 21. RAPID ID METHODS ▸Fluorescence in-situ hybridization (FISH) ■ PNA FISH ● Peptide nucleic acid probe ● No amplification ● ID in 1.5-3 hours 21 ■ Verigene ● Amplifies nucleic acid target ● Hybridize with oligonucleoti ● ID in 2.5 hours
- 22. RAPID ID METHODS(con’t) ▸ MALDI-TOF: ○ Matrix-Assisted Laser Desorption/ Ionization- Time-of-Flight Mass Spectrometry ▸ Identifies a wide variety of organisms in a matter of minutes including: ▸ Fungi, fastidious bacteria, and anaerobes ▸ organisms must first be subcultured from the blood and isolated in pure culture ▸ Direct detection from blood is not yet FDA approved. ▸ There is no way to determine antimicrobial susceptibility by using this method. 22
- 23. TREATMENT AND PREVENTION ▸TREATMENT ○ Antimicrobial therapy ■ Initially, broad spectrum antibiotic ■ After ID/AST, therapy is targeted ○ Antisepsis therapy ■ Anticoagulants to prevent activation of coagulation cascade ■ Glucocorticoids to treat adrenal insufficiency ▸ PREVENTION ○ Immunizations ■ Pneumococcal vaccine-prevent S.pneumoniae ■ Hib vaccine-prevents H.influenzae ■ Influenza/varicella vaccine-prevent secondary bacteremias ○ Healthcare associated bacteremias ■ Prevent iatrogenic infections from 23
- 24. REFERENCES 24 • Broyles, M.(2013, June). A Closer Look at Sepsis. ADVANCE for Medical Laboratory Professionals, 25(5), 12-13. • http://www.achats-publics.fr/Fournisseurs/BIOMERIEUX.htm http://www.bd.com/ds/productCenter/212536.asp • http://www.bd.com/ds/productCenter/445718.asp • http://www.temple.edu/medicine/microbiology_lab.htm • Kiser, K. M., Payne, W. C., & Taff, T. A. (2011). Clinical Laboratory Microbiology: A Practical Approach . Upper Saddle River, NJ: Pearson Education. • Mahon, C. R., Lehman, D. C., & Manuselis, G. (2014). Textbook of Diagnostic Microbiology (5th ed.). Maryland Heights, MO: Saunders.
Editor's Notes
- #1 For this unit, we will review general concepts related to bacteremic infections including definitions of conditions related to bacteremia, how conditions manifest, epidemiologic risk factors and pathogenesis, diagnostic lab procedures, and treatment. Bacteremia currently occurs in more than 250,000 hospitalized patients and was the 11th leading cause of death overall in the U.S in 2019.
- #2 Read the definitions.
- #3 Bacteremia may be classified by its site of origin. Primary bacteremia occurs when the bacteria are present in an endovascular source such as an infected cardiac valve or an infected intravenous (IV) catheter, whereas secondary bacteremia occurs when the bacteria come from an infected extravascular source, such as lungs in patients with pneumonia. A case in which the source of bacteremia remains undefined is termed bacteremia of unknown origin. Classification in this manner has important clinical consequences because it determines the appropriate therapy and prognosis. For example, a secondary bacteremia from an infected focus, such as an abscess, may require surgical therapy to remove the abscess or source of infection, in addition to antimicrobials to eliminate the infection. Bacteremia of unknown origin generally has a poorer prognosis than primary or secondary bacteremia.
- #4 Bacteremia may also be categorized by the general class of microorganism or specific pathogen that has invaded the bloodstream. Gram-positive bacteremia is caused by such organisms as S. pneumoniae, Staphylococcus aureus, or Enterococcus faecium, whereas gram-negative bacteremia is caused by such organisms as Escherichia coli or Pseudomonas aeruginosa. Anaerobic bacteremia is caused by such organisms as Bacteroides fragilis, whereas polymicrobial bacteremia is caused by a mixture of organisms. General classification of bacteremia in this fashion can provide initial clues to the underlying source of a bacteremia and guide therapy, even before organisms have been identified.
- #5 Bacteremia can also be categorized by its place of acquisition. Community-acquired bacteremia, as the term suggests, occurs in individuals living in the general community, whereas healthcare associated occurs in patients who are hospitalized or living in a nursing home or other health care facility. To avoid misclassification of bacteremia that began at the time of hospital admission as nosocomial when it is, in fact, community acquired, nosocomial bacteremia is conventionally defined as any bacteremia occurring more than 72 hours after hospital admission. Certain bacteremias are more often community acquired. For example, more than 90% of cases of S. pneumoniae bacteremia are acquired in the community. Others, such as those caused by P. aeruginosa or Enterococcus spp., are more likely to be nosocomial. The place of acquisition may thus be extremely significant in guiding initial therapy. For example, nosocomial bacteremia is more likely to be caused by drug-resistant organisms that express β-lactamases or other resistance factors that inactivate first-line antimicrobial agents, although this distinction is currently blurred in the case of hospital-acquired and community-acquired methicillin-resistant Staphylococcus aureus (MRSA).
- #6 Bacteremia may also be classified by the duration of a bacteremic episode. Bacteremic episodes may be transient, intermittent, or continuous. The frequency, time, and number of blood cultures to be collected may depend on the type of bacteremic episode that the patient is experiencing. Transient bacteremia usually occurs after a procedural manipulation of a specific body site colonized by indigenous microbiota, causing the organisms to enter blood. Such sites include the mouth and the GI and urogenital tracts. Transient bacteremia may appear for a brief period following a dental, colonoscopic, or cystoscopic procedure. The organisms involved are normally rapidly cleared by the host immune defense, so their presence is rarely symptomatic. Intermittent bacteremia can occur because of the presence of abscesses somewhere in the body or as a clinical manifestation of certain types of infections, such as meningococcemia, gonococcemia, or pneumonia. In intermittent bacteremia, organisms are periodically released from the primary site of infection into blood. Continuous bacteremia occurs when the organisms are coming from an intravascular source and are consistently present in the bloodstream. Infective endocarditis is the most common clinical manifestation associated with continuous bacteremia, although other endovascular sources, such as infected intravascular catheters or septic thrombi, can also result in continuous bacteremia.
- #7 The increased incidence of bacteremia in the United States during the past 30 years is likely a result of the following factors, some of which have changed dramatically over this period: Decreased immune competency of selected patient populations: Bacteremias are more frequent among persons with hematologic malignancies, those receiving immunosuppressive chemotherapy, and those undergoing bone marrow transplantations. Persons with other chronic underlying diseases (e.g., diabetes, cirrhosis) and those receiving immunosuppressive therapy (e.g., those receiving glucocorticoids for rheumatoid arthritis, stem cell or solid organ transplant recipients) are also at increased risk for bacteremia. Infection with human immunodeficiency virus (HIV) predisposes patients to increased risk of bacteremias because of the immunosuppression caused by the virus. Increased use of invasive procedures: The increased use of indwelling devices, respirators, and invasive diagnostic procedures may be a factor in the occurrence of bacteremia. The widespread use of semi-permanent vascular catheters, to administer chemotherapy to patients with cancer or to provide vascular access for hemodialysis in patients with end-stage renal disease, increases the risk of bacteremia by breaking the normal integrity of the skin and permitting colonization of a foreign body in direct contact with the bloodstream. Surgery involving the urinary, GI, and biliary tracts may also result in bacteremia because of the disruption of mucosal barriers that normally function to block the spread of resident microbiota. Age of the patient: Bacteremias are more prevalent in people at the extremes of age, with infants, young children, and adults older than 55 years being most susceptible. The presence of comorbid conditions, such as diabetes, hypertension, chronic obstructive pulmonary disease, and congestive heart failure in older adults, and neoplastic disorders, HIV infection, and low and very low birth weights in neonates and infants, significantly increases the incidence of bacteremia in these groups. Antimicrobial resistance: The indiscriminate administration of broad-spectrum antimicrobials reduces susceptible normal microbiota and favors colonization and invasion by resistant bacteria. An increasing population of antimicrobial-resistant organisms results in bacteremias that are harder to treat, leading to increased morbidity and mortality.
- #8 Numerous sources can give rise to bacteremia. The most common sites associated with bacteremia and sepsis are infected intravascular catheters, urinary tract, lung, and abdomen. Semi-permanent catheters (Hickman, Broviac, Quinton, or Tenckhoff catheters, so named after their developers) are placed in central veins and remain in place for weeks or months to administer chemotherapy and/or parenteral nutrition to patients with cancer or to perform hemodialysis in patients with end-stage renal disease. Still other catheters are placed in arteries or, via central veins, the pulmonary artery or right-sided chambers of the heart, where they may be left for days for hemodynamic monitoring. Infection of the upper urinary tract (acute pyelonephritis) leads to bacteremia in as many as 40% of affected patients. Common organisms in cases of pneumonia that produce a concurrent bacteremia include S. pneumoniae, S. aureus, P. aeruginosa, and Klebsiella/Enterobacter spp. Primary peritonitis, which frequently occurs in patients with cirrhosis, is associated with bacteremia in 75% of cases involving aerobic bacteria. Common pathogens include E. coli, K. pneumoniae, and enterococci. Cellulitis caused by S. aureus, Streptococcus pyogenes, or Streptococcus agalactiae can lead to bacteremia. Skin breakdown in bedridden patients (bed sores) or peripheral vascular disease from diabetes is a common cause of infected skin ulcers, which can provide a portal of entry for bacterial invasion of the bloodstream, often resulting in polymicrobial bacteremia. Transient bacteremia (from dental procedures or a superficial skin infection) can seed cardiac valves with bacteria. Acute osteomyelitis is often associated with transient bacteremia caused by S. aureus; frequently the portal of entry is an otherwise unnoticed minor skin infection. The organisms seed end loop capillaries in bone, where blood flow is slow, and begin to multiply, causing destruction of bone and giving rise to intermittent bacteremia in about 50% of cases. Acute bacterial meningitis is generally the result of transient bacteremia caused by S. pneumoniae or Neisseria meningitidis.
- #9 The pathogenesis of bacteremia depends in part on the Infecting pathogen, portal of entry, and immune status of the patient. Bacteremia often occurs because of disruption of normal skin or mucosal barriers, leading to bacterial invasion of the bloodstream. Once bacteremia occurs, the patient’s immune system attempts to control infection via antibodies that opsonize organisms and activate complement-mediated killing by phagocytosis and filtering mechanisms in the body, e.g., spleen, that may sequester organisms and facilitate their destruction by phagocytic cells
- #10 The classic signs and symptoms of bacteremia include abrupt onset of shaking chills, fever, or hypothermia. About 14% of patients with bacteremia will have hypotension, and about 40% of patients experience prostration and diaphoresis (profuse sweating). Tachypnea (abnormal rapid breathing) is an early sign of bacteremia, and adult respiratory distress syndrome occurs in 18% of patients with culture-positive septic shock. Other symptoms may include delirium, stupor, or agitation (evidence of decreased central nervous system perfusion), along with nausea and vomiting. As many as 38% of patients with bacteremia and sepsis will have acute renal failure, with oliguria or anuria. Ecthyma gangrenosum, a central necrotic area surrounded by an erythematous base, is typically associated with Pseudomonas bacteremia. The failure of the body to mount an elevated temperature is associated with increased mortality in newborns and older adults. Clinical conditions with altered laboratory values that may be indicative of bacteremia include the following: • Thrombocytopenia • Leukocytosis or leukopenia • Lactic acidosis • Hypoglycemia or hyperglycemia • Abnormal liver function test results (especially hyperbilirubinemia) • Coagulopathy • DIC • Elevations in C-reactive protein (CRP), haptoglobin, and fibrinogen levels
- #11 Because of the potential of a serious negative outcome of a septicemia or bacteremia, a blood culture is one of the most important cultures to obtain rapid accurate results. As such, a positive blood culture is a critical value. Critical values are those laboratory test results that are outside the reference range and indicate a potentially fatal outcome. The primary care provider needs to be notified immediately of all critical values to provide immediate treatment. In the case of a positive blood culture, the Gram stain result should be called for immediately. It is important to remember that even though antiseptic technique is used in the collection of blood, somewhere between 1% and 3% of blood cultures become contaminated with such organisms as CoNS, resulting in pseudobacteremia. Blood for blood culture should be obtained by venipuncture and not from indwelling IV or intraarterial lines. There is more risk of isolating skin biota from indwelling lines rather than via venipuncture. If blood is collected from an indwelling line, a second sample collected via venipuncture should be cultured for comparison. For patients who have IV lines through which they are getting fluids and/or medications, blood must be drawn below the line because blood drawn above the line will be diluted with the fluid being transfused. The best practice, however, is not to draw blood from the extremity that has an IV line but to perform venipuncture on an extremity that does not have an indwelling line.
- #12 One study clearly demonstrated the advantage of culturing 10 mL of blood per blood culture bottle compared with 5 mL of blood per bottle, finding that 7.2% more cases of bacteremias were detected in the bottle inoculated with 10 mL of blood and that the organisms were detected sooner than in the bottles inoculated with only 5 mL of blood. Unfortunately, a number of studies have found that it is common practice to inoculate blood culture bottles with less than the recommended volumes of blood. If inoculating more blood into the blood culture bottle increases the sensitivity and rate of detection, why not inoculate even more blood than recommended into each bottle? Taking excessive amounts of blood from a patient can induce anemia in the patient, and according to at least one study, inoculating more than 10 mL per bottle did not increase the rate of positivity and decreased it instead. According to several recent studies determining best practices for timing and blood volume collection that yield the highest percentage of positive cultures, at least 60 mL of blood should be collected in a 24-hour period, with 10 mL of blood inoculated into each bottle; three sets of two bottles each will satisfy the volume requirement and will likely result in the detection of bacteremia, if present. Another reason for inoculating multiple bottles with blood collected from multiple separate venipunctures is to aid in the determination of whether an isolated organism is a true pathogen or a contaminant. The collection of one single sample should be strongly discouraged because the volume of blood cultured is not sufficient for detecting some infections, as noted.
- #13 As noted, a blood culture set typically includes a bottle designed for recovery of aerobic microorganisms and another bottle for recovery of anaerobic microorganisms. The typical aerobic culture bottle contains a medium that is nutritionally enriched, such as soybean casein digest broth, peptone broth, tryptic or trypticase soy broth, brain-heart infusion broth, Brucella broth, or Columbia broth base. Although anaerobic broth may contain the same types of basic media as the aerobic culture systems, 0.5% cysteine may be added to permit the growth of certain thiol-requiring organisms, and the media may be prereduced to decrease the oxidation-reduction potential to help support the growth of anaerobes. Newer anaerobic broth media (e.g., F/X; Becton, Dickinson, Sparks, MD) contain blood-lysing agents that reduce the time to detection of anaerobes by lysing red blood cells (RBCs), which provides added nutrients, and WBCs, which releases phagocytized organisms. Unvented blood culture bottles generally can be used to support anaerobic organisms. In specimens from patients receiving of β-lactam antimicrobial agents, penicillinase may be added to the medium to inactivate these agents, although this is rarely done today. More commonly, many commercially available automated blood culture systems have blood culture bottles containing an antimicrobial removal device (ARD), a resin that nonspecifically absorbs any antimicrobial agent present in the patient's blood, whereas other systems incorporate activated charcoal for this purpose. The yield of bacteria and yeasts increases with the incorporation of these inhibitors into the culture medium.
- #14 When blood is collected for culture, it is critical that the blood not be allowed to clot. The formation of a clot will trap the bacteria and reduce the ability to detect them. Thus blood should be inoculated directly into blood culture bottles or, if necessary, into a tube containing anticoagulants. Tubes containing heparin, ethylenediaminetetraacetic acid (EDTA), and sodium citrate have been shown to inhibit the growth of many different organisms and should not be used. Tubes containing 0.025% to 0.050% sodium polyanetholsulfonate (SPS) are better tubes to use for collecting blood for culture, although SPS can inhibit a few organisms as well, notably Peptostreptococcus anaerobius and some strains of Neisseria spp. and Streptobacillus moniliformis. Intermediate collection tubes are therefore not recommended. SPS, one of the commonly used additives, performs the following functions: • Anticoagulation (effective at a 0.03% concentration) • Neutralization of the bactericidal activity (i.e., complement and lysozyme) of human serum • Prevention of phagocytosis • Inactivation of certain antimicrobial agents (e.g., streptomycin, kanamycin, gentamicin, polymyxin B) Despite its usefulness in blood culture media, SPS inhibits the growth of certain organisms, notably P. anaerobius, N. gonorrhoeae, N. meningitidis, and Gardnerella vaginalis. If these organisms are suspected, 1.2% gelatin added to the blood culture bottle may help neutralize the inhibitory effect of SPS.
- #15 Although only three manual blood culture systems are currently marketed and sporadically used in the United States, they are easy to use, inexpensive, and generally adequate for the detection of common bacteria and fungi. Septi-Chek consists of a slide paddle containing chocolate (CHOC), MacConkey (MAC), and malt extract agars (selective for yeast and fungi) attached to the top of a standard broth bottle. Once these bottles have been inoculated, they should be tipped daily or at least twice weekly to bathe the slide paddle with the broth culture medium, thereby allowing frequent blind subcultures without the use of needles and syringes. Most organisms will grow within 48 hours of inoculation, but the bottles are incubated for 7 days before they are discarded and reported as negative. Disadvantages include a slightly higher cost of materials and contamination rate, and they are labor-intensive. An additional unvented bottle is still required for adequate isolation of anaerobes. The Oxoid Signal system (Thermo Fisher Scientific, Waltham, MA) is a manual blood culture system in which blood is inoculated into a bottle containing a liquid medium that will support the growth of aerobes, anaerobes, and microaerophiles. A clear plastic signal device is then attached to the top of the bottle. The Signal device has a long needle that extends down into the bottle below the level of the liquid. When microorganisms grow in the bottle, they generate CO2, which accumulates in the head space of the bottle. The increase of gas in the atmosphere of the bottle increases pressure on the liquid, forcing it up through the needle and into the clear plastic Signal device. The presence of fluid in the Signal device can be seen by the microbiologist during daily inspection of the bottles and indicates the growth of bacteria. The fluid from the Signal device can then be removed for Gram staining and plating. The inoculated Oxoid bottles are incubated at 35° ± 2° C, agitated on a shaker, for the first 24 hours. Bottles are held for a total of 7 days, with a terminal blind subculture performed and examined before the culture is reported as negative.
- #16 The lysis centrifugation method (Isolator/Isostat; Wampole, Inverness Medical Professional Diagnostics, Princeton, NJ) has been shown to provide optimal recovery of unusual fastidious bacteria such as Bartonella, yeasts, filamentous and dimorphic fungi, and mycobacteria that are causing systemic infections. This method produces a concentrated sample of blood for direct inoculation onto appropriate solid agar media, as opposed to the two systems described earlier, in which blood is inoculated and incubated in a broth medium. The Isolator tube contains a mixture of saponin, propylene glycol, SPS, and EDTA. This mixture causes lysis of WBCs and RBCs, releasing intracellular organisms, prevents clotting, and neutralizes complement. Microorganisms are concentrated through high-speed centrifugation (3000g for 30 minutes). The sediment containing the organisms is directly inoculated onto a solid culture medium that includes fungal and mycobacterial media.
- #17 Bactec is a noninvasive, continuous-monitoring blood culture instruments that uses fluorescence to detect CO2. When microorganisms grow in the bottle, the CO2 that they produce is detected by a gas-permeable sensor on the bottom of each vial. When a bottle is placed into the instrument, a baseline reading of the sensor is taken; this reading is used as a reference for subsequent readings. Carbon dioxide produced by an organism diffuses into the sensor, generating hydrogen ions. The increase in hydrogen ion concentration increases the fluorescence output of the sensor. Using photodetectors, the instrument measures the amount of fluorescence every 10 minutes, which corresponds to the amount of CO2 produced by the microorganism. A computer program then interprets these data using several algorithms to determine when to flag a bottle as positive. The instrument alerts the microbiologist to the presence of a positive vial by displaying a message on the computer monitor and with an audible alarm. The picture in the top right corner shows the BACTEC 9000 schematic.
- #18 VersaTREK differs from the other continuous-monitoring systems in that it detects the consumption or production of multiple gases (CO2, hydrogen [H2], and O2) by organisms growing in the culture medium. These gases are detected by monitoring changes in head space pressure. The advantages to this system include the detection of different gases that may be produced or consumed by an organism present in the bottle, not just production of CO2, and earlier detection of growth by detecting the consumption of gas as organisms enter the log phase of growth, before they start to produce CO2. An internal computer algorithm monitors the changes in pressure, plots the pressure against time to derive a growth curve, and determines when to flag the bottle as positive.
- #19 A fully automated, nonradiometric blood culture system, the BacT/ALERT 3D system, consists of aerobic and anaerobic bottles with pH-sensitive membranes placed in the bottom of the bottles. Microbial growth causes a release of CO2, which changes the pH in the sensor, as indicated by a change in color from gray to yellow. The color change is measured by reflected light. The instrument measures CO2 production colorimetrically without entering the bottles. An advantage of this system is that the changes in the color of the sensor can be detected and verified, if necessary. Another advantage of this system is that in 2003, bioMérieux (Durham, NC) released gas-impermeable plastic blood culture bottles that are safer and lighter than traditional glass bottles and do not interfere with microorganism growth or metabolism.
- #20 Routine blood cultures are usually held for 5-7 days depending on the facility. If the physician suspects endocarditis, cultures should be held for two weeks to along for group. Some organisms, such as Brucella, fungi, and HACEK, should be held 3-4 weeks to along for growth of slow growing organisms. If blood culture bottles do not have growth at 24 hours, an initial report of: “no growth at 24 hours” is posted on the patient chart. Preliminary reports are updated on a daily basis if no growth of organisms is seen. If by day(s) 5-7, no growth is still the status, a final report should be posted of “No growth day 5”. At the point where a bottle is marked positive by the instrument, the technician should gram stain the bottle, then call the results to the practitioner. The bottles should them be subbed to plate media. The plates inoculated will depend on the bottle which is positive and the type of organism(s) growing in the bottle. From this point, identification of the organism will be performed. Potential pathogens include S. aureus, S. pneumoniae, H. influenzae, and many more. Contaminants include coagulase negative staph, Bacillus spp., and diptheroids.
- #21 Fluorescence in situ hybridization targets ribosomal ribonucleic acid (rRNA) in an organism by using an oligonucleotide or peptide nucleic acid (PNA) probe with a fluorescent label. Although this is a molecular method, there is no amplification of nucleic acid involved. The procedure takes 1.5 to 3 hours to perform; the sensitivity of species-specific probes in one study was reported to be 97%, with 95% specificity. Different probes are available that target common organisms isolated from blood cultures, such as S. aureus, Enterococcus spp., and Candida spp. The Verigene system (Nanosphere, Northbrook, IL) amplifies nucleic acid targets that are then detected by hybridization of oligonucleotides bound to nanosphere particles with a silver staining process.
- #22 MALDI-TOF, which stands for Matrix-Assisted Laser Desorption/ Ionization-Time-of-Flight Mass Spectrometry identifies a wide variety of organisms in a matter of minute. Some of the organisms it can identify include fungi, fastidious bacteria, and anaerobes. Organisms must first be subcultured from the blood and isolated in pure culture. Direct detection from blood is not yet FDA approved. Currently, there is no way to determine antimicrobial susceptibility by using this method.
- #23 Sepsis being such a complex disorder, it is expected that many therapeutic options are needed to alleviate this devastating clinical event. Several treatment modalities have been tried, but some are not always successful. Antimicrobial agents remain the mainstay of treatment of bacteremia. Because of the potentially devastating consequences of untreated bacteremia, therapy is frequently instituted empirically on the basis of clinical signs and symptoms, before bacteremia has been confirmed by a positive blood culture or before the causative agent has been definitively identified. Broad-spectrum antimicrobial agents are frequently used for initial empiric therapy, and a combination of agents may be used to ensure coverage of several possible pathogens. Because of the high mortality of septic shock, whether or not accompanied by bacteremia, a number of therapies aimed at blocking the cascade of events that result in sepsis, shock, and death have been studied and are being evaluated. These therapies, described here, are invariably used in combination with antimicrobial agents. Unfortunately, even with treatment, 30% to 50% of patients with sepsis die, usually because of underlying illnesses in addition to their sepsis. Prevention of community-acquired bacteremias has been aided by immunizations. Pneumococcal and Hib vaccination have been effective in preventing invasive infections caused by S. pneumoniae and H. influenzae, respectively, and vaccination for viral pathogens (e.g., influenza virus, varicella virus) has helped lower the incidence of invasive secondary infections caused by bacterial pathogens, such as S. aureus and S. pyogenes. In the hospital setting, prevention of healthcare associated bacteremias revolves around minimizing iatrogenic infections from indwelling intravascular catheters and other invasive devices by following recommended infection control practices.