Burn Wound Biopsy Bacterial Quantitation: A Statistical Analysis
F. Jerry Volenec, PhD, Kansas Gary M. Clark, PhD, Kansas Mani M. Mani, MD, Kansas
City, Missouri
City, Kansas City, Kansas
Loren J. Humphrey, MD, PhD, Kansas
City, Missouri
Quantitative bacteriologic monitoring techniques for open wounds and burns have been devised to assess the extent of wound colonization and invasive infection. Among these, the quantitative biopsy culture [l-3] and the quantitative swab culture [4] have predominated. Such techniques have suggested that a concentration of lo5 organisms/g or greater of biopsy tissue or lo6 organisms/swab culture is a critjcal level of bacterial colonization indicative of potential graft or wound closure failure or increased risk of wound sepsis [5-91. Consideration must be given in such measurements to (1) the error inherent within the test system, and (2) the variation in the number of organisms at different locations within a wound. This study was undertaken to statistically evaluate these sources of variation in the burn wound biopsy procedure. Material and Methods Burn Culture Methodology. Paired 4 mm punch biopsy samples were taken on alternate days from three separate locations on each of four burn patients. These patients had flame burns covering 40 to 67 per cent of the total body surface area. Wounds were washed free of topical antimicrobial before sampling. The paired biopsy specimens were taken from an area of 1 to 2 cm2. Biopsy size dissected free of eschar ranged from 0.014 to 0.057 g (mean 0.028). The mean difference in size between the paired samples was 0.006 g. The tissue samples were weighed and homogenized to a 1 per cent suspension in tryptic soy broth using a motor From the University of Missouri at Kansas City School of Medicine, Kansas City, Missouri, and the University of Kansas Medical Center, Kansas City, Kansas. Reprint requests should be addressed to F. Jerry Volenec, Department of Medicine, University of Missouri at Kansas City School of Medicine, 2411 Holmes, Kansas City, Missouri 64108.
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driven Ten Broeck grinder. Tissue specimens were not flamed or pretreated before homogenization. Serial IO-fold dilutions were prepared and inoculated onto each of two tryptic soy agar plates and incubated overnight at 37°C. Colonies were then counted and the number of organisms per gram of tissue calculated for each biopsy in each pair. A total of 36 paired samples were evaluated by this procedure. Eight of the pairs were eliminated from further consideration because the number of organisms was too small to count (the lowest sample dilution possible was log lo-‘). One pair was eliminated because the number of organisms was too large to count. Each of the counts in the remaining 27 pairs were then converted t,o logarithms to the base 10. Statistical Methodology. Statistical analyses were performed under the assumption that the logarithm of the number of organisms per gram in a burn biopsy site approximately follows a normal distribution. It is further assumed that the mean of the distribution may vary among biopsy sites but that the standard deviation remains constant. Each of the 27 samples of size two yields an estimate of the common standard deviation having 1 degree of freedom so that the composite or pooled estimate, sp, will have 27 degrees of freedom. Once a single observation, x, is obtained from a given biopsy site, a 100 (1 - 01) per cent confidence interval for the mean of that site can be computed as follows: x * ti;-:-“‘“’s
1)
(Equation
1)
where t,&-(Vfi)1sthe 100 (1 - a/2) percentage point of Student’s t distribution with 27 degrees of freedom. If (Y= 0.05, this point is 2.052. Results Organisms isolated frequency of recovery
from biopsy samples and the were as follows: Staphylococ-
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Volenec et al
TABLE I
Logarithms of Counts Per Gram
TABLE II 95 Per Cent Confidence Intervals (Cl) Based on Single Determinations
Patient No.
Set No.,
Sample A
Sample B
Range
SD
1
1 1 2 2 2 3 3
6.23 6.54 3.00 3.00 5.20 3.18 3.96
4.59 6.72 3.65 3.90 5.23 3.48 3.74
1.64 0.18 0.65 0.90 0.03 0.30 0.22
1.16 0.13 0.46 0.64 0.02 0.21 0.16
1 1
3.00 3.85 4.45 4.04 3.48 6.04 4.00 4.71 6.08
4.11 3.00 6.23 5.23 5.62 4.26 4.40 4.78 5.30
1.11 0.85 1.78 1.19 2.14 1.78 0.40 0.07 0.78
0.78 0.60 1.26 0.85 1.51 1.26 0.28 0.05 0.55
4.18 3.86 3.87 3.81 3.57 4.61
4.20 3.40 3.70 3.62 4.39 4.87
0.02 0.46 0.03 0.19 0.82 0.26
0.01 0.33 0.02 0.13 0.58 0.18
4.04 4.85 4.68 5.20 5.11
4.00 3.70 4.63 4.62 4.72
0.04 1.15 0.05 0.58 0.39
0.28 0.81 0.04 0.41 0.28
4.38
4.45
0.67
0.64”
2
2 2 2 3 3 3 3
1
2 2 2 4
1 2 2 2
Mean
The mean standard deviation (SD) is computed as the square root of the mean variance. l
cus epidermidis, 50.0 per cent; Pseudomonas aeruginosa, 39.5 per cent; Staphylococcus aureus, 37.5 per cent; Escherichia coli, 22.9 per cent; Klebsiella species, 21.8 per cent; Enterococcus species, 18.7 per cent; Enterobacter species, 17.7 per cent; Corynebacterium species, 15.4 per cent; and Candida species, 14.5 per cent. Other organisms isolated at less than 10 per cent frequency were Proteus, Streptococcus, Serratia, and Acinetobacter species. Table I gives the logarithms of the counts per gram from the two samples for each of the 27 pairs of determinations. The range and standard deviation for each pair are also listed. The absolute difference of the logarithms ranged from 0.02 to 1.51 (mean 0.67). In an attempt to verify the assumption of a common standard deviation, correlations were calculated between the mean of the paired observations and both the variance and the standard deviation. The values obtained were 0.24‘and 0.16, respectively, each far from being significant. The estimate of the pooled standard deviation was 0.64. Using equation 1, the 95 per cent confidence
who
Counts/g 5x 1x 5x 1x 5x 1x 5x
10s 104 104 105 10s 10s 10s
(counts/g) 3.7 4.0 4.7 5.0 5.7 6.0 6.7
95% Cl 2.4, 2.7, 3.4, 3.7, 4.4, 4.7, 5.4,
5.0 5.3 6.0 6.3 7.0 7.3 8.0
interval based on any single observation, x, is x f 1.31. Table II lists the 95 per cent confidence intervals for various values of x. Comments
The importance of the quantitative biopsy in monitoring burn wound bacterial concentration lies in the confidence one has in the resulting data. Other types of quantitative wound culture techniques such as contact plates or gauze techniques failed because the resulting data could not accurately account for the presence of deep tissue infection [IO-121. The quantitative biopsy, in contrast to these procedures, does enumerate bacteria in subeschar tissue. This study indicated that in comparing paired biopsies from adjacent sites, the 95 per cent confidence interval is logic 1.31. This value undoubtedly is the result of the error inherent in the test system and in variations of bacterial density between even directly adjacent sites. This value agrees favorably with the 95 per cent confidence interval value of logic 1.7 reported for the quantitative swab technique [4]. Thus, the quantitative biopsy does provide a reliable approximation of the number of organisms present in a burn wound site at a given time. Studies quantitating bacteria in wounds indicated that logrc 5.0 organisms/g of tissue resulted in graft failure [6,7] or in the failure of secondary closure of wounds [8,9]. Thus the value logre 5.0 organisms/g has been taken as a critical value indicative of invasive bacterial infection. Consideration must be given, however, to factors other than the number of bacteria in a biopsy specimen. The body surface area burned, distribution of high organism concentrations, duration of high organism concentration, and type of organism must all contribute to the overall effect on the patient. As demonstrated by patient 1 in Table I, while counts greater than logrc 5.0 organisms/g were obtained from three to four sites in the first set of biopsies, counts of this magnitude were evident in only two to six samples taken 2 days later and in none
The American Journal of Surgery
Burn Biopsy Bacterial Quantitation
of four samples taken after a second 2 day interval. Therefore, in order to accurately reflect the differences of the infectious process in this patient, biopsies from diverse sites and over a period of time are required. Care must also be taken in the identification of the genus of organisms colonizing a burn wound extensively. Certainly, Pseudomonas or Staphylococcus aureus represent more severe consequences than equal colonization of Escherichia species or Staphylococcus epidermidis, for example. Thus the burn wound biopsy provides a reliable procedure for quantitating organisms colonizing a burn wound. When used as a routine monitoring technique at diverse sites in burn wounds, changes in sequential samples give an indication of the dynamics of infection in the burn patient.
Summary
Sequential paired punch biopsy samples were taken from three separate locations on each of four burn patients and were quantitated for the number of viable bacteria per gram of tissue. The range (log10 0.02 to loglo 1.51) and the standard deviation (log10 0.67) were determined for each pair. The 95 per cent confidence interval based on any single observation, x, was determined to be x f 1.31. It is concluded that the burn wound biopsy is a reliable procedure for
Volume 138, November 1979
quantitating organisms in a burn wound and that changes in sequential samples give an indication of the dynamics of infection in the burn patient. References 1. Lawrence JC, Lilly HA: A quantitative method for investigating the bacteriology of skin: its application to burns. Br J Exp Pathol53: 550, 1972. 2. Loebl EC, Marvin JA, Heck EL, Curreri PW, Baxter CR: The use of quantitative biopsy cultures in bacteriologic monitoring of burn patients. J Surg Res 16: 1, 1974. 3. Robson NC, Heggers JP: Bacterial quantification on open wounds. Milit Med 134: 19, 1969. 4. Levine NS, Lindberg RB, Mason AD, Pruitt BA: The quantitative swab culture and smear: a quick, simple method for determining the number of viable aerobic bacteria on open wounds. J Trauma 16: 89, 1976. 5. Duke WF, Robson NC, Krizek TJ: Civilian wounds, their bacterial flora and rate of infection. Surg forum 23: 513, 1972. 6. Krizek TJ, Robson MC, Kho E: Bacterial growth and skin graft survival. Surg Forum 18: 518, 1967. 7. Robson MC, Krizek TJ: Predicting skin graft survival. J Trauma 13: 213, 1973. 8. Robson MC, Lea CE, Dalton JB, Heggers JP: Quantitative bacteriology and delayed wound closure. Surg Forum 19: 501, 1968. 9. Robson MC, Shaw RC, Heggers JP: The reclosure of postoperative incisional abscesses based on bacterial quantification of the wound. Ann Surg 171: 279, 1970. 10. Bretano L, Gravens AC: A method for the quantitation of bacteria in burn wounds. Appl Microbial 15: 670, 1967. 11. Clarkson JG, Ward CG, Polk HC: Quantitative bacteriologic study of the burn wound surface. Surg Forum 18: 506, 1967. 12. Georgiade NG, Lucas MC, Osterhouk SA: A comparison of methods for quantitation of bacteria in burn wounds. II. Clinical evaluation. Am J C/in Pafhol3: 40, 1969.
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