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The importance of volume of blood cultured in the detection of bacteremia and fungemia
DIAGNMICROBIOLINFECTDIS 1983;1:107-110
107
The Importance of Volume of Blood Cultured in the Detection of Bacteremia and Fungemia Duane M. Ilstrup and John A. Washington II
An analysis was made on the basis of 5,389 isolates from 5,008 positive blood cultures of the relative yields from 10, 20, and 30 ml of blood. Average yields from cultures of 20 and 30 ml of blood were, respectively, 38 and 61% greater than that from 10 ml of blood.
INTRODUCTION As emphasized recently in a revised Cumitech on blood cultures by Relier et al. (1982), the volume of blood cultured appears relatively more important than medium or atmosphere of incubation in the detection of septicemia. In controlled evaluations of two sample sizes of blood cultured in separate bottles containing the same medium and incubated under identical atmospheric conditions, Tenney et al. (1982) have demonstrated a 29% greater yield of organisms from a 7-ml sample than from a 2ml sample, and we have demonstrated a 25% greater yield of organisms from a 15ml sample than from a 5-ml sample (Washington, 1978). Data on the effects of culturing larger volumes of blood on yield are limited for various reasons, including the physiological and ethical restrictions on the volume of blood collected for culture, the necessity of diluting blood on a 1:5 to 1:10 basis in broth, and different atmospheres of incubation favoring growth of certain microorganisms and, as a consequence, the requirement for two blood culture bottles permitting aerobic and anaerobic incubation (Relier et al., 1982). Assuming, however, that each of two or more blood culture bottles is inoculated with equivalent volumes of blood, that the effects on organism yield of different types of media are minimal, and that the number of isolates favored by aerobic incubation conditions is approximately equal to those favored by anaerobic incubation conditions, it should be possible to plot the cumulative effects on yield of culturing blood in two or more media. Thus, in a small study of 174 positive blood culture vials, Salventi et al. (1979) demonstrated the following percentage increments in organism yield per volume of blood cultured: 5-10 ml, 13%; 10-20 ml, 13%; 20-30 ml, 7%; and 30-40 ml, 5%. The purpose of this study was to examine the cumulative effects of culturing 30 ml of blood from adults with clinically suspected sepsis at the Mayo Clinic and affiliated hospitals between 1976 and 1981.
From the Mayo Clinic and Mayo Foundation, Rochester, Minnesota. Address reprint requests to: John A. Washington II, M.D., Head Section of Clinical Micro-
biology, Mayo Clinic, Rochester, MN 55905. Received November 1, 1982; accepted January 12, 1983. © 1983 Elsevier Science Publishing Co., Inc., 52 Vanderbflt Avenue, New York, NY 10017
0732-8893/83/03.00
108
D.M. Ilstrup and J.A. Washington III
METHODS Blood Culture Procedure During the period of study, 30 ml of blood was collected by phlebotomists for each culture; 10 ml was inoculated into each of three blood culture bottles. For purposes of this study, isolates of Staphylococcus epidermidis, Bacillus, Corynebacterium, and Propionibacterium were excluded, and the analysis was limited to those cultures in which each of the following three blood culture bottles was inoculated. Bottles 1 and 2 contained 100 ml of soybean-casein digest (Tryptic Soy Broth, Difco Laboratories) with 0.025% sodium polyanetholsulfonate (SPS) under vacuum with 10% CO2. Bottle 1 remained unvented, while bottle 2 was transiently vented prior to incubation. Bottle 3 contained different media being evaluated in succession: 100 ml of transiently vented soybean-casein digest (Trypticase Soy Broth, BBL Microbiology Systems), brucella broth (Difco Laboratories), and brain heart infusion broth (Difco Laboratories) with 0.025% SPS under vacuum with 10% CO2 or a transiently vented bottle containing biphasic brain-heart infusion medium with SPS and prepared in our laboratory. Evaluations of each of these media showed their performance to have been equivalent to that in bottle no. 2 (Hall et al., 1978, 1979}. Each bottle was examined after approximately 4-6 hrs of incubation, daily for 7 days, and finally on the 14th day of incubation. With the exception of the biphasic medium bottle, which was tipped at the time of each examination so that the blood-broth mixture flowed over the agar slant, routine subcultures were performed between 6-17 hrs and after 48 hrs of incubation onto chocolate blood agar plates which were incubated at 35°C in an atmosphere containing 5-10% CO2 for 48 hrs.
Data Analysis Since three bottles were always inoculated simultaneously, the results for 2 bottles versus 1 bottle, 3 bottles versus 1 bottle, and 3 bottles versus 2 bottles had to be evaluated as averages of the possible combinations of the three bottles. For example, the effect of using 3 bottles (30 ml) versus 2 bottles (20 ml) is presented as the average of: i)
yield of bottles 1, 2, and 3 x 100 yield of bottles 1 and 2
ii)
yield of bottles 1, 2, and 3 yield of bottles 1 and 3 x 100
iii)
yield of bottles 1, 2, and 3 yield of bottles 2 and 3 x 100.
RESULTS An analysis was made of the frequency of positivity of each bottle alone and in combination with each or both of the other two bottles (Table 1). A total of 5,389 organisms was isolated from 5,008 positive cultures. The reasons for the smaller number of isolates from bottle 3 alone remain unknown since the phlebotomists followed no fixed order of inoculation of bottles and since there had been no statistically significant differences between the number of isolates overall recovered by the media represented in bottle 3 and those recovered from bottle 2 (Hall et al., 1978, 1979).
Detection of Bacteremia a n d Fungemia
109
TABLE 1. N u m b e r of Isolates from Blood Culture Bottles Bottle"
No. of isolates
1 alone 2 alone 3 alone 1 + 2 1 + 3 2 + 3 1 + 2 + 3
3,457 3,784 2,891 4,724 4,435 4,639 5,389
"Each of the 3 bottles was inoculated with 10 ml of blood. Bottle 1 = TSB, 100 ml with 0.025% SPS, under vacuumwith 10% CO2,unvented; 2 = same as no. 1 excepttransientlyvented;3 = varioustransiently vented media with 0.025% SPS (see text). If the yield from culture of 10 ml of blood in any single bottle was taken as 100%, then the average yields from cultures of 20 ml in two and 30 ml i n three bottles were, respectively, 138% and 162%. If the yield from any c o m b i n a t i o n of two bottles (i.e., 20 ml of blood) was taken as 100%, the yield from all three bottles (30 ml) averaged 117%. A n alternative analysis is to estimate the percent of cultures in w h i c h c o m b i n a t i o n s of only one or two bottles w o u l d be "false negatives" w h e n the three bottle combination is the standard of comparison. With our data, the average percent of false negatives with only one bottle w o u l d be 37% and with two bottles w o u l d be 15%. DISCUSSION Although our results must be qualified by differences among the three bottles inoculated i n composition of media, atmospheres of incubation, a n d yield, it should nonetheless be possible, on the basis of our data, to plot a general relationship between the v o l u m e of blood cultured and yield [Figure 1). Other studies (Tenney et al., 1982; Washington, 1978; Salventi, 1979] have also s h o w n increased yield with increased volume. FIGURE l. Yield of organisms relative to volume of blood cultured. 170
Relative Yield (%of Isolates With Only 10 ml. Of Blood)
160 m
150 m
140 m
130 B
120 B
100
m
I
I
10
20
Volume of Blood Cultured (ml.)
30
110
D.M. Ilstrup and J.A. Washington III
The unexplained lower frequency of positive cultures from the various media represented over time in bottle 3 is bothersome; however, the fact remains that nearly 3,000 positive cultures were obtained from this bottle only. Had the number of positive cultures in bottle 3 been equivalent to that in each of the other two bottles, there would have been a further increase in yield per volume of blood cultured. It should be stressed that the volume of blood cultured refers to the volume of blood obtained from a single venipuncture for a single culture, rather than a cumulative volume obtained by separate venipunctures for several blood cultures. Regardless of the volume of blood obtained for a single culture, it is recommended that cultures of two to three separately collected blood samples be performed since many bacteremias and fungemias are transient or intermittent (Relier et al., 1982). It is also stressed that the results here apply only when three separate blood culture bottles are each inoculated with 10 ml of the 30 ml blood sample drawn and not to the inoculation of 30 ml into a single bottle. Therefore, the increased yield is not only a function of culture of an increased volume, but also of inoculation of an increased number of blood culture bottles. While culture of a 10 ml sample of blood has been suggested as a reasonable lower limit per culture (Relier et al., 1982), it should be apparent from the relative yields shown in Figure 1 that culture of a larger volume will significantly increase the number of bacteremias and fungemias detected in adults. REFERENCES Hall MM, llstrup DM, Washington JAII (1978) Comparison of three blood culture media with tryptic soy broth. J Clin Microbiol 8:299. Hall MM, Mneske CA, Ilstrup DM, Washington JAII (1979) Evaluation of a biphasic medium for blood cultures. J Clin Micrabiol 10:673. Relier LB, Murray PR, MacLowry JD (1982) Cumitech 1A. In: Blood cultures II. Coordinating ed. JA Washington II. Washington: American Society for Microbiology; pp. 1-11. Salventi JF, Davies TA, Randall EL, Whitaker S, Waters JR (1979) Effect of blood dilution on recovery of organisms from clinical blood cultures in medium containing sodium polyanethol sulfonate. J Clin Microbiol 9:248. Tenney JH, Relier LB, Mirrett S, Wang W-LL, Weinstein MP (1982) Controlled evaluation of the volume of blood cultured in detection of bacteremia and fungemia. J Clin Microbiol 15:558. Washington JA II (1978) Conventional approaches to blood culture. In: The Detection of Septicemia. Ed. JA Washington II. West Palm Beach: CRC Press, Inc., pp. 41-87.
107
The Importance of Volume of Blood Cultured in the Detection of Bacteremia and Fungemia Duane M. Ilstrup and John A. Washington II
An analysis was made on the basis of 5,389 isolates from 5,008 positive blood cultures of the relative yields from 10, 20, and 30 ml of blood. Average yields from cultures of 20 and 30 ml of blood were, respectively, 38 and 61% greater than that from 10 ml of blood.
INTRODUCTION As emphasized recently in a revised Cumitech on blood cultures by Relier et al. (1982), the volume of blood cultured appears relatively more important than medium or atmosphere of incubation in the detection of septicemia. In controlled evaluations of two sample sizes of blood cultured in separate bottles containing the same medium and incubated under identical atmospheric conditions, Tenney et al. (1982) have demonstrated a 29% greater yield of organisms from a 7-ml sample than from a 2ml sample, and we have demonstrated a 25% greater yield of organisms from a 15ml sample than from a 5-ml sample (Washington, 1978). Data on the effects of culturing larger volumes of blood on yield are limited for various reasons, including the physiological and ethical restrictions on the volume of blood collected for culture, the necessity of diluting blood on a 1:5 to 1:10 basis in broth, and different atmospheres of incubation favoring growth of certain microorganisms and, as a consequence, the requirement for two blood culture bottles permitting aerobic and anaerobic incubation (Relier et al., 1982). Assuming, however, that each of two or more blood culture bottles is inoculated with equivalent volumes of blood, that the effects on organism yield of different types of media are minimal, and that the number of isolates favored by aerobic incubation conditions is approximately equal to those favored by anaerobic incubation conditions, it should be possible to plot the cumulative effects on yield of culturing blood in two or more media. Thus, in a small study of 174 positive blood culture vials, Salventi et al. (1979) demonstrated the following percentage increments in organism yield per volume of blood cultured: 5-10 ml, 13%; 10-20 ml, 13%; 20-30 ml, 7%; and 30-40 ml, 5%. The purpose of this study was to examine the cumulative effects of culturing 30 ml of blood from adults with clinically suspected sepsis at the Mayo Clinic and affiliated hospitals between 1976 and 1981.
From the Mayo Clinic and Mayo Foundation, Rochester, Minnesota. Address reprint requests to: John A. Washington II, M.D., Head Section of Clinical Micro-
biology, Mayo Clinic, Rochester, MN 55905. Received November 1, 1982; accepted January 12, 1983. © 1983 Elsevier Science Publishing Co., Inc., 52 Vanderbflt Avenue, New York, NY 10017
0732-8893/83/03.00
108
D.M. Ilstrup and J.A. Washington III
METHODS Blood Culture Procedure During the period of study, 30 ml of blood was collected by phlebotomists for each culture; 10 ml was inoculated into each of three blood culture bottles. For purposes of this study, isolates of Staphylococcus epidermidis, Bacillus, Corynebacterium, and Propionibacterium were excluded, and the analysis was limited to those cultures in which each of the following three blood culture bottles was inoculated. Bottles 1 and 2 contained 100 ml of soybean-casein digest (Tryptic Soy Broth, Difco Laboratories) with 0.025% sodium polyanetholsulfonate (SPS) under vacuum with 10% CO2. Bottle 1 remained unvented, while bottle 2 was transiently vented prior to incubation. Bottle 3 contained different media being evaluated in succession: 100 ml of transiently vented soybean-casein digest (Trypticase Soy Broth, BBL Microbiology Systems), brucella broth (Difco Laboratories), and brain heart infusion broth (Difco Laboratories) with 0.025% SPS under vacuum with 10% CO2 or a transiently vented bottle containing biphasic brain-heart infusion medium with SPS and prepared in our laboratory. Evaluations of each of these media showed their performance to have been equivalent to that in bottle no. 2 (Hall et al., 1978, 1979}. Each bottle was examined after approximately 4-6 hrs of incubation, daily for 7 days, and finally on the 14th day of incubation. With the exception of the biphasic medium bottle, which was tipped at the time of each examination so that the blood-broth mixture flowed over the agar slant, routine subcultures were performed between 6-17 hrs and after 48 hrs of incubation onto chocolate blood agar plates which were incubated at 35°C in an atmosphere containing 5-10% CO2 for 48 hrs.
Data Analysis Since three bottles were always inoculated simultaneously, the results for 2 bottles versus 1 bottle, 3 bottles versus 1 bottle, and 3 bottles versus 2 bottles had to be evaluated as averages of the possible combinations of the three bottles. For example, the effect of using 3 bottles (30 ml) versus 2 bottles (20 ml) is presented as the average of: i)
yield of bottles 1, 2, and 3 x 100 yield of bottles 1 and 2
ii)
yield of bottles 1, 2, and 3 yield of bottles 1 and 3 x 100
iii)
yield of bottles 1, 2, and 3 yield of bottles 2 and 3 x 100.
RESULTS An analysis was made of the frequency of positivity of each bottle alone and in combination with each or both of the other two bottles (Table 1). A total of 5,389 organisms was isolated from 5,008 positive cultures. The reasons for the smaller number of isolates from bottle 3 alone remain unknown since the phlebotomists followed no fixed order of inoculation of bottles and since there had been no statistically significant differences between the number of isolates overall recovered by the media represented in bottle 3 and those recovered from bottle 2 (Hall et al., 1978, 1979).
Detection of Bacteremia a n d Fungemia
109
TABLE 1. N u m b e r of Isolates from Blood Culture Bottles Bottle"
No. of isolates
1 alone 2 alone 3 alone 1 + 2 1 + 3 2 + 3 1 + 2 + 3
3,457 3,784 2,891 4,724 4,435 4,639 5,389
"Each of the 3 bottles was inoculated with 10 ml of blood. Bottle 1 = TSB, 100 ml with 0.025% SPS, under vacuumwith 10% CO2,unvented; 2 = same as no. 1 excepttransientlyvented;3 = varioustransiently vented media with 0.025% SPS (see text). If the yield from culture of 10 ml of blood in any single bottle was taken as 100%, then the average yields from cultures of 20 ml in two and 30 ml i n three bottles were, respectively, 138% and 162%. If the yield from any c o m b i n a t i o n of two bottles (i.e., 20 ml of blood) was taken as 100%, the yield from all three bottles (30 ml) averaged 117%. A n alternative analysis is to estimate the percent of cultures in w h i c h c o m b i n a t i o n s of only one or two bottles w o u l d be "false negatives" w h e n the three bottle combination is the standard of comparison. With our data, the average percent of false negatives with only one bottle w o u l d be 37% and with two bottles w o u l d be 15%. DISCUSSION Although our results must be qualified by differences among the three bottles inoculated i n composition of media, atmospheres of incubation, a n d yield, it should nonetheless be possible, on the basis of our data, to plot a general relationship between the v o l u m e of blood cultured and yield [Figure 1). Other studies (Tenney et al., 1982; Washington, 1978; Salventi, 1979] have also s h o w n increased yield with increased volume. FIGURE l. Yield of organisms relative to volume of blood cultured. 170
Relative Yield (%of Isolates With Only 10 ml. Of Blood)
160 m
150 m
140 m
130 B
120 B
100
m
I
I
10
20
Volume of Blood Cultured (ml.)
30
110
D.M. Ilstrup and J.A. Washington III
The unexplained lower frequency of positive cultures from the various media represented over time in bottle 3 is bothersome; however, the fact remains that nearly 3,000 positive cultures were obtained from this bottle only. Had the number of positive cultures in bottle 3 been equivalent to that in each of the other two bottles, there would have been a further increase in yield per volume of blood cultured. It should be stressed that the volume of blood cultured refers to the volume of blood obtained from a single venipuncture for a single culture, rather than a cumulative volume obtained by separate venipunctures for several blood cultures. Regardless of the volume of blood obtained for a single culture, it is recommended that cultures of two to three separately collected blood samples be performed since many bacteremias and fungemias are transient or intermittent (Relier et al., 1982). It is also stressed that the results here apply only when three separate blood culture bottles are each inoculated with 10 ml of the 30 ml blood sample drawn and not to the inoculation of 30 ml into a single bottle. Therefore, the increased yield is not only a function of culture of an increased volume, but also of inoculation of an increased number of blood culture bottles. While culture of a 10 ml sample of blood has been suggested as a reasonable lower limit per culture (Relier et al., 1982), it should be apparent from the relative yields shown in Figure 1 that culture of a larger volume will significantly increase the number of bacteremias and fungemias detected in adults. REFERENCES Hall MM, llstrup DM, Washington JAII (1978) Comparison of three blood culture media with tryptic soy broth. J Clin Microbiol 8:299. Hall MM, Mneske CA, Ilstrup DM, Washington JAII (1979) Evaluation of a biphasic medium for blood cultures. J Clin Micrabiol 10:673. Relier LB, Murray PR, MacLowry JD (1982) Cumitech 1A. In: Blood cultures II. Coordinating ed. JA Washington II. Washington: American Society for Microbiology; pp. 1-11. Salventi JF, Davies TA, Randall EL, Whitaker S, Waters JR (1979) Effect of blood dilution on recovery of organisms from clinical blood cultures in medium containing sodium polyanethol sulfonate. J Clin Microbiol 9:248. Tenney JH, Relier LB, Mirrett S, Wang W-LL, Weinstein MP (1982) Controlled evaluation of the volume of blood cultured in detection of bacteremia and fungemia. J Clin Microbiol 15:558. Washington JA II (1978) Conventional approaches to blood culture. In: The Detection of Septicemia. Ed. JA Washington II. West Palm Beach: CRC Press, Inc., pp. 41-87.