CLINICAL MICROBIOLOGY REVIEWS, JUly 1990, p. 269-279 0893-8512/90/030269-11$02.00/0 Copyright i© 1990, American Society for Microbiology

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Quantitative Aspects of Septicemia PABLO YAGUPSKY' AND FREDERICK S. NOLTE2* Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York 14642,1 and Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia 303222 INTRODUCTION ....................................................... 269 DEVELOPMENT OF QUANTITATIVE BLOOD CULTURE METHODS ........................................ 269 CLINICAL SIGNIFICANCE OF QUANTITATIVE BLOOD CULTURES ........................................ 271 271 Severity of Infection in Children and Adults ....................................................... 272 Efficacy of Antibiotic Therapy ....................................................... Endocarditis ....................................................... 273 Catheter-Related Sepsis (CRS) ....................................................... 274 Contaminants versus True Pathogens in Positive Blood Cultures ..................................................276 CONCLUSIONS ....................................................... 276 LITERATURE CITED ....................................................... 276

INTRODUCTION

DEVELOPMENT OF QUANTITATIVE BLOOD CULTURE METHODS

Blood cultures are a crucial part of the evaluation of patients with suspected sepsis, and a positive blood culture is the best criterion for defining that condition. In most clinical microbiology laboratories, routine blood cultures are accomplished by using broth media. Although broth-based blood culture systems provide a sensitive means for recovering microorganisms from blood, they provide no information about the number of microorganisms present in the original blood sample. In contrast, inoculation of blood onto solid media results in growth of individual colonies which can be enumerated and the result can be used to calculate the concentration of bacteria per milliliter of blood. Until recently, the methods available for quantitative blood cultures have been cumbersome and labor intensive and did not find widespread use in clinical microbiology laboratories. In 1976, Dorn et al. (18, 19) described a blood culture technique based on lysis of cellular elements in blood followed by concentration of microorganisms by centrifugation and plating of the sediment on solid media. Improvements were made in the original technique, and a blood culture system based on this method (Isolator Microbial Tube) was made commercially available by E. I. du Pont de Nemours & Co., Inc., Wilmington, Del. A recent survey of blood culture methods used in 288 clinical microbiology laboratories revealed that 13% used the Isolator system for routine bacteriologic culture and 30% used it for fungal culture (K. S. C. Kehl, Clin. Microbiol. Newsl. 8:127-133, 1986). The availability of the Isolator system has sparked a renewed interest in the quantitation of microorganisms in patient blood. In this article, we review the development of quantitative blood culture methods and published applications of these methods. We also critically evaluate the available data on possible diagnostic and prognostic significance of quantitative blood culture results and identify areas for future research. The subject of quantitative blood cultures has also been reviewed recently by Kiehn (42).

The first quantitative blood cultures were performed by the pour plate or spread plate techniques. The pour plate technique involves mixing blood and molten agar; with the spread plate technique, blood is spread over the surface of an agar plate. The number of colonies that grow either in or on the agar is enumerated, and the CFU per milliliter of blood are calculated. Both methods suffer because only a small volume, usually 1 ml of blood, can be inoculated. To maximize the recovery of microorganisms, it is recommended that at least 10 ml of blood be cultured from adult patients (36, 89, 94). As a result, these methods are of practical value only when the quantity of bacteria in blood is relatively large. The magnitude of bacteremia in infants and children is generally greater than that in adults so cultures of as little as 0.5 to 1.0 ml of blood are considered adequate in most cases (94). Direct plating methods have been used in pediatrics to enhance the recovery of commonly encountered pathogens such as Haemophilus influenza and Neisseria meningitidis and to study the correlation between the magnitude of bacteremia and clinical manifestations (52, 53). Quantitative blood culture studies in pediatrics have used several methods, including pour plate (1, 25); spread plate (75); quantitative direct plating, using heparin tubes (53); and lysis direct plating, using the Isolator 1.5 Microbial Tube (10-12, 98). La Scolea et al. (53) described a quantitative direct plating method in which 0.5 to 1.0 ml of blood was collected into a heparin tube; upon arrival in the laboratory, the blood was inoculated onto agar plates. The quantitative direct plating method was compared with broth methods for detection of bacteremia in children (53). It detected 83% of the cultures positive for H. influenza and 100% of those positive for N. meningitidis, but only 50% of the cultures positive for Streptococcus pneumoniae. The number of other pathogens recovered was too small for evaluation. Of those cultures positive by both quantitative direct plating and a broth procedure, 55% were positive first by quantitative direct plating. The Isolator 1.5 Microbial Tube was designed for culturing small-volume (0.5- to 1.5-ml) pediatric blood samples directly to agar media. It contains 0.96 mg of the anticoagulant sodium polyanetholsulfonate and 0.1 ml of a saponin-con-

*

Corresponding author. 269

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training solution to lyse the cellular components of the blood. The lysis step allows the recovery of viable phagocytized microorganisms. The Isolator 1.5 tubes have been compared with conventional and radiometric broth methods in several clinical trials (10, 12, 98). In none of the trials did the Isolator system recover significantly more organisms than the comparative method, and in only one trial did the Isolator system provide positive results more rapidly. However, in each trial there were substantial reductions in the time required to obtain isolated colonies with the Isolator system. Large increases in the contamination rate for Isolator cultures were reported in each evaluation and averaged 10.9%. All of the direct plating methods described above are practical only for small volumes of blood. Concentrationdirect plating methods were developed to maximize the detection of low-magnitude bacteremia or fungemia and to remove inhibitory factors that may be present in blood. The basic concept behind these methods is that blood cells are lysed, microorganisms are concentrated either by membrane filtration or centrifugation, and, finally, the concentrate is plated directly to solid media. Since the concentration procedures effectively separate microorganisms from inhibitory substances in blood, these methods potentially improve the recovery of microorganisms. Brown and Kelsh (9) were the first to report use of a membrane filter technique for recovery of Brucella spp. from the blood. Tidwell and Gee (88) found that detection of experimental bacteremia in rabbits was faster and more reliable with a filter technique than with a conventional broth method. In another study (69), the kinetics of Salmonella typhi bacteremia during therapy were followed with a membrane filtration technique. These early studies used cumbersome and time-consuming methods in which only small volumes of blood were processed for culture. Winn et al. (104) and Finegold et al. (27) reported a differential membrane filtration technique which allowed the processing of larger blood volumes and separate culture of different blood components. The formed elements of blood were separated by differential centrifugation and plasma only was passed through a membrane filter. The filter was placed on the surface of an agar plate, and the erythrocytes and leukocytes were cultured separately on solid media. Isolated colonies were available after 24 to 48 h of incubation, even in patients treated with antibiotics. The technique provided quantitative information and also facilitated the diagnosis of polymicrobic bacteremia. The basic membrane filtration technique was modified by Stanaszek (79), Sullivan et al. (82-84), and Zierdt et al. (106) to include a solution that lysed blood cells before filtration. By using this modified technique, transient bacteremia following common urologic procedures was demonstrated, as well as superior recovery of gram-positive bacteria in both experimental and human clinical studies. Komorowski and Farmer (48) found that a lysis-filtration technique was superior to a conventional broth culture for detection of candidemia. Lamberg et al. (50) described an improved filtration method that did not require lysis of blood cells or sophisticated equipment. The blood is applied to a Ficoll-Hypaque gradient, centrifuged, and filtered. In a study with seeded blood samples, this system had a sensitivity equivalent to the broth method used for comparison. In spite of these promising advances, membrane filtration techniques remain cumbersome, expensive, and time-consuming, and no commercially available system is based on this technique. In 1976, Dorn et al. (18, 19) described a concentration technique for blood cultures termed lysis-centrifugation. In

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this two-step process, the blood was lysed in one tube and the lysate was transferred to a second tube, where microorganisms were concentrated by centrifugation onto a stabilizing density layer composed of 50% sucrose and 1.5% gelatin. After centrifugation, most of the supernatant was removed with a needle and syringe. Aliquots of the sediment were then plated to agar media. Dorn et al. used the new technique in a study of 1,000 blood cultures from patients suspected of having bacteremia (18). The blood was also cultured in Trypticase soy broth and in pour plates. Of 176 significant positive cultures, the lysis-centrifugation technique recovered 73% and the pour plate and broth methods recovered 49 and 38%, respectively. The lysis-centrifugation technique demonstrated enhanced recovery of fungi, grampositive cocci, and Pseudomonas spp. The superior performance of the lysis-centrifugation technique could be due to the high theoretical dilution of antimicrobial factors (1:400) or to protection provided to cell-wall-damaged bacteria by the hypertonic sucrose-gelatin cushion. Although this new technique showed promise, it was cumbersome and had a high (9.3%) rate of contamination. In 1978, Dom and Smith (22) described a new centrifugation blood culture device. It was essentially a doublestoppered evacuated glass tube containing 0.3 ml of a dense hydrophobic cushion, 1.2 ml of an aqueous solution of sodium polyanetholsulfonate, a lysing agent, and an antifoaming agent. Blood was introduced into the tube and mixed thoroughly, and the tube was centrifuged at 3,000 x g for 30 min in a fixed-angle rotor. After centrifugation, the majority of the supernatant was removed and discarded. The remaining 1.5 ml of solution was equally dispersed to five agar plates. A clinical trial of this new blood culture device was conducted with 3,335 blood cultures (20). All blood samples were cultured concurrently in supplemented peptone broth with sodium polyanetholsulfonate under a C02-rich atmosphere. The centrifugation technique detected 80% of positive cultures compared with 67% for the broth method. Superior recovery of Staphylococcus aureus, Pseudomonas spp., and yeasts was demonstrated, and the time required for detection of a positive blood culture was shorter for the centrifugation method. The contamination rate observed with the new method, 1.4%, was comparable to that of the broth method. A lysis-centrifugation device similar to that developed by Dom et al. is commercially available as the Isolator 7.5- or 10-ml Microbial Tubes. The Isolator system has been compared with both conventional and radiometric broth and biphasic blood cultures in many clinical trials and overall had demonstrated good performance (5-7, 33, 34, 38-40, 46, 63). Certain common findings emerged from these evaluations of the Isolator system. The lysis-centrifugation method recovered significantly more fungi than conventional broth, radiometric broth, or biphasic brain heart infusion bottles. The superiority of the lysis-centrifugation method for the recovery of mycobacteria (30, 45), especially Mycobacterium avium-M. intracellulare complex (31, 43, 44, 58, 104), Cryptococcus neoformans (5, 6), and Histoplasma capsulatum (5, 100), from blood has been useful in diagnosis of these infections in immunocompromised patients. Enhanced isolation of staphylococci and members of the family Enterobacteriaceae was also found in several studies (6, 33, 34, 38, 39, 46,63). However, the Isolator system may not be optimal for recovery of Streptococcus pneumoniae, Pseudomonas aeruginosa, and anaerobic bacteria (6, 33, 34, 39, 46, 63). The Isolator system was superior to conventional broth or

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biphasic broth cultures in terms of speed of detection of positive cultures but, on average, was no faster than the radiometric method. Positive cultures with the lysis-centrifugation system occur as colonies on agar plates, and therefore this system offers potential advantages in the time required to identify and determine antibiotic susceptibility of isolates. However, for laboratories performing direct identification and antibiotic susceptibility tests from positive blood culture bottles, this advantage may be less meaningful. The incidence of polymicrobic bacteremia has been increasing steadily during the last decades, possibly as a result of the expanding population of immunocompromised patients, aggressive surgery, and widespread use of intravascular medical devices (41, 95, 96). Detection of polymicrobic bacteremia is extremely important to select the most appropriate antimicrobial therapy. Blood cultures on solid media have some potential advantages over broth cultures because overgrowth of cultures by one organism is less likely to occur on solid media, and morphologic differences between isolated colonies are easily recognized (34). Clinical trials have confirmed the superiority of blood cultures performed on solid media for recovery of multiple organisms from blood. In one study, 14 of 15 episodes of polymicrobic bacteremia were detected by lysis-centrifugation as opposed to only 3 of 15 detected by the broth culture method (40). In a second study that included 25 episodes of polymicrobic bacteremia, the Isolator system detected 21 episodes and the biphasic bottle detected only 17 (39). Blood cultures on solid media have also been shown to be superior to broth methods for diagnosis of polymicrobic bacteremia in catheter-related infections (70, 72). One significant disadvantage of the Isolator system is the relatively high contamination rate. This occurs because the lysis-centrifugation method requires additional processing steps and the use of petri plates rather than closed bottles, which offer greater opportunity to introduce contaminants. Reported contamination rates for the Isolator system range from 0.3 to 13.8% (81). Rates in different laboratories depend on definition of contamination, experience with the system, use of relatively dry solid media, and inoculation of plates in a laminar flow hood. It appears that use of a laminar flow cabinet is a critical factor for reducing contamination rates to an acceptable level. In one study, the contamination rate dropped from 9.5 to 6.2% (87) and in another study it dropped from 9.6 to 2.5% (94) when lysis-centrifugation blood cultures were processed in a laminar flow cabinet. An important concern for laboratories that use direct plating methods is the length of time that blood is held in the tube before processing. Cashman et al. (13) conducted a study with blood cultures seeded with low numbers of 26 different bacterial species and demonstrated that a holding time of 15 h might be feasible for the Isolator tube. However, the authors observed substantial increases in numbers of some species and significant declines in others over the 15-h period. The quantity of bacteria recovered from specimens held for this length of time would bear little relationship to the quantity of bacteria originally present in the blood. Stockman et al. (80), in a prospective study of 5,125 fungal blood cultures, found that lysis-centrifugation tubes processed within 9 h showed a significantly higher yield of yeasts, filamentous fungi, and bacteria than did those processed after 9 h of hold time. With any of the direct plating blood culture methods, including the Isolator system, specimens should be processed as quickly as possible to enhance

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recovery of pathogens and to ensure that colony counts truly reflect the quantity of bacteria in vivo. CLINICAL SIGNIFICANCE OF QUANTITATIVE BLOOD CULTURES Severity of Infection in Children and Adults The clinical spectrum of bacteremia is extremely wide, ranging from a self-limited septic event after trauma or manipulation of colonized mucosal surfaces to overwhelming and often fatal infections. It has been estimated that every year in the United States half of the 200,000 patients with septicemia die in spite of the progress achieved in antibiotic and supportive therapy (92, 94). Great efforts have been made to identify factors that contribute to this high case mortality rate. For more than 30 years, several authors have used quantitative blood cultures to investigate the magnitude of bacteremia in septic patients and its correlation with the occurrence of complications and death. The results of these studies have shown that most episodes of clinically significant bacteremia in adults are characterized by low numbers of circulating bacteria. Werner et al. (99) found that 54% of blood cultures from patients with staphylococcal and streptococcal endocarditis contained between 1 and 30 CFU/ml of blood. In a study of adults, Henry et al. found 10 bacteria per ml of blood. It is apparent that the magnitude of bacteremia in adult patients correlates with mortality rate. However, because bacteremia usually results from breakdown of defense mechanisms that control infection, underlying debilitating diseases appear to be a more important factor than the number of CFU per milliliter of blood in determining the ultimate prognosis. Intravascular foci of infection such as endocarditis and catheter-related sepsis may be important exceptions to these generalizations about the relationship between the magnitude of bacteremia and outcome. Whimbey et al. (101) analyzed colony counts in 15 patients with central venous catheter-related Staphylococcus aureus bacteremia and found that the magnitude of bacteremia was directly related to the incidence of metastatic complications. Six of 7 patients with 2 100 CFU/ml in blood from a vessel had metastatic foci of infection, whereas none of 7 patients with c 10 CFU/ml had metastatic complications (P < 0.01). The remaining patient had 20 CFU/ml of blood and did not develop metastatic infection. Three patients with, and no patients without, metastatic infections died. Correlation between magnitude of bacteremia and severity of clinical disease in pediatric patients has been firmly established. Using the pour plate technique, Dietzman et al. (16) studied the quantitative aspects of E. coli septicemia in 30 neonates and found a bimodal distribution of bacterial density: 65% of the patients had 1,000 CFU/ml. Only 1 of 9 patients with 1,000 CFU/ml. In a study of 83 H. influenzae type b bacteremic episodes, it was demonstrated that children with high-density bacteremia had a significantly shorter duration of illness before presentation (median, 1 day) than did low-grade bacteremic patients (median, 3 days), and such children had a higher rate of complications including secondary foci of infection (62). Other studies involving older children with bacteremic

infections caused by three major pediatric pathogens, H. influenza type b, Streptococcus pneumoniae, and N. meningitidis, also demonstrated a clear relationship between severity of disease and bacterial counts (3, 25, 54, 55, 62, 75, 85, 86, 98). For H. influenza, the combined results of these studies show that 50 of 72 (69.4%) patients with meningitis and 19 of 28 (67.9%) patients with epiglottitis had bacterial counts of >100 CFU/ml, but only 8 of 42 (19.0%) patients with less severe infections such as pneumonia, cellulitis, osteomyelitis, arthritis, otitis media, or bacteremia without demonstrable focus had bacterial densities as great (3, 54, 55, 62, 86, 98). Among the patients with S. pneumoniae bacteremia, 6 of 8 (75%) with meningitis, but only 3 of 55 (5.4%) with less severe diseases, had counts of >100 CFU/ ml (3, 25, 86, 98). The combined results for children with N. meningitidis infections show that 15 of 24 (62.5%) patients with meningitis and half of those with WaterhouseFriderichsen syndrome have >100 CFU/ml. Bacterial counts of 100 CFU/ml (P < 0.001). Febrile diseases of infectious origin are more common in early childhood than during any other period of human life. Although most febrile children suffer from self-limited ill-

ness, 3 to 15% of febrile children between the ages of 6 and 24 months have bacteremic infections that may result in severe complications and death (3, 37). Clinical signs are not always reliable criteria for the identification of bacteremia. Occasionally, an ill child managed as an outpatient with a "benign" infection is already bacteremic and will later

develop meningitis (86). Identification of febrile children at risk of complications of bacteremia is important to ensure appropriate evaluation and antibiotic therapy. In one report, four pediatric outpatients who presented with seemingly mild respiratory infections and otitis media had blood cultures obtained because of high fever (85). Three developed meningitis due to S. pneumoniae or N. meningitidis, and the fourth developed H. influenza epiglottitis. In each case,

CFU/ml. In a second study, three children initially admitted without evidence of focal infection, in whom N. meningitidis was recovered from blood in excess of 500 CFU/ml, developed counts on the initial blood culture were >100

meningitis (85). Modern quantitative blood culture techniques allow both rapid detection of bacteremia and provide information on its magnitude. When high bacterial counts in the blood of a child are found, careful reevaluation is necessary to identify and treat potential life-threatening complications especially in those children sent home after initial evaluation. Although a relationship has been established between the severity of disease and bacterial density in the blood for H. influenza, S. pneumoniae, and N. meningititis, more studies are needed to establish this relationship for other pathogens such as group B beta-hemolytic streptococci and Staphylococcus aureus.

Efficacy of Antibiotic Therapy Detection of bacteremia by a positive blood culture followed by antibiotic susceptibility testing of the isolate are crucial for confirmation of the diagnosis and rational selection of antibiotics. It is accepted that administration of

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appropriate antibiotic therapy should result in negative blood cultures. Persistence of positive blood cultures is interpreted as evidence of treatment failure, and changes in antibiotic therapy are usually made. However, in certain clinical situations such as infections in immunocompromised patients, M. avium-M. intracellulare complex infections in patients with acquired immunodeficiency syndrome, or the slowly resolving bacteremia of typhoid fever, prompt sterilization of the blood does not always occur. Serial quantitative blood cultures have potential value as an in vivo test of antibiotic efficacy. Using a membrane-filtration technique, Randriambololona and Dodin (69) demonstrated that administration of chloramphenicol to patients with typhoid fever results in gradual decrease in the magnitude of bacteremia over 3 to 8 days, followed by negative cultures. Normalization of body temperature occurs close to the time of clearance of Salmonella typhi from the blood. Marshall and Bell (62) recently reported the results of a quantitative blood culture study in 81 bacteremic children. They showed that children with partially treated H. influenzae meningitis had lower bacterial counts in blood than untreated controls, and another report (102) demonstrated that, in five appropriately treated patients with persistently positive blood cultures, broth cultures concealed a prompt decline in the number of bacteria circulating in the blood seen in quantitative blood cultures. The declining colony counts supported the clinical assessment that patients responded to therapy. In 1982, Fojtasek and Kelly (30) reported that quantitative blood cultures were useful for monitoring the response to antibiotic therapy in a renal transplant recipient with M. chelonae bacteremia. Mycobacterial counts initially ranged from 50 to 70 CFU/ml and dropped to 45 CFU/ml after 2 weeks of amikacin therapy. Cefotaxime was added to the antibiotic regimen and the density dropped to 5 CFU/ml after several days of combined therapy. The counts rose to 25 to 30 CFU/ml when cefotaxime alone was continued. By this time, MIC data were available and showed that the organism was resistant to cefotaxime and susceptible to amikacin and cefoxitin. The antibiotic therapy was changed to cefoxitin; the level of mycobacteremia decreased (0.2 to 2 CFU/ml) and remained at that level for several weeks. Blood cultures became negative after removal of the transplanted kidney and one of the arteriovenous fistulas used for hemodialysis. With the emergence of acquired immunodeficiency syndrome, disseminated M. avium-M. intracellulare complex infections have become common. The disease is characterized by heavy infiltration of the reticuloendothelial system by the bacterium and by prolonged high-level mycobacteremia (44, 58, 105) resembling that seen in lepromatous leprosy (23). Due to difficulties in assessing the clinical response to antibiotic therapy, serial quantitative blood cultures have been used to monitor the effectiveness of antibiotic treatment (58, 105). In some patients bacterial counts decreased or blood cultures became negative with appropriate therapy, but in other patients counts remained stable or even increased. Regardless of the quantitative changes in the magnitude of bacteremia, diffuse infiltration of various organs by mycobacteria was observed in all patients at autopsy, indicating that, as in lepromatous leprosy, controlling bacteremia does not guarantee control of tissue infection (105). Some patients with septicemia deteriorate clinically in spite of antibiotic therapy that is highly effective against the infecting organism in vitro. Using quantitative blood cultures and a Limulus lysate gelation assay, Shenep et al. (76) showed that the amount of total circulating endotoxin in

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patients with gram-negative septicemia was related to the magnitude of bacteremia and that the level of free endotoxin rose sharply after administration of antibiotics. They postulated that destruction of bacterial cells by antimicrobial agents releases endotoxin and other bacterial products sequestered within the intact bacterial cell into the bloodstream, aggravating the host cell injury and worsening the clinical disease. The serial quantitation of plasma endotoxin levels and bacteremia helps explain the paradoxical response sometimes seen in patients treated appropriately for gramnegative sepsis. However, neither the level of bacteremia nor the level of endotoxemia alone determined the clinical severity of gram-negative sepsis in this study. Quantitative blood cultures can provide useful information about in vivo efficacy of antibiotic therapy in patients with persistently positive blood cultures; however, further studies are needed to extend these observations to larger groups of patients with septicemia. Serial quantitative blood cultures obtained from patients receiving antibiotics could be used to study the in vivo relevance of certain in vitro phenomenon, such as antibiotic tolerance and antibiotic synergy. Endocarditis Bacterial endocarditis, a uniformly fatal disease before the antibiotic era, was the subject of several early quantitative blood culture studies. More than 70 years ago, Warren and Herrick (90) used the pour plate technique to show that a broad range of bacterial counts, from one to several hundred CFU per milliliter, occurred in the blood of patients with endocarditis. In 1925, Libman demonstrated that magnitude of bacteremia in a particular patient remains fairly constant during the course of the disease (56). Seven years later, Weiss and Ottenberg (97) studied the relationship between number of circulating bacteria and febrile response in patients with endocarditis. The results showed that there was a constant and relatively uniform number of bacteria released into the blood from vegetations, with periodic liberation of larger numbers of bacteria into the circulation ("bacterial showers"). In four cases of subacute endocarditis, the rise and fall in the magnitude of bacteremia was followed by corresponding changes in patient body temperature. However, fever spikes lagged behind the peak levels of bacteremia by several hours. The authors suggested that diagnostic blood cultures should be obtained at periods of low temperature just prior to the next expected temperature rise to take advantage of the large number of bacteria present in blood and thus maximize the chances to detect the septic episode. They also confirmed previous observations that the severity of disease, measured by patient survival period, was not related to bacterial counts in peripheral blood. A unique study was performed by Beeson et al. (2) shortly before the advent of effective antibiotic therapy for endocarditis when the disease course was not substantially modified by available therapy. Patients frequently suffered for months before dying from cardiac or embolic complications, allowing the opportunity for studies that provided important clues for understanding the pathophysiology of bacteremia. Blood was drawn simultaneously from catheterized femoral arteries and veins, hepatic and renal veins, superior vena cava, and right auricle in a group of six patients. In general, bacterial counts in the femoral vein were slightly lower than in the corresponding arterial cultures, indicating that some circulating bacteria were retained in the peripheral vascular bed, a fact consistent with the

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frequent occurrence of embolic lesions in the extremities of patients with endocarditis. The greatest reduction in bacterial counts was found in the hepatic veins (97% decrease), showing the importance of the liver and spleen as sites of removal of circulating microorganisms. In another study, the sensitivity of arterial, venous, and bone marrow cultures in the detection of bacteremia was examined in 88 patients with endocarditis due to viridans streptococci (74). By using the pour plate technique, it was shown that bacterial counts in arterial and venous blood were approximately the same magnitude. In this study, cultures of bone marrow were superior to arterial and venous cultures in detecting bacteremia, but no quantitative information for bone marrow cultures was provided. In 1967, Werner et al. (99) published a retrospective review of 206 cases of bacterial endocarditis seen over 17 years at the New York Hospital-Cornell Medical Center. They showed that the magnitude of bacteremia in streptococcal and staphylococcal endocarditis was generally of low order and the number of bacteria per milliliter of blood was relatively constant. In this study, the cumulative detection rates of the first two cultures was 98.5% in cases of streptococcal endocarditis and 100% in cases caused by other organisms. The clinical implication is that, in patients with endocarditis, blood cultures should be persistently positive; therefore, it is seldom necessary to obtain more than two or three blood cultures over a 24-h period to make the diagnosis. Quantitative blood cultures have also been used to localize the site of infection in patients with endocarditis, and especially in those with involvement of the right side of the heart. At the time when these studies were conducted, modern sonographic techniques were not available, and diagnosis of right-sided endocarditis was particularly difficult. In one study, published in 1973, Reyes et al. performed intraoperative quantitative blood cultures from each cardiac chamber during a valvulotomy on a grossly infected tricuspid valve (71). Culture of the right ventricle yielded 19 CFU of P. aeruginosa per ml, while all other cultures were negative. In 1974, Pazin et al. performed quantitative blood cultures of cardiac chambers and great vessels during the course of cardiac catheterization of two intravenous drug abusers with bacteremia (68). Finding of unusually high bacterial counts distal to a valve was considered diagnostic of involvement of that particular valve in the disease. The technique succeeded in confirming endocarditis as the cause of patient bacteremia, in excluding other potential sources of sepsis, and in localizing the site of the infection. Quantitative blood cultures have contributed to our understanding of the basic pathophysiology of bacterial endocarditis. The bacteremia is continuous, with the number of bacteria per milliliter of blood usually low and relatively constant. Fever spikes lag behind the peak levels of bacteremia by several hours in patients with endocarditis. The early quantitative studies of bacteremia in endocarditis helped form the basis for the current recommendations for the number and timing of blood cultures (89, 91, 93). The use of serial quantitative blood cultures to monitor the efficacy of antibiotic therapy of endocarditis should be investigated.

Catheter-Related Sepsis (CRS) Intravenous infusion therapy has become a cornerstone of modern medical management. Total parenteral nutrition, prolonged chemotherapy for cancer, and intensive care of critically ill patients have led to the development of new

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techniques for establishing access to central veins. In 1973, Broviac et al. reported the use of a silicone-rubber catheter that was inserted into a central vein but exited at a distant site after passing through a subcutaneous tunnel (8). A small cuff encircling the catheter at the exit site became fixed in place by fibrotic tissue and provided mechanical stability and a barrier to infection. Unfortunately, emergence of a new sort of intravascular infection has been one of the untoward effects of central venous catheterization. Infections of these tunneled, cuffed silicone catheters are categorized into three nonexclusive groups: septic infections, exit site infections, and tunnel infections. Although the risk of death from CRS is low due to the low pathogenicity of the microorganisms usually involved, infection of intravascular devices is today's leading cause of nosocomial bacteremia (15). Catheter-related exit site and tunnel infections are readily diagnosed on the basis of local inflammation, purulence, fever, and leukocytosis. The diagnosis of septic infections, however, is more difficult due to the lack of localizing signs and the fact that patients requiring central venous access are among the most debilitated patients, often with multiple potential sources of bacteremia. Several techniques have been developed to help diagnose CRS in these cases, including the semiquantitative catheter tip culture pioneered by Maki et al. (60), the use of Gram stain or impression smears of the catheter, and quantitative cultures of the catheter obtained by flushing the catheter with nutrient broth (14). A major drawback with these techniques is that the catheter must be removed from the patient before the diagnosis can be made. In one report, more than two-thirds of the central venous catheters removed because of suspected CRS were found to be sterile or proved not to be the source of the septicemia (64). Quantitative blood cultures have been advocated for diagnosis of CRS. In 1979, Wing and colleagues (103) documented CRS in a patient with a Broviac catheter, using pour plate blood cultures. Blood drawn from a peripheral vein grew 25 CFU of Enterobacter cloacae and Citrobacter freundii per ml. Blood drawn from the catheter grew >10,000 CFU of the same organisms per ml. The catheter was removed and found to have an adherent clot present at the tip. Culture of the clot also grew E. cloacae and C. freundii. Whimbey et al. (102) reported two cases in which large differences in colony counts in blood obtained simultaneously from a peripheral vein and the Broviac catheter were used to identify the catheter as the source of infection. In one series of 28 children with Broviac catheters, 100 surveillance blood cultures were obtained through the catheter in afebrile, clinically well patients and from both a peripheral vein and the central catheter during 30 febrile episodes (70). All blood samples were cultured both by the spread plate method and in nutrient broth. Of the 100 surveillance cultures, 79 were negative by both methods. Eighteen cultures showed growth, but the catheters were not considered infected because 100 CFU of Bacillus cereus per ml. Although no illness resulted, the catheter was removed and a Bacillus species and P. aeruginosa were recovered from the tip. These findings were considered consistent with contamination of the cultures or asymptomatic catheter colonization. In the group of febrile patients, CRS was diagnosed nine

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times in seven children. In each case, colony counts in the blood drawn through the catheter were at least 10 times greater than those found in peripheral blood. In six cases, blood obtained from the catheter contained >2,000 CFU/ml, of which four were >5,000 CFU/ml. Quantitative cultures in two patients with septicemia not attributable to the catheter had low colony counts in blood from the catheter. Surveillance blood cultures from the catheters were not helpful in predicting subsequent septicemia in any patients. In a short communication by Myint and Lowes (S. Myint and A. Lowes, Lancet ii:269-270, 1985), bacterial counts of >1,000 CFU of coagulase-negative staphylococci and Enterococcus faecium per ml were found in culture of the central venous catheters in two different patients while the peripheral cultures yielded 100 and 1 CFU/ml, respectively. In another report, quantitative blood cultures were obtained from paired peripheral veins and central catheters in 63 infants with umbilical artery indwelling catheters or Broviac central venous catheters in whom sepsis was suspected (72). Infants in whom both central and peripheral cultures were positive were considered to have proven sepsis, whereas infants with positive central catheter cultures but negative peripheral cultures were diagnosed as having colonized catheters without sepsis. In 10 of 21 children with confirmed sepsis, colony counts in cultures drawn from central lines were >50% higher than the corresponding peripheral counts, suggesting CRS. Paired colony counts were approximately equal in one case, suggesting that sepsis was not related to the catheter. Confluent growth in the remaining 10 pairs prohibited precise colony counts and any conclusions about the source of bacteremia. In 1987, Mosca et al. (64) reported their experience with the use of quantitative blood cultures in 26 intravascular devices suspected of being the source of sepsis. A fivefold or greater central-to-peripheral bacterial count ratio was considered confirmatory of CRS. In the eight instances in which the diagnostic criterion was fulfilled, removal of the catheter resulted in prompt defervescence of the septic patients. In half of the remaining 18 cases in which the central venous cultures were sterile or the differences in colony counts were small, the catheter was removed but the patients' conditions did not improve. The remaining nine catheters were left in place and another source of sepsis was ultimately found. In the studies of CRS cited in the preceding paragraphs, the central venous catheter/peripheral bacterial counts ratio used to identify CRS ranged from 1.5- to several hundredfold. Establishing an appropriate ratio is complicated by the fact that central venous blood passes through the lungs before it reaches the peripheral veins. Since bacteria are removed from the blood by reticuloendothelial cells present in the lung, the final concentration of microorganisms in peripheral blood is less than the concentration in catheter blood even in those patients with sepsis from other sources. To address this problem, Flynn et al. (29) used simultaneous quantitative blood cultures of samples obtained from the superior vena cava and the femoral vein of rabbits with non-catheter-associated bacteremia. As expected, significantly larger bacterial concentrations were found in blood from the superior vena cava compared with concentrations in peripheral blood. Based on data collected in this animal model, the authors calculated that 99% of non-catheterrelated bacteremia should show differences of 100 fold (28, 29). In spite of the potential advantages of quantitative blood cultures as a means of identifying CRS and evaluating the efficacy of antibiotic therapy without removing the catheter, few studies comparing quantitative blood cultures and the semiquantitative method of Maki et al. (60) have been published. In 1983, Moyer et al. (66) compared results of peripheral nonquantitative blood cultures, quantitative cultures obtained through the catheter, and semiquantitative cultures of the catheter tips. Among nonbacteremic patients, all central line blood cultures were negative or grew 15 colonies. Among the five bacteremic patients, four catheter blood cultures grew >25 CFU/ml and >15 colonies were recovered from all catheter tips. In a prospective study of total parenteral nutrition-related infections, a pour plate method was used to culture blood from the central line and the semiquantitative technique of Maki et al. (59) was used to culture intravascular line segments when it was removed (78). Blood from the catheter was collected twice a week for pour plate cultures. Twelve of 100 courses of total parenteral nutrition resulted in infections: five patients had sepsis and seven had local infections. In 5 of 12 patients, pour plate cultures were positive, with 8 CFU of Candida parapsilosis per ml and 2 25 CFU/ml when bacteria were recovered. In 9 of 12, cultures of the intravascular segments were positive and grew >50 CFU/plate. All of the septic patients had positive line segment cultures (50 to >100 CFU/plate), but only two of the five had positive pour plate cultures of central line blood. No quantitative peripheral blood cultures were done. Despite the infrequent positive pour plate cultures results, the authors concluded that these cultures were helpful in implicating the total parenteral nutrition line in three instances because the pour plate culture results antedated positive peripheral blood culture results or removal of the line. The correlation between the results of quantitative blood culture technique and the semiquantitative catheter tip culture method for the diagnosis of CRS has been more recently examined by Fan et al. (26) and Paya et al. (67). In the study by Fan et al., semiquantitative tip cultures and quantitative blood cultures performed by the pour plate method were compared with 24 catheters removed from patients with suspected CRS. CRS was defined as a central-to-peripheral bacterial count ratio of >7 or growth of >15 colonies from the tip of the catheter (26). Seven of the nine catheters positive by the semiquantitative method were also positive by the quantitative blood culture criterion. For none of the patients with sterile catheters was the central-to-peripheral bacterial count ratio significant. In the study by Paya et al. (67) that involved 52 intravascular cannulae from patients with sepsis, CRS was defined as the isolation of >15 CFU from the removed cannulae. The quantitative blood culture method detected only 7 of the 15 cases in which >15 CFU were isolated from the cannula. On the other hand, a high central-to-peripheral bacterial count ratio was found in four cases that did not fulfill the criterion for the diagnosis of CRS. The authors suggested the following explanations for the conflicting results: (i) colonization of intravascular devices may occur intraluminally with no involvement of the outer surface or the tip (77); (ii) blood may be contaminated during phlebotomy or, alternatively, the catheter tip may be contaminated during its removal through an infected exit site; and (iii) lysis-centrifugation

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may be sensitive enough to detect low-grade bacteremias that are associated with cannula tip counts of