Bacteria

The organisms most commonly isolated from blood are gram-positive cocci, including coagulase-negative staphylococci, Staphylococcus aureus, and Enterococcus spp., and other organisms likely to be inhabitants of the hospital environment that colonize the skin, oropharynx, and gastrointestinal tract of patients. Some of the most common, clinically significant bacteria isolated from blood cultures are listed in Box 68-1. In general, the number of fungi and coagulase-negative staphylococci has increased, whereas the number of clinically significant anaerobic isolates has decreased since the early 2000s.

Of importance, the laboratory isolation of certain bacterial species from blood can indicate the presence of an underlying, occult, or undiagnosed neoplasm. Alterations in local conditions at the site of the neoplasm allowing bacteria to proliferate and seed the bloodstream have been suggested as a potential mechanism for the association between bacteremia and cancer. Another possible mechanism is reduced killing of bacterial cells by the host phagocytes. Organisms associated with neoplastic disease include Clostridium septicum and other uncommonly isolated clostridial species, Streptococcus galldyticus, Aeromonas hydrophila, Plesiomonas shigelloides, and Campylobacter spp. Finally, if Streptococcus anginosis group bacteria are isolated from blood, the possibility of an abscess should be considered.

Fungi

Fungemia (the presence of fungi in blood) is usually a serious condition, occurring primarily in immunosuppressed patients and in those with serious or terminal illness. Candida albicans is by far the most common species, but Malassezia furfur can often be isolated in patients, particularly neonates, receiving lipid-supplemented parenteral nutrition. Candida spp. account for approximately 8% to 10% of all nosocomial bloodstream infections.

Except for Histoplasma, which multiply in leukocytes (white blood cells), fungi do not invade blood cells, but their presence in the blood usually indicates a focus of infection elsewhere in the body. Fungi in the bloodstream can disseminate (be carried) to all organs of the host, where they may grow, invade normal tissue, and produce toxic products. Fungi gain entrance to the circulatory system via loss of integrity of the gastrointestinal or other mucosa; through damaged skin; from primary sites of infection, such as the lung or other organs; or by means of intravascular catheters.

Systemic fungal infections begin as pneumonia and may disseminate from the lungs, which serve as the portal of entry. Arthroconidia of Coccidioides immitis and microconidia of Histoplasma capsulatum and Blastomyces dermatitidis are ingested by alveolar macrophages in the lung. These macrophages carry the fungi to nearby lymph nodes, usually the hilar nodes. The fungi multiply within the node tissue and ultimately are released into the circulating blood, from which they are capable of seeding other organs or are destroyed by the body’s defenses. Molds are particularly insensitive to host defenses such as antibody and phagocytic cells because of their large size and their sterol containing cell wall structure.

Parasites

Eukaryotic parasites may be found transiently in the bloodstream as they migrate to other tissues or organs. Their presence, however, cannot be considered consistent with a state of good health. For example, tachyzoites of the parasite Toxoplasma gondii may be found in circulating blood. They invade cells within lymph nodes and other organs, including the lungs, liver, heart, brain, and eyes. The resulting cellular destruction accounts for the manifestations of toxoplasmosis. Also, microfilariae are seen in peripheral blood during infection with Dipetalonema, Mansonella, Loa loa, Wuchereria, or Brugia.

Malarial parasites invade host erythrocytes and hepatic parenchymal cells. The significant anemia and subsequent tissue hypoxia (reduction in oxygen levels) may result from destruction of red blood cells by the parasite. Vascular trapping of normal erythrocytes by the infected red blood cells, which are less flexible and tend to clog small capillaries, is a major cause of morbidity. The host’s immunologic response is to remove the parasites and damaged red blood cells; the immune response may also have deleterious effects.

Parasites in the bloodstream are usually detected by direct visualization. Those parasites for which traditional diagnosis is dependent on observation of the organism in peripheral blood smears include Plasmodium, Trypanosoma, and Babesia. Patients with malaria or filariasis may display a periodicity in their episodes of fever that allows the physician to time the collection of blood for microscopic examination intended for optimal detection. Rapid serological methods and molecular methods are currently used to detect malaria, babesiosis, and trypanosomiasis. These tests are described in Chapter 49.

Viruses

Although many viruses do circulate in the peripheral blood at some stage of disease, the primary pathology relates to infection of the target organ or cells. Those viruses that preferentially infect blood cells are Epstein-Barr virus (invades lymphocytes), cytomegalovirus (invades monocytes, polymorphonuclear cells, and lymphocytes), and human immunodeficiency virus (HIV) (involves only certain T lymphocytes and perhaps macrophages) and other human retroviruses that attack lymphocytes. The pathogenesis of viral diseases of the blood is the same as that for viral diseases of any organ; by diverting the cellular machinery to create new viral components or by other means, the virus may prevent the host cell from performing its normal function. The cell may be destroyed or damaged by viral replication, and immunologic responses of the host may also contribute to the pathogenesis.

Although many viral diseases have a viremic stage, recovery of virus particles or detection of circulating viruses is used in the diagnosis of only a few diseases. Chapter 66 discusses the recovery of viruses from blood in greater detail.

Intravascular Infections

Intravascular infections include infective endocarditis, mycotic aneurysm, suppurative thrombophlebitis, and intravenous (IV), catheter-associated bacteremia. Because these infections are within the vascular system, organisms are present in the bloodstream at a fairly constant rate (i.e., a continuous bacteremia). These infections in the cardiovascular system are extremely serious and considered life threatening.

Infective Endocarditis.

The development of infective endocarditis (infection of the endocardium most commonly caused by bacteria) is believed to involve several independent events. Cardiac abnormalities, such as congenital valvular diseases that lead to turbulence in blood flow or direct trauma from IV catheters, can damage cardiac endothelium. This damage to the endothelial surface results in the deposition of platelets and fibrin. If bacteria transiently gain access to the bloodstream (this can occur after an innocuous procedure such as brushing the teeth) after alteration of the capillary endothelial cells, the organisms may stick to and then colonize the damaged cardiac endothelial cell surface. After colonization, the surface will rapidly be covered with a protective layer of fibrin and platelets. This protective environment is favorable to further bacterial multiplication. This web of platelets, fibrin, inflammatory cells, and entrapped organisms is called a vegetation (Figure 68-1). The resulting vegetations ultimately seed bacteria into the blood at a slow but constant rate.

The primary causes of infective endocarditis are the viridans streptococci, comprising several species (Box 68-2). These organisms are normal inhabitants of the oral cavity, often gaining entrance to the bloodstream as a result of gingivitis, periodontitis, or dental manipulation. Heart valves, especially those previously damaged, present convenient surfaces for attachment of these bacteria. Streptococcus sanguis and Streptococcus mutans are frequently isolated in streptococcal endocarditis. Gram-negative bacilli, known as the AACEK group, Aggregatibacter aphrophilus, Actinobacillus actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae, can also be associated with endocarditis.

Box 68-2   Agents of Infective Endocarditis

Aggregatibacter aprophilus

Viridans streptococci*

Nutritionally deficient streptococci (Abiotrophia spp. and Granulicatella spp.)

Enterococci*

Streptococcus bovis

Staphylococcus aureus*

Staphylococci (coagulase-negative)

Enterobacteriaceae

Pseudomonas spp. (usually in drug users)

Haemophilus spp.

Unusual gram-negative bacilli (e.g., Actinobacillus, Cardiobacterium, Eikenella, Coxiella burnetii)

Yeast

Other (including polymicrobial infectious endocarditis)

---

^*Most common organisms associated with native valve endocarditis in non-drug-using adults.

With the ever-increasing use of IV catheters, arterial lines, and vascular prostheses, organisms considered normal or hospital-acquired inhabitants of the human skin are able to gain access to the bloodstream and attach to various surfaces, including heart valves and vascular endothelium. It has been estimated that more than 200,000 nosocomial infections (bloodstream) occur annually in the United States in adults and children. The majority of these infections are caused by the use of intravascular catheters. Staphylococcus epidermidis and other coagulase-negative staphylococci have been increasingly implicated as the cause of infection associated with intravascular catheters. S. epidermidis is the most common etiologic agent identified in prosthetic valve endocarditis, with S. aureus being the second most common. S. aureus is an important cause of septicemia without endocarditis and is found in association with other foci, such as abscesses, wound infections, and pneumonia, as well as sepsis related to indwelling intravascular catheters.

Mycotic Aneurysm and Suppurative Thrombophlebitis.

Two other intravascular infections, mycotic aneurysms and suppurative thrombophlebitis, result from damage to the endothelial cells lining blood vessels. With respect to mycotic aneurysm, an infection causes inflammatory damage and weakening of an arterial wall; this weakening causes a bulging of the arterial wall (i.e., aneurysm) that can eventually rupture. The etiologic agents are similar to those that cause endocarditis.

Suppurative thrombophlebitis is an inflammation of a vein wall. The pathogenesis of this intravascular infection involves an alteration in the vein’s endothelial lining followed by clot formation. The site is then seeded with organisms, thereby establishing a primary site of infection. Suppurative thrombophlebitis represents a frequent complication of hospitalized patients caused by the increasing use of IV catheters.

Intravenous Catheter–Associated Bacteremia.

IV catheters are an integral part of the care for many hospitalized patients. More than 3 million central venous catheters are used annually in the United States. For example, central venous catheters are used to administer fluids, blood products, medications, antibiotics, and nutrition, and for hemodynamic monitoring. A short-term, triple-lumen (channel opening within a tube) central venous catheter is shown in Figure 68-2. Unfortunately, a major consequence of these medical devices is colonization of the catheter by either bacteria or fungi, which can lead to catheter infection and serious bloodstream infection. This consequence is a major nosocomial source of illness and even death.

IV catheter–associated bacteremia (or fungemia) is believed to occur primarily by two routes (Figure 68-3). The first route involves the movement of organisms from the catheter entry site through the patient’s skin and down the external surface of the catheter to the catheter tip within the bloodstream. After arriving at the tip, the organisms multiply and may cause a bacteremia. The second way that IV catheter–associated bacteremia may occur is by migration of organisms along the inside of the catheter (the lumen) to the catheter tip. The catheter’s hub, where tubing connects into the IV catheter, is considered the site at which organisms gain access to the patient’s bloodstream through the catheter lumen. The most common etiologic agents for IV catheter–associated bloodstream infections, regardless of the route of infection, are organisms found on the skin (Box 68-3). Certain strains of S. epidermidis appear to be uniquely suited for causing catheter-related infections because of their ability to produce a biofilm or “slime” that consists of complex sugars (polysaccharides) believed to help the organism adhere to the catheter’s surface. The initial attachment of S. epidermidis to the catheter’s polystyrene surface is related to a cell surface protein. Once attached, the organism proliferates, subsequently forming a biofilm. Uncommon routes of IV catheter–tip infection include contaminated fluids or blood-borne seeding from another infection site.

Box 68-3   Common Agents of IV Catheter–Associated Bacteremia

Staphylococcus epidermidis

Other coagulase-negative staphylococci

Staphylococcus aureus

Enterobacteriaceae

Pseudomonas aeruginosa

Candida spp.

Corynebacterium spp.

Other gram-negative rods

Extravascular Infections

Except for intravascular infections, bacteria usually enter the circulation through the lymphatic system. Most cases of clinically significant bacteremia are a result of extravascular infection. When organisms multiply at a local site of infection such as the lung, they are drained by the lymphatics and reach the bloodstream. In most individuals, organisms in the bloodstream are effectively and rapidly removed by the reticuloendothelial system in the liver, spleen, and bone marrow and by circulating phagocytic cells. Depending on the extent of immunologic control of the infection, the organism may be circulated more widely, thereby causing a bacteremia or fungemia.

The most common portals of entry for bacteremia are the genitourinary tract (25%), respiratory tract (20%), abscesses (10%), surgical wound infections (5%), biliary tract (5%), miscellaneous sites (10%), and uncertain sites (25%). For the most part, the probability of bacteremia occurring from an extravascular site depends on the site of infection, its severity, and the organism. For example, any organism producing meningitis is likely to produce bacteremia at the same time. Of importance, certain organisms causing extravascular infections commonly invade the bloodstream; some of these organisms are listed in Table 68-1. In addition to these organisms, a large number of other bacteria and fungi that cause extravascular infections are also capable of invading the bloodstream. Whether these organisms invade the bloodstream depends on the host’s ability to control the infection and the organism’s pathogenic potential. Some of the organisms associated with potential bloodstream infections from a localized site include members of the family Enterobacteriaceae, Streptococcus pneumoniae, Staphylococcus aureus, Neisseria gonorrhoeae, anaerobic cocci, Bacteroides, Clostridium, beta-hemolytic streptococci, and Pseudomonas. These are only some of the organisms frequently isolated from blood. Almost every known bacterial species and many fungal species have been implicated in extravascular bloodstream infections.

Antisepsis.

Once a vein is selected, the skin site is defatted (fat removal) with 70% isopropyl alcohol and an antiseptic is applied to kill surface and subsurface bacteria. Regardless of the antiseptic used, it is critical to follow the manufacturer’s recommendation for the length of time the antiseptic is allowed to remain on the skin. Available data indicate that iodine tincture (iodine in alcohol) and chlorhexidine are equivalent for skin preparation before drawing blood cultures. The steps necessary for drawing blood for culture are given in Procedure 68-1, which can be found on the Evolve site.

As part of ongoing quality assurance, laboratories should determine the rate of blood culture contamination by clinically evaluating patients’ conditions in conjunction with the organism isolated from culture. Laboratories that recover contaminants at rates greater than 3% should suspect improper phlebotomy techniques and should institute measures to educate the phlebotomists in proper skin preparation methods.

Precautions.

Standard precautions require that phlebotomists wear gloves for blood drawing. Because blood for culture must be obtained aseptically, it is important that contaminated surfaces that might come in contact with the disinfected venipuncture site be disinfected. For example, if the site must be touched after preparation, the phlebotomist must disinfect the gloved fingers used for palpation. Also, if the rubber stopper or septum of the container into which blood is to be inoculated (e.g., test tubes or commercial culture bottles) is potentially contaminated, the phlebotomist must disinfect the septum.

Specimen Volume

Number of Blood Cultures

Because periodicity of microorganisms in the bloodstream may be characteristic for some diseases, continuous for some and random in others, patterns of bacteremia must be considered in establishing standards for the timing and number of blood cultures. If the volume of blood is adequate, usually two or three blood cultures are sufficient to achieve the optimum blood culture sensitivity. In patients with endocarditis who have not received antibiotics, a single blood culture is positive in 90% to 95% of the cases, whereas a second blood culture establishes the diagnosis in at least 98% of patients, depending on the study. For patients who have received prior antibiotic therapy, three separate blood collections of 16 to 20 mL each, and an additional blood culture or two taken on the second day, if necessary, detects most etiologic agents of endocarditis. This presumes use of a culture system adequate for growth of the organism involved, which often entails extending the incubation period. Similarly, for patients without infective endocarditis, 65.1% are detected in the first culture, 80% by the first two cultures, and 95.7% were detected in the first three blood cultures.

Timing of Collection

The timing of cultures is not as important as other factors in patients with intravascular infections because organisms are released into the bloodstream at a fairly constant rate. Because the timing of intermittent bacteremia is unpredictable, it is generally accepted that two or three blood cultures be spaced an hour apart. However, a study found no significant difference in the yield between multiple blood cultures obtained simultaneously or those obtained at intervals. The authors concluded that the overall volume of blood cultured was more critical to increasing organism yield than timing.

When a patient’s condition requires therapy to be initiated as rapidly as possible, little time is available to collect multiple blood culture samples over a timed interval. An acceptable compromise is to collect 40 mL of blood at one time, 20 mL from each of two separate venipuncture sites, using two separate needles and syringes before the patient is given antimicrobial therapy. Regardless, blood should be transported immediately to the laboratory and placed into the incubator or instrument as soon as possible. With blood culture instrumentation, a delay beyond 2 hours can delay the detection of positive cultures.

Miscellaneous Matters

Conventional Blood Cultures

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 68-4) 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 subcultured 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.

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 68-5). 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.

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 subcultured 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 68-6). 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.

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 subcultured 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 microbiology 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 performed 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 presumptive 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 isolates 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:

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

Leptospira

Leptospirosis can be diagnosed by isolating the causative spirochete from blood during the first 4 to 7 days of illness. Leptospires will grow 1 to 3 cm below the surface, usually within 2 weeks. The organisms remain viable in blood with SPS for 11 days, allowing for transport of specimens from distant locations. Direct dark-field examination of peripheral blood is not recommended because many artifacts are present that resemble spirochetes. If blood must be shipped to a reference laboratory for culture, blood may be collected into heparin, oxalate, or citrate tubes and maintained at ambient temperature. One to two drops of blood are inoculated into semi-solid oleic acid-albumin medium at the patient’s bedside. Various commercial mediums are available, such as Fletcher’s medium (BD Diagnostics, Sparks, MD). Multiple cultures are recommended to improve recovery of the organisms. Due to the organism’s failure to grow in conventional blood culture systems, molecular assays may improve detection of the organism, as well as the use of serological markers for rapid diagnosis. (Further information about Borrelia and Leptospira is provided in Chapter 46.)