CLINICAL MICROBIOLOGY REVIEWS, JUly 1990, p. 269-279 Vol. 3, No. 30893-8512/90/030269-11$02.00/0Copyright i© 1990, American Society for Microbiology

Quantitative Aspects of SepticemiaPABLO YAGUPSKY' AND FREDERICK S. NOLTE2*

Department of Microbiology and Immunology, University ofRochester Medical Center, Rochester, New York 14642,1 andDepartment ofPathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia 303222

INTRODUCTION ....................................................... 269DEVELOPMENT OF QUANTITATIVE BLOOD CULTURE METHODS ........................................269CLINICAL SIGNIFICANCE OF QUANTITATIVE BLOOD CULTURES ........................................271

Severity of Infection in Children and Adults ....................................................... 271Efficacy of Antibiotic Therapy....................................................... 272Endocarditis....................................................... 273Catheter-Related Sepsis (CRS) ....................................................... 274Contaminants versus True Pathogens in Positive Blood Cultures ..................................................276

CONCLUSIONS ....................................................... 276LITERATURE CITED ....................................................... 276

INTRODUCTION

Blood cultures are a crucial part of the evaluation ofpatients with suspected sepsis, and a positive blood cultureis the best criterion for defining that condition. In mostclinical microbiology laboratories, routine blood cultures areaccomplished by using broth media. Although broth-basedblood culture systems provide a sensitive means for recov-ering microorganisms from blood, they provide no informa-tion about the number of microorganisms present in theoriginal blood sample. In contrast, inoculation of blood ontosolid media results in growth of individual colonies whichcan be enumerated and the result can be used to calculate theconcentration of bacteria per milliliter of blood.

Until recently, the methods available for quantitativeblood cultures have been cumbersome and labor intensiveand did not find widespread use in clinical microbiologylaboratories. In 1976, Dorn et al. (18, 19) described a bloodculture technique based on lysis of cellular elements in bloodfollowed by concentration of microorganisms by centrifuga-tion and plating of the sediment on solid media. Improve-ments were made in the original technique, and a bloodculture system based on this method (Isolator MicrobialTube) was made commercially available by E. I. du Pont deNemours & Co., Inc., Wilmington, Del. A recent survey ofblood culture methods used in 288 clinical microbiologylaboratories revealed that 13% used the Isolator system forroutine bacteriologic culture and 30% used it for fungalculture (K. S. C. Kehl, Clin. Microbiol. Newsl. 8:127-133,1986). The availability of the Isolator system has sparked arenewed interest in the quantitation of microorganisms inpatient blood.

In this article, we review the development of quantitativeblood culture methods and published applications of thesemethods. We also critically evaluate the available data onpossible diagnostic and prognostic significance of quantita-tive blood culture results and identify areas for futureresearch. The subject of quantitative blood cultures has alsobeen reviewed recently by Kiehn (42).

* Corresponding author.

DEVELOPMENT OF QUANTITATIVE BLOODCULTURE METHODS

The first quantitative blood cultures were performed bythe pour plate or spread plate techniques. The pour platetechnique involves mixing blood and molten agar; with thespread plate technique, blood is spread over the surface ofan agar plate. The number of colonies that grow either in oron the agar is enumerated, and the CFU per milliliter ofblood are calculated. Both methods suffer because only asmall volume, usually 1 ml of blood, can be inoculated. Tomaximize the recovery of microorganisms, it is recom-mended that at least 10 ml of blood be cultured from adultpatients (36, 89, 94). As a result, these methods are ofpractical value only when the quantity of bacteria in blood isrelatively large.The magnitude of bacteremia in infants and children is

generally greater than that in adults so cultures of as little as0.5 to 1.0 ml of blood are considered adequate in most cases(94). Direct plating methods have been used in pediatrics toenhance the recovery of commonly encountered pathogenssuch as Haemophilus influenza and Neisseria meningitidisand to study the correlation between the magnitude ofbacteremia and clinical manifestations (52, 53).

Quantitative blood culture studies in pediatrics have usedseveral methods, including pour plate (1, 25); spread plate(75); quantitative direct plating, using heparin tubes (53); andlysis direct plating, using the Isolator 1.5 Microbial Tube(10-12, 98). La Scolea et al. (53) described a quantitativedirect plating method in which 0.5 to 1.0 ml of blood wascollected into a heparin tube; upon arrival in the laboratory,the blood was inoculated onto agar plates. The quantitativedirect plating method was compared with broth methods fordetection of bacteremia in children (53). It detected 83% ofthe cultures positive for H. influenza and 100% of thosepositive for N. meningitidis, but only 50% of the culturespositive for Streptococcus pneumoniae. The number ofother pathogens recovered was too small for evaluation. Ofthose cultures positive by both quantitative direct platingand a broth procedure, 55% were positive first by quantita-tive direct plating.The Isolator 1.5 Microbial Tube was designed for culturing

small-volume (0.5- to 1.5-ml) pediatric blood samples di-rectly to agar media. It contains 0.96 mg of the anticoagulantsodium polyanetholsulfonate and 0.1 ml of a saponin-con-

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training solution to lyse the cellular components of the blood.The lysis step allows the recovery of viable phagocytizedmicroorganisms. The Isolator 1.5 tubes have been comparedwith conventional and radiometric broth methods in severalclinical trials (10, 12, 98). In none of the trials did the Isolatorsystem recover significantly more organisms than the com-parative method, and in only one trial did the Isolator systemprovide positive results more rapidly. However, in each trialthere were substantial reductions in the time required toobtain isolated colonies with the Isolator system. Largeincreases in the contamination rate for Isolator cultures werereported in each evaluation and averaged 10.9%.

All of the direct plating methods described above arepractical only for small volumes of blood. Concentration-direct plating methods were developed to maximize thedetection of low-magnitude bacteremia or fungemia and toremove inhibitory factors that may be present in blood. Thebasic concept behind these methods is that blood cells arelysed, microorganisms are concentrated either by membranefiltration or centrifugation, and, finally, the concentrate isplated directly to solid media. Since the concentration pro-cedures effectively separate microorganisms from inhibitorysubstances in blood, these methods potentially improve therecovery of microorganisms.Brown and Kelsh (9) were the first to report use of a

membrane filter technique for recovery of Brucella spp. fromthe blood. Tidwell and Gee (88) found that detection ofexperimental bacteremia in rabbits was faster and morereliable with a filter technique than with a conventional brothmethod. In another study (69), the kinetics of Salmonellatyphi bacteremia during therapy were followed with a mem-brane filtration technique. These early studies used cumber-some and time-consuming methods in which only smallvolumes of blood were processed for culture. Winn et al.(104) and Finegold et al. (27) reported a differential mem-brane filtration technique which allowed the processing oflarger blood volumes and separate culture of different bloodcomponents. The formed elements of blood were separatedby differential centrifugation and plasma only was passedthrough a membrane filter. The filter was placed on thesurface of an agar plate, and the erythrocytes and leukocyteswere cultured separately on solid media. Isolated colonieswere available after 24 to 48 h of incubation, even in patientstreated with antibiotics. The technique provided quantitativeinformation and also facilitated the diagnosis of polymicro-bic 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 fol-lowing common urologic procedures was demonstrated, aswell as superior recovery of gram-positive bacteria in bothexperimental and human clinical studies. Komorowski andFarmer (48) found that a lysis-filtration technique was supe-rior to a conventional broth culture for detection of candi-demia. Lamberg et al. (50) described an improved filtrationmethod that did not require lysis of blood cells or sophisti-cated equipment. The blood is applied to a Ficoll-Hypaquegradient, centrifuged, and filtered. In a study with seededblood samples, this system had a sensitivity equivalent to thebroth method used for comparison. In spite of these prom-ising advances, membrane filtration techniques remain cum-bersome, expensive, and time-consuming, and no commer-cially available system is based on this technique.

In 1976, Dorn et al. (18, 19) described a concentrationtechnique for blood cultures termed lysis-centrifugation. In

this two-step process, the blood was lysed in one tube andthe lysate was transferred to a second tube, where microor-ganisms were concentrated by centrifugation onto a stabiliz-ing density layer composed of50% sucrose and 1.5% gelatin.After centrifugation, most of the supernatant was removedwith a needle and syringe. Aliquots of the sediment werethen plated to agar media. Dorn et al. used the new tech-nique in a study of 1,000 blood cultures from patientssuspected of having bacteremia (18). The blood was alsocultured in Trypticase soy broth and in pour plates. Of 176significant positive cultures, the lysis-centrifugation tech-nique recovered 73% and the pour plate and broth methodsrecovered 49 and 38%, respectively. The lysis-centrifugationtechnique demonstrated enhanced recovery of fungi, gram-positive cocci, and Pseudomonas spp. The superior perfor-mance of the lysis-centrifugation technique could be due tothe high theoretical dilution of antimicrobial factors (1:400)or to protection provided to cell-wall-damaged bacteria bythe hypertonic sucrose-gelatin cushion. Although this newtechnique showed promise, it was cumbersome and had ahigh (9.3%) rate of contamination.

In 1978, Dom and Smith (22) described a new centrifuga-tion blood culture device. It was essentially a double-stoppered evacuated glass tube containing 0.3 ml of a densehydrophobic cushion, 1.2 ml of an aqueous solution ofsodium polyanetholsulfonate, a lysing agent, and an anti-foaming agent. Blood was introduced into the tube andmixed thoroughly, and the tube was centrifuged at 3,000 x gfor 30 min in a fixed-angle rotor. After centrifugation, themajority of the supernatant was removed and discarded. Theremaining 1.5 ml of solution was equally dispersed to fiveagar plates.A clinical trial of this new blood culture device was

conducted with 3,335 blood cultures (20). All blood sampleswere cultured concurrently in supplemented peptone brothwith sodium polyanetholsulfonate under a C02-rich atmo-sphere. The centrifugation technique detected 80% of posi-tive cultures compared with 67% for the broth method.Superior recovery of Staphylococcus aureus, Pseudomonasspp., and yeasts was demonstrated, and the time required fordetection of a positive blood culture was shorter for thecentrifugation method. The contamination rate observedwith the new method, 1.4%, was comparable to that of thebroth method.A lysis-centrifugation device similar to that developed by

Dom et al. is commercially available as the Isolator 7.5- or10-ml Microbial Tubes. The Isolator system has been com-pared with both conventional and radiometric broth andbiphasic blood cultures in many clinical trials and overall haddemonstrated good performance (5-7, 33, 34, 38-40, 46, 63).

Certain common findings emerged from these evaluationsof the Isolator system. The lysis-centrifugation methodrecovered significantly more fungi than conventional broth,radiometric broth, or biphasic brain heart infusion bottles.The superiority of the lysis-centrifugation method for therecovery of mycobacteria (30, 45), especially Mycobacte-rium avium-M. intracellulare complex (31, 43, 44, 58, 104),Cryptococcus neoformans (5, 6), and Histoplasma capsula-tum (5, 100), from blood has been useful in diagnosis of theseinfections in immunocompromised patients. Enhanced iso-lation of staphylococci and members of the family Entero-bacteriaceae was also found in several studies (6, 33, 34, 38,39, 46,63). However, the Isolator system may not be optimalfor recovery of Streptococcus pneumoniae, Pseudomonasaeruginosa, and anaerobic bacteria (6, 33, 34, 39, 46, 63).The Isolator system was superior to conventional broth or

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QUANTITATIVE ASPECTS OF SEPTICEMIA 271

biphasic broth cultures in terms of speed of detection ofpositive cultures but, on average, was no faster than theradiometric method. Positive cultures with the lysis-centrif-ugation system occur as colonies on agar plates, and there-fore this system offers potential advantages in the timerequired to identify and determine antibiotic susceptibilityof isolates. However, for laboratories performing directidentification and antibiotic susceptibility tests from posi-tive blood culture bottles, this advantage may be less mean-ingful.The incidence of polymicrobic bacteremia has been in-

creasing steadily during the last decades, possibly as a resultof the expanding population of immunocompromised pa-tients, aggressive surgery, and widespread use of intravas-cular medical devices (41, 95, 96). Detection of polymicrobicbacteremia is extremely important to select the most appro-priate antimicrobial therapy. Blood cultures on solid mediahave some potential advantages over broth cultures becauseovergrowth of cultures by one organism is less likely tooccur on solid media, and morphologic differences betweenisolated colonies are easily recognized (34). Clinical trialshave confirmed the superiority of blood cultures performedon solid media for recovery of multiple organisms fromblood. In one study, 14 of 15 episodes of polymicrobicbacteremia were detected by lysis-centrifugation as opposedto only 3 of 15 detected by the broth culture method (40). Ina second study that included 25 episodes of polymicrobicbacteremia, the Isolator system detected 21 episodes and thebiphasic bottle detected only 17 (39). Blood cultures on solidmedia have also been shown to be superior to broth methodsfor diagnosis of polymicrobic bacteremia in catheter-relatedinfections (70, 72).One significant disadvantage of the Isolator system is the

relatively high contamination rate. This occurs because thelysis-centrifugation method requires additional processingsteps and the use of petri plates rather than closed bottles,which offer greater opportunity to introduce contaminants.Reported contamination rates for the Isolator system rangefrom 0.3 to 13.8% (81). Rates in different laboratories dependon definition of contamination, experience with the system,use of relatively dry solid media, and inoculation of plates ina laminar flow hood. It appears that use of a laminar flowcabinet is a critical factor for reducing contamination rates toan acceptable level. In one study, the contamination ratedropped from 9.5 to 6.2% (87) and in another study itdropped from 9.6 to 2.5% (94) when lysis-centrifugationblood 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 thetube before processing. Cashman et al. (13) conducted astudy with blood cultures seeded with low numbers of 26different bacterial species and demonstrated that a holdingtime of 15 h might be feasible for the Isolator tube. However,the authors observed substantial increases in numbers ofsome species and significant declines in others over the 15-hperiod. The quantity of bacteria recovered from specimensheld for this length of time would bear little relationship tothe quantity of bacteria originally present in the blood.Stockman et al. (80), in a prospective study of 5,125 fungalblood cultures, found that lysis-centrifugation tubes proc-essed within 9 h showed a significantly higher yield of yeasts,filamentous fungi, and bacteria than did those processedafter 9 h of hold time. With any of the direct plating bloodculture methods, including the Isolator system, specimensshould be processed as quickly as possible to enhance

recovery of pathogens and to ensure that colony counts trulyreflect the quantity of bacteria in vivo.

CLINICAL SIGNIFICANCE OF QUANTITATIVEBLOOD 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 ormanipulation of colonized mucosal surfaces to overwhelm-ing and often fatal infections. It has been estimated thatevery year in the United States half of the 200,000 patientswith septicemia die in spite of the progress achieved inantibiotic and supportive therapy (92, 94). Great efforts havebeen made to identify factors that contribute to this high casemortality rate.For more than 30 years, several authors have used quan-

titative blood cultures to investigate the magnitude of bac-teremia in septic patients and its correlation with the occur-rence of complications and death. The results of thesestudies have shown that most episodes of clinically signifi-cant bacteremia in adults are characterized by low numbersof circulating bacteria. Werner et al. (99) found that 54% ofblood cultures from patients with staphylococcal and strep-tococcal endocarditis contained between 1 and 30 CFU/ml ofblood. In a study of adults, Henry et al. found <1 CFU/ml ofblood in 27% of episodes of bacteremia due to Staphylococ-cus aureus, in 62% of those caused by Escherichia coli, andin 55% of patients with septicemia due to P. aeruginosa (34).Kreger et al. (49) found that 73% of 77 patients withgram-negative bacteremia had blood cultures which con-tained <10 bacteria per ml of blood.

In children, the magnitude of bacteremia is usually muchhigher than in adults and in general is inversely related to thechild's age, with the highest numbers of bacteria found in theblood of septic neonates. In neonatal E. coli sepsis, Dietz-man et al. (16) demonstrated that 78% of patients had >5CFU/ml of blood and one-third had bacterial counts inexcess of 1,000 CFU/ml. Studies performed in children withbacteremic diseases caused by encapsulated bacteria haveshown bacterial counts higher than 100 CFU/ml in 43% ofthe patients (3, 51, 52, 54, 62, 86, 98). Marshall and Bell (62)used quantitative blood cultures to study 83 episodes of H.influenzae bacteremia in children and found that thosepatients with high bacterial densities tended to be youngerthan those with low-grade bacteremia. A similar relationshipbetween age and magnitude of H. influenzae bacteremia wasdemonstrated by Moxon and Ostrow in the infant rat model(65).The relationship between magnitude of bacteremia and

severity of clinical picture has also been extensively inves-tigated. In a series of 20 adults with bacteremia and septicshock, Hall and Gold (32) found that all six patients with>100 CFU/ml of blood died while only 41% of those withlesser bacteremia died. Although the number of microorgan-isms in blood showed a moderate correlation with theoccurrence and severity of septic shock, some patients withbacterial counts as high as 300 CFU/ml of blood did notsuffer shock. In another study, Weil and Spink found acorrelation between magnitude of bacteremia in patientswith gram-negative sepsis and fatal outcome (95). The mor-tality rate for eight patients with <5 CFU/ml of blood was50%, while in 19 cases in which >5 CFU were isolated frompatients' blood, mortality increased to 84%.Although in a recently published study no correlation

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between magnitude of bacteremia and mortality was foundamong 55 patients with Staphylococcus aureus septicemia(61), data on gram-negative sepsis have consistently shownthat high-magnitude bacteremia is associated with poorprognosis. DuPont and Spink (24) reviewed 860 cases ofgram-negative bacteremia at the University of MinnesotaMedical Center from 1958 to 1966. Among the 655 adults,there was a definitive correlation between the number ofcolonies counted in pour plates and mortality. This correla-tion was seen in patients suffering from underlying diseasesof relatively good or intermediate prognosis, whereas pa-tients with poor prognosis usually died regardless of bacte-rial counts. Similar results were reported in a study of 242bacteremic patients seen at the University of MarylandHospital from 1965 to 1972 (47). A statistically significantcorrelation was found between mortality and magnitude ofgram-negative bacteremia, while a similar trend was foundamong patients with gram-positive and polymicrobic bacte-remia. The authors also stressed the role played by thenature and severity of the underlying disease in determiningthe clinical outcome.The relationship among gram-negative bacteremia, under-

lying disease, and mortality rate was also investigated byKreger et al. (49). They found that patients with more severeunderlying disease did not necessarily have higher bacterialcounts in the blood. However, the density of bacteremia wasrelated to fatality rates among patients with underlyingdiseases of similar severity, and of all patients combined, themortality rate was higher in those with > 10 bacteria per mlof blood.

It is apparent that the magnitude of bacteremia in adultpatients correlates with mortality rate. However, becausebacteremia usually results from breakdown of defense mech-anisms that control infection, underlying debilitating dis-eases appear to be a more important factor than the numberof CFU per milliliter of blood in determining the ultimateprognosis. Intravascular foci of infection such as endocardi-tis and catheter-related sepsis may be important exceptionsto these generalizations about the relationship between themagnitude 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 wasdirectly related to the incidence of metastatic complications.Six of 7 patients with 2 100 CFU/ml in blood from a vesselhad metastatic foci of infection, whereas none of 7 patientswith c 10 CFU/ml had metastatic complications (P < 0.01).The remaining patient had 20 CFU/ml of blood and did notdevelop 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 firmlyestablished. Using the pour plate technique, Dietzman et al.(16) studied the quantitative aspects of E. coli septicemia in30 neonates and found a bimodal distribution of bacterialdensity: 65% of the patients had <200 CFU/ml, and 31% hadbacterial counts of >1,000 CFU/ml. Only 1 of 9 patients with<1,000 CFU/ml had meningitis as compared with 6 of 11with colony counts of >1,000 CFU/ml. In a study of 83 H.influenzae type b bacteremic episodes, it was demonstratedthat children with high-density bacteremia had a significantlyshorter duration of illness before presentation (median, 1day) than did low-grade bacteremic patients (median, 3days), and such children had a higher rate of complicationsincluding 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. men-ingitidis, also demonstrated a clear relationship betweenseverity of disease and bacterial counts (3, 25, 54, 55, 62, 75,85, 86, 98). For H. influenza, the combined results of thesestudies show that 50 of 72 (69.4%) patients with meningitisand 19 of 28 (67.9%) patients with epiglottitis had bacterialcounts of >100 CFU/ml, but only 8 of 42 (19.0%) patientswith less severe infections such as pneumonia, cellulitis,osteomyelitis, arthritis, otitis media, or bacteremia withoutdemonstrable focus had bacterial densities as great (3, 54,55, 62, 86, 98). Among the patients with S. pneumoniaebacteremia, 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 withN. meningitidis infections show that 15 of 24 (62.5%) pa-tients with meningitis and half of those with Waterhouse-Friderichsen syndrome have >100 CFU/ml. Bacterial countsof <100 CFU/ml were observed in 7 of 10 (70%) cases withmild focal meningococcal disease or occult bacteremia (85,86). In summary, 71 of 104 (68.3%) children with meningitisdue to H. influenza, S. pneumoniae, or N. meningitidis butonly 37 of 145 (25.5%) with other bacteremic diseases causedby these organisms had bacterial counts of >100 CFU/ml(P < 0.001).

Febrile diseases of infectious origin are more common inearly 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 and24 months have bacteremic infections that may result insevere complications and death (3, 37). Clinical signs are notalways reliable criteria for the identification of bacteremia.Occasionally, an ill child managed as an outpatient with a"benign" infection is already bacteremic and will laterdevelop meningitis (86). Identification of febrile children atrisk of complications of bacteremia is important to ensureappropriate evaluation and antibiotic therapy. In one report,four pediatric outpatients who presented with seeminglymild respiratory infections and otitis media had blood cul-tures obtained because of high fever (85). Three developedmeningitis due to S. pneumoniae or N. meningitidis, and thefourth developed H. influenza epiglottitis. In each case,counts on the initial blood culture were >100 CFU/ml. In asecond study, three children initially admitted without evi-dence of focal infection, in whom N. meningitidis wasrecovered from blood in excess of 500 CFU/ml, developedmeningitis (85).Modern quantitative blood culture techniques allow both

rapid detection of bacteremia and provide information on itsmagnitude. When high bacterial counts in the blood of achild are found, careful reevaluation is necessary to identifyand treat potential life-threatening complications especiallyin those children sent home after initial evaluation. Althougha relationship has been established between the severity ofdisease and bacterial density in the blood for H. influenza,S. pneumoniae, and N. meningititis, more studies areneeded to establish this relationship for other pathogenssuch as group B beta-hemolytic streptococci and Staphylo-coccus aureus.

Efficacy of Antibiotic TherapyDetection of bacteremia by a positive blood culture fol-

lowed by antibiotic susceptibility testing of the isolate arecrucial for confirmation of the diagnosis and rational selec-tion of antibiotics. It is accepted that administration of

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appropriate antibiotic therapy should result in negativeblood cultures. Persistence of positive blood cultures isinterpreted as evidence of treatment failure, and changes inantibiotic therapy are usually made. However, in certainclinical situations such as infections in immunocompromisedpatients, M. avium-M. intracellulare complex infections inpatients with acquired immunodeficiency syndrome, or theslowly resolving bacteremia of typhoid fever, prompt steril-ization of the blood does not always occur. Serial quantita-tive blood cultures have potential value as an in vivo test ofantibiotic efficacy. Using a membrane-filtration technique,Randriambololona and Dodin (69) demonstrated that admin-istration of chloramphenicol to patients with typhoid feverresults in gradual decrease in the magnitude of bacteremiaover 3 to 8 days, followed by negative cultures. Normaliza-tion of body temperature occurs close to the time of clear-ance of Salmonella typhi from the blood.

Marshall and Bell (62) recently reported the results of aquantitative blood culture study in 81 bacteremic children.They showed that children with partially treated H. influ-enzae meningitis had lower bacterial counts in blood thanuntreated controls, and another report (102) demonstratedthat, in five appropriately treated patients with persistentlypositive blood cultures, broth cultures concealed a promptdecline in the number of bacteria circulating in the bloodseen in quantitative blood cultures. The declining colonycounts supported the clinical assessment that patients re-sponded to therapy. In 1982, Fojtasek and Kelly (30) re-ported that quantitative blood cultures were useful for mon-itoring the response to antibiotic therapy in a renal transplantrecipient with M. chelonae bacteremia. Mycobacterialcounts initially ranged from 50 to 70 CFU/ml and dropped to45 CFU/ml after 2 weeks of amikacin therapy. Cefotaximewas added to the antibiotic regimen and the density droppedto 5 CFU/ml after several days of combined therapy. Thecounts rose to 25 to 30 CFU/ml when cefotaxime alone wascontinued. By this time, MIC data were available andshowed that the organism was resistant to cefotaxime andsusceptible to amikacin and cefoxitin. The antibiotic therapywas changed to cefoxitin; the level of mycobacteremiadecreased (0.2 to 2 CFU/ml) and remained at that level forseveral weeks. Blood cultures became negative after re-moval of the transplanted kidney and one of the arteriove-nous fistulas used for hemodialysis.With the emergence of acquired immunodeficiency syn-

drome, disseminated M. avium-M. intracellulare complexinfections have become common. The disease is character-ized by heavy infiltration of the reticuloendothelial systemby the bacterium and by prolonged high-level mycobactere-mia (44, 58, 105) resembling that seen in lepromatous leprosy(23). Due to difficulties in assessing the clinical response toantibiotic therapy, serial quantitative blood cultures havebeen used to monitor the effectiveness of antibiotic treat-ment (58, 105). In some patients bacterial counts decreasedor blood cultures became negative with appropriate therapy,but in other patients counts remained stable or even in-creased. Regardless of the quantitative changes in the mag-nitude of bacteremia, diffuse infiltration of various organs bymycobacteria was observed in all patients at autopsy, indi-cating that, as in lepromatous leprosy, controlling bactere-mia does not guarantee control of tissue infection (105).Some patients with septicemia deteriorate clinically in

spite of antibiotic therapy that is highly effective against theinfecting organism in vitro. Using quantitative blood culturesand a Limulus lysate gelation assay, Shenep et al. (76)showed that the amount of total circulating endotoxin in

patients with gram-negative septicemia was related to themagnitude of bacteremia and that the level of free endotoxinrose sharply after administration of antibiotics. They postu-lated that destruction of bacterial cells by antimicrobialagents releases endotoxin and other bacterial products se-questered within the intact bacterial cell into the blood-stream, aggravating the host cell injury and worsening theclinical disease. The serial quantitation of plasma endotoxinlevels and bacteremia helps explain the paradoxical responsesometimes seen in patients treated appropriately for gram-negative sepsis. However, neither the level of bacteremianor the level of endotoxemia alone determined the clinicalseverity of gram-negative sepsis in this study.

Quantitative blood cultures can provide useful informationabout in vivo efficacy of antibiotic therapy in patients withpersistently positive blood cultures; however, further stud-ies are needed to extend these observations to larger groupsof patients with septicemia. Serial quantitative blood cul-tures obtained from patients receiving antibiotics could beused to study the in vivo relevance of certain in vitrophenomenon, such as antibiotic tolerance and antibioticsynergy.

Endocarditis

Bacterial endocarditis, a uniformly fatal disease before theantibiotic era, was the subject of several early quantitativeblood culture studies. More than 70 years ago, Warren andHerrick (90) used the pour plate technique to show that abroad range of bacterial counts, from one to several hundredCFU per milliliter, occurred in the blood of patients withendocarditis. In 1925, Libman demonstrated that magnitudeof bacteremia in a particular patient remains fairly constantduring the course of the disease (56). Seven years later,Weiss and Ottenberg (97) studied the relationship betweennumber of circulating bacteria and febrile response in pa-tients with endocarditis. The results showed that there was aconstant and relatively uniform number of bacteria releasedinto the blood from vegetations, with periodic liberation oflarger numbers of bacteria into the circulation ("bacterialshowers"). In four cases of subacute endocarditis, the riseand fall in the magnitude of bacteremia was followed bycorresponding changes in patient body temperature. How-ever, fever spikes lagged behind the peak levels of bactere-mia by several hours. The authors suggested that diagnosticblood cultures should be obtained at periods of low temper-ature just prior to the next expected temperature rise to takeadvantage of the large number of bacteria present in bloodand thus maximize the chances to detect the septic episode.They also confirmed previous observations that the severityof disease, measured by patient survival period, was notrelated 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 en-docarditis when the disease course was not substantiallymodified by available therapy. Patients frequently sufferedfor months before dying from cardiac or embolic complica-tions, allowing the opportunity for studies that providedimportant clues for understanding the pathophysiology ofbacteremia. Blood was drawn simultaneously from catheter-ized femoral arteries and veins, hepatic and renal veins,superior vena cava, and right auricle in a group of sixpatients. In general, bacterial counts in the femoral veinwere slightly lower than in the corresponding arterial cul-tures, indicating that some circulating bacteria were retainedin the peripheral vascular bed, a fact consistent with the

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frequent occurrence of embolic lesions in the extremities ofpatients with endocarditis. The greatest reduction in bacte-rial counts was found in the hepatic veins (97% decrease),showing the importance of the liver and spleen as sites ofremoval of circulating microorganisms.

In another study, the sensitivity of arterial, venous, andbone marrow cultures in the detection of bacteremia wasexamined in 88 patients with endocarditis due to viridansstreptococci (74). By using the pour plate technique, it wasshown that bacterial counts in arterial and venous bloodwere approximately the same magnitude. In this study,cultures of bone marrow were superior to arterial andvenous cultures in detecting bacteremia, but no quantitativeinformation for bone marrow cultures was provided.

In 1967, Werner et al. (99) published a retrospectivereview of 206 cases of bacterial endocarditis seen over 17years at the New York Hospital-Cornell Medical Center.They showed that the magnitude of bacteremia in strepto-coccal and staphylococcal endocarditis was generally of loworder and the number of bacteria per milliliter of blood wasrelatively constant. In this study, the cumulative detectionrates of the first two cultures was 98.5% in cases of strepto-coccal endocarditis and 100% in cases caused by otherorganisms. The clinical implication is that, in patients withendocarditis, blood cultures should be persistently positive;therefore, it is seldom necessary to obtain more than two orthree blood cultures over a 24-h period to make the diagno-sis.

Quantitative blood cultures have also been used to localizethe site of infection in patients with endocarditis, and espe-cially in those with involvement of the right side of the heart.At the time when these studies were conducted, modernsonographic techniques were not available, and diagnosis ofright-sided endocarditis was particularly difficult. In onestudy, published in 1973, Reyes et al. performed intraoper-ative quantitative blood cultures from each cardiac chamberduring 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. In1974, Pazin et al. performed quantitative blood cultures ofcardiac chambers and great vessels during the course ofcardiac catheterization of two intravenous drug abusers withbacteremia (68). Finding of unusually high bacterial countsdistal to a valve was considered diagnostic of involvement ofthat particular valve in the disease. The technique succeededin confirming endocarditis as the cause of patient bactere-mia, in excluding other potential sources of sepsis, and inlocalizing the site of the infection.

Quantitative blood cultures have contributed to our under-standing of the basic pathophysiology of bacterial endocardi-tis. The bacteremia is continuous, with the number ofbacteria per milliliter of blood usually low and relativelyconstant. Fever spikes lag behind the peak levels of bacter-emia by several hours in patients with endocarditis. Theearly quantitative studies of bacteremia in endocarditishelped form the basis for the current recommendations forthe number and timing of blood cultures (89, 91, 93). The useof serial quantitative blood cultures to monitor the efficacy ofantibiotic therapy of endocarditis should be investigated.

Catheter-Related Sepsis (CRS)

Intravenous infusion therapy has become a cornerstone ofmodern medical management. Total parenteral nutrition,prolonged chemotherapy for cancer, and intensive care ofcritically ill patients have led to the development of new

techniques for establishing access to central veins. In 1973,Broviac et al. reported the use of a silicone-rubber catheterthat was inserted into a central vein but exited at a distantsite after passing through a subcutaneous tunnel (8). A smallcuff encircling the catheter at the exit site became fixed inplace by fibrotic tissue and provided mechanical stability anda barrier to infection. Unfortunately, emergence of a newsort of intravascular infection has been one of the untowardeffects of central venous catheterization. Infections of thesetunneled, cuffed silicone catheters are categorized into threenonexclusive 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, infec-tion of intravascular devices is today's leading cause ofnosocomial bacteremia (15). Catheter-related exit site andtunnel infections are readily diagnosed on the basis of localinflammation, purulence, fever, and leukocytosis. The diag-nosis of septic infections, however, is more difficult due tothe lack of localizing signs and the fact that patients requiringcentral venous access are among the most debilitated pa-tients, often with multiple potential sources of bacteremia.Several techniques have been developed to help diagnoseCRS in these cases, including the semiquantitative cathetertip culture pioneered by Maki et al. (60), the use of Gramstain or impression smears of the catheter, and quantitativecultures of the catheter obtained by flushing the catheterwith nutrient broth (14). A major drawback with thesetechniques is that the catheter must be removed from thepatient before the diagnosis can be made. In one report,more than two-thirds of the central venous catheters re-moved because of suspected CRS were found to be sterile orproved not to be the source of the septicemia (64).

Quantitative blood cultures have been advocated for diag-nosis of CRS. In 1979, Wing and colleagues (103) docu-mented CRS in a patient with a Broviac catheter, using pourplate blood cultures. Blood drawn from a peripheral veingrew 25 CFU of Enterobacter cloacae and Citrobacterfreundii per ml. Blood drawn from the catheter grew>10,000 CFU of the same organisms per ml. The catheterwas removed and found to have an adherent clot present atthe tip. Culture of the clot also grew E. cloacae and C.freundii. Whimbey et al. (102) reported two cases in whichlarge differences in colony counts in blood obtained simul-taneously from a peripheral vein and the Broviac catheterwere used to identify the catheter as the source of infection.

In one series of 28 children with Broviac catheters, 100surveillance blood cultures were obtained through the cath-eter in afebrile, clinically well patients and from both aperipheral vein and the central catheter during 30 febrileepisodes (70). All blood samples were cultured both by thespread plate method and in nutrient broth. Of the 100surveillance cultures, 79 were negative by both methods.Eighteen cultures showed growth, but the catheters were notconsidered infected because <10 colonies grew on theplates, multiple organisms or organisms of doubtful clinicalsignificance were recovered, or concordance between platesand broths was lacking. Subsequent catheter surveillancecultures were negative in all 18 patients. The remaining threepositive surveillance cultures were from the same child andgrew >100 CFU of Bacillus cereus per ml. Although noillness resulted, the catheter was removed and a Bacillusspecies and P. aeruginosa were recovered from the tip.These findings were considered consistent with contamina-tion 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 theblood drawn through the catheter were at least 10 timesgreater 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 intwo patients with septicemia not attributable to the catheterhad low colony counts in blood from the catheter. Surveil-lance blood cultures from the catheters were not helpful inpredicting subsequent septicemia in any patients.

In a short communication by Myint and Lowes (S. Myintand A. Lowes, Lancet ii:269-270, 1985), bacterial counts of>1,000 CFU of coagulase-negative staphylococci and En-terococcus faecium per ml were found in culture of thecentral venous catheters in two different patients while theperipheral cultures yielded 100 and 1 CFU/ml, respectively.

In another report, quantitative blood cultures were ob-tained from paired peripheral veins and central catheters in63 infants with umbilical artery indwelling catheters orBroviac central venous catheters in whom sepsis was sus-pected (72). Infants in whom both central and peripheralcultures were positive were considered to have provensepsis, whereas infants with positive central catheter cul-tures but negative peripheral cultures were diagnosed ashaving colonized catheters without sepsis. In 10 of 21children with confirmed sepsis, colony counts in culturesdrawn from central lines were >50% higher than the corre-sponding peripheral counts, suggesting CRS. Paired colonycounts were approximately equal in one case, suggestingthat sepsis was not related to the catheter. Confluent growthin the remaining 10 pairs prohibited precise colony countsand any conclusions about the source of bacteremia.

In 1987, Mosca et al. (64) reported their experience withthe use of quantitative blood cultures in 26 intravasculardevices suspected of being the source of sepsis. A fivefold orgreater central-to-peripheral bacterial count ratio was con-sidered confirmatory of CRS. In the eight instances in whichthe diagnostic criterion was fulfilled, removal of the catheterresulted in prompt defervescence of the septic patients. Inhalf of the remaining 18 cases in which the central venouscultures were sterile or the differences in colony counts weresmall, the catheter was removed but the patients' conditionsdid not improve. The remaining nine catheters were left inplace 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 ratioused to identify CRS ranged from 1.5- to several hundred-fold. Establishing an appropriate ratio is complicated by thefact that central venous blood passes through the lungsbefore it reaches the peripheral veins. Since bacteria areremoved from the blood by reticuloendothelial cells presentin the lung, the final concentration of microorganisms inperipheral blood is less than the concentration in catheterblood even in those patients with sepsis from other sources.To address this problem, Flynn et al. (29) used simulta-

neous quantitative blood cultures of samples obtained fromthe superior vena cava and the femoral vein of rabbits withnon-catheter-associated bacteremia. As expected, signifi-cantly larger bacterial concentrations were found in bloodfrom the superior vena cava compared with concentrationsin peripheral blood. Based on data collected in this animalmodel, the authors calculated that 99% of non-catheter-related bacteremia should show differences of <fivefold inbacterial concentrations of central and peripheral blood andproposed this criterion for diagnosing CRS. In two clinicalstudies they found that, in patients with CRS, the observed

central-to-peripheral bacterial count ratio far exceeded theproposed value and ranged from 57 to >100 fold (28, 29).

In spite of the potential advantages of quantitative bloodcultures as a means of identifying CRS and evaluating theefficacy of antibiotic therapy without removing the catheter,few studies comparing quantitative blood cultures and thesemiquantitative method of Maki et al. (60) have beenpublished. In 1983, Moyer et al. (66) compared results ofperipheral nonquantitative blood cultures, quantitative cul-tures obtained through the catheter, and semiquantitativecultures of the catheter tips. Among nonbacteremic patients,all central line blood cultures were negative or grew <24CFU/ml, but 15 of 68 catheter tips grew >15 colonies.Among the five bacteremic patients, four catheter bloodcultures grew >25 CFU/ml and >15 colonies were recoveredfrom all catheter tips.

In a prospective study of total parenteral nutrition-relatedinfections, a pour plate method was used to culture bloodfrom the central line and the semiquantitative technique ofMaki et al. (59) was used to culture intravascular linesegments when it was removed (78). Blood from the catheterwas collected twice a week for pour plate cultures. Twelveof 100 courses of total parenteral nutrition resulted in infec-tions: five patients had sepsis and seven had local infections.In 5 of 12 patients, pour plate cultures were positive, with 8CFU of Candida parapsilosis per ml and 2 25 CFU/ml whenbacteria were recovered. In 9 of 12, cultures of the intravas-cular segments were positive and grew >50 CFU/plate. Allof the septic patients had positive line segment cultures (50to >100 CFU/plate), but only two of the five had positivepour plate cultures of central line blood. No quantitativeperipheral blood cultures were done. Despite the infrequentpositive pour plate cultures results, the authors concludedthat these cultures were helpful in implicating the totalparenteral nutrition line in three instances because the pourplate culture results antedated positive peripheral bloodculture results or removal of the line.The correlation between the results of quantitative blood

culture technique and the semiquantitative catheter tip cul-ture method for the diagnosis of CRS has been more recentlyexamined by Fan et al. (26) and Paya et al. (67). In the studyby Fan et al., semiquantitative tip cultures and quantitativeblood cultures performed by the pour plate method werecompared with 24 catheters removed from patients withsuspected CRS. CRS was defined as a central-to-peripheralbacterial count ratio of >7 or growth of >15 colonies fromthe tip of the catheter (26). Seven of the nine catheterspositive by the semiquantitative method were also positiveby the quantitative blood culture criterion. For none of thepatients with sterile catheters was the central-to-peripheralbacterial count ratio significant.

In the study by Paya et al. (67) that involved 52 intravas-cular cannulae from patients with sepsis, CRS was defined asthe isolation of >15 CFU from the removed cannulae. Thequantitative blood culture method detected only 7 of the 15cases in which >15 CFU were isolated from the cannula. Onthe other hand, a high central-to-peripheral bacterial countratio was found in four cases that did not fulfill the criterionfor the diagnosis of CRS. The authors suggested the follow-ing explanations for the conflicting results: (i) colonization ofintravascular devices may occur intraluminally with noinvolvement of the outer surface or the tip (77); (ii) bloodmay be contaminated during phlebotomy or, alternatively,the catheter tip may be contaminated during its removalthrough an infected exit site; and (iii) lysis-centrifugation

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may be sensitive enough to detect low-grade bacteremiasthat are associated with cannula tip counts of <15 CFU.

In the past it was assumed that CRS could not be cured aslong as the catheter remained in place (59, 73). However, ineight reported series of Broviac-type catheter infectionsreviewed by Decker and Edwards (15), eradication of theorganism causing sepsis was accomplished without removalof the central line in 75% of the infections and, morerecently, in 61% of episodes reported by Benezra et al. (4).Most of the current methods require that the catheter beremoved to confidently characterize a septic episode ascatheter related. However, identifying the catheter as thesource of the bacteremia is immaterial if clinical manage-ment and prognosis are the same regardless of the source.Comparison of paired quantitative central catheter and

peripheral venous blood cultures shows promise as a meansof diagnosing CRS, and it is the only available method thatdoes not require removal of the catheter. However, for thisapproach to gain universal acceptance, additional studiescomparing this method with other techniques are needed. Inaddition, larger numbers of actual catheter infections need tobe studied to determine the range of central-to-peripheralbacterial count ratios that occur so that the sensitivity,specificity, and predictive values of this method for thediagnosis of CRS can be established. Processing bloodcultures by quantitative methods provides additional infor-mation, but that information has not yet been demonstratedto be necessary to management or to correlate with outcomeof CRS (15).

Contaminants versus True Pathogens in PositiveBlood Cultures

Isolation of "classic" pathogens such as Streptococcuspneumoniae or Salmonella typhi from blood culture isalways considered evidence of true bacteremia. However,when microorganisms of low virulence, especially skin flora,are recovered from blood cultures, they are traditionallydismissed as contaminants (57). Advances in medical thera-peutics have resulted in an increasing population of immu-nocompromised patients due to antineoplastic chemother-apy, organ transplantation, and survival of extremelypremature neonates. In addition, the use of intravascularcatheters and prosthetic devices has become part of standardmedical practice (17). As a result of these changes, micro-organisms that were once considered nonpathogenic haveemerged increasingly as causes of disease. At present,coagulase-negative staphylococci are leading causes of bac-teremia in neonatal intensive care units and in patients withinfected prosthetic heart valves, orthopedic prostheses, andventriculoperitoneal shunts (17). A definition of contamina-tion based on clinical criteria, the number of positive cul-tures, and the identity of the microorganism can often beused in immunocompetent patients, but in the immunocom-promised host, bacteremia due to an opportunistic pathogencannot be easily excluded. Finding reliable criteria to distin-guish true positive and contaminated blood cultures wouldbe of great value. False-positive blood cultures can result inwasted laboratory effort, the overuse of antibiotics, unto-ward drug effects, and prolonged hospitalization.

Quantitative information provided by the lysis-centrifuga-tion method has been proposed as an objective means todistinguish true bacteremia from contamination. For coagu-lase-negative staphylococci, Propionibacterium acnes, andaerobic diphtheroids, Dorn et al. (21) found that colonycounts of <1 CFU/ml of blood were invariably associated

with contamination, as determined after evaluation of thepatients' medical records. However, 16% of contaminatedblood cultures contained >1 CFU/ml. In a study of neonatesand young infants with umbilical and Broviac catheters,isolation of skin flora from blood cultures or recovery ofmicroorganisms after day 3 of incubation were consideredevidence of contamination (72). Colony counts of <10 CFU/ml were observed in 6 of 7 contaminated cultures but in only1 of 21 blood cultures from septic patients. McLaughlin et al.(63) found 1 CFU/ml of blood in 98 of 138 (71%) bloodcultures contaminated with coagulase-negative staphylo-cocci, alpha-hemolytic streptococci, or diphtheroids, whilethe majority of true pathogens were isolated at a level of >1CFU/ml. However, not all cultures from which <1 CFU/mlwere isolated represented contamination. Similar resultswere obtained by Hill et al. (35), Welch et al. (98), Kelly etal. (40), and Campos and Spainhour (10).

In summary, a clear trend exists toward low colony countsin contaminated blood cultures compared with true positivecultures. However, no quantitative criterion can invariablydistinguish contamination from bacteremia. Available datasuggest that quantitative blood cultures are useful in inter-preting positive blood culture results when considered alongwith clinical criteria and the identity of the microorganism.

CONCLUSIONS

For many years quantitative blood cultures found onlylimited use in the diagnosis and management of septicemiabecause the available methods were cumbersome, laborintensive, and practical only for small blood volumes. De-spite limitations, early studies of the quantitative aspects ofbacteremia added to our understanding of sepsis and itscomplications. The development and commercial availabil-ity of lysis-centrifugation and lysis-direct plating methodshave provided clinical microbiologists convenient and sen-sitive methods for quantitative blood cultures and haverenewed interest in the quantitation of microorganisms inpatient blood. Recent attention has been focused on thediagnostic and prognostic significance of determining themagnitude of bacteremia in various clinical circumstancesand patient populations. Although no study has shownunequivocally that quantitative blood culture results arenecessary for accurate diagnosis or appropriate managementof septic patients, there are certain circumstances in whichbacterial counts can be helpful. Two of the most promisingareas for additional research are for diagnosis of centralvenous catheter-associated bacteremia and assessment ofthe efficacy of antibiotic therapy. There are several funda-mental issues that need to be addressed before small differ-ences in the concentration of bacteria in blood can beinterpreted meaningfully. The reproducibility of the colonycounts obtained with the quantitative blood culture methodsand the normal variation in the level of bacteremia thatoccurs over time need to be defined. In addition, theseobservations need to be extended to larger groups of septicpatients before the utility of quantitative blood cultureresults can be adequately assessed.

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