A Sequential, Prospective Analysis of Immunologic Abnormalities and Infection Following Severe Thermal Injury J. WESLEY ALEXANDER, M.D., Sc.D., CORA K. OGLE, PH.D., J. DWIGHT STINNETT, PH.D., BRUCE G. MACMILLAN, M.D. A sequential, prospective analysis of humoral and cellular immune function was performed on 20 burn patients with injuries involving a45% total body surface area. Infected patients had significantly worse neutrophil bactericidal activity against Staphylococcus aureus 502A than did noninfected patients. Chemotaxis of neutrophils correlated poorly with infection although chemotaxis was frequently abnormal. The opsonic index of serum was depressed early after the burn but returned to nearly normal values by the fourth to the fourteenth postburn day. There was no difference between infected and noninfected patients. Serum levels of IgG, properdin and C3, while initially low, remained within the normal range after the ninth postburn day in both groups. Factor B levels rose rapidly during the first three weeks after injury to more than double normal levels in many patients. Suggestive evidence for consumption of opsonic protein occurred with five of 19 episodes of bacteremia. The responsiveness of isolated lymphocytes to PHA was normal. However, patients' sera were shown to significantly inhibit the responsiveness of normal lymphocytes to PHA. Analysis of immunologic profiles for individual patients indicates that abnormalities of neutrophil function are the most important acquired defect predisposing patients to the development of bacteremia following major thermal injury; abnormalities of opsonic action play a secondary but important role.
A MAJOR THERMAL BURN is the most severe form of
A injury that man can sustain and survive. Infection
has always been the leading cause of death following major burn injuries, and multiple lines of both clinical and laboratory evidence have indicated that an increased susceptibility to infection exists over and above that simply associated with loss of the skin barrier. Indeed, virtually every variable of immunologic resistance to infection has been studied and reported to be abnormal following severe thermal injury.' However, these studies have examined only isolated variables, and most were performed before the current practice of intensive nutritional therapy was available. This study was designed to examine the spectrum of immunologic resistance to infection to determine which variables are of primary importance. Supported by USPHS Grants AI-R936-01A1 and 5P01-GM15428-09. Submitted for publication: April 4, 1978.
From the Surgical Immunobiology Laboratory of the Department of Surgery, the Cincinnati Shriners Burns Institute, and the Paul I. Hoxworth Blood Center, University of Cincinnati Medical Center, Cincinnati, Ohio
Methods
Patient Material and Analysis
Twenty consenting patients with burn injuries involving -45% total body surface area were studied over a period of 18 months. Thirteen were children treated at the Shriners Burns Institute (SBI) and seven were adults treated at the Cincinnati General Hospital (CGH). The average burn size was 59.5% total with 44% 30 (burn index = 52). The patients were admitted to study up to 21 days following burn (the average was 13 days, half of the patients were admitted within four days). Informed consent was obtained before each patient was entered. Therapy of the patients was not altered by their participation in this study. All patients received routine topical therapy, initially with silver sulfadiazine with alterations as indicated by surface cultures. Systemic antibiotics were administered only on specific indications, primarily bacteremia. The caloric requirements to reflect basal plus anticipated hypermetabolic demands were estimated for each of the patients, depending on the size of the burn, and an attempt was made to provide these calories by continuous nasogastric instilfation in patients unable to voluntarily ingest enough to meet their requirements. In most patients (SBI), daily measurements were made of weight and caloric intake. In 14 patients for whom adequate data were available for the calculation, the average maximum weight loss from preburn weight was 12%. In each patient, there was routine monitoring for evidence of infection, and blood cultures were obtained whenever such existed. Blood was taken for study three times per week for six weeks or until closure of the wound or death of the patient. At each study interval, the following tests
0003-4932/78/1200/0809 $00.90 © J. B. Lippincott Company
809
810
ALEXANDER AND OTHERS
were done: measurement of the antibacterial function of neutrophils using S. aureus and E. coli; chemotaxis of neutrophils; serum levels of albumin, C3(B), IgG, properdin and factor B; measurement of the opsonic activity of serum; lymphocyte response to phytohemagglutinin (PHA); and measurement of inhibition of PHA response of normal lymphocytes in the presence of patient serum. Tests of cellular function were performed on the day of collection, and serum related tests were performed in groups. Patient serum was procured within two hours of collection. Half was divided into aliquots and quickly frozen (-90°) and the other half was dialyzed at 40 for 24 hours against normal saline before division and freezing (-90°). A single large pool of normal human serum (PNHS) from ten normal adults was similarly processed at the beginning of the study to serve as standards and controls throughout the study. Patients were divided into two groups, infected and noninfected, based upon the presence or absence of a positive blood culture during the period of study. For each test, pooled data were then analyzed comparing infected versus noninfected groups. All of the data available were plotted individually for each of the patients, including major clinical events such as surgery or anesthesia, the administration of blood and blood products, weight, temperature, caloric intake, positive blood cultures and systemic antibiotics. Tests Performed
The antibacterial function of neutrophils was measured by the technique of Alexander and Meakins3 using S. aureus 502A as the test organism. Results were recorded as a neutrophil bactericidal index (NBI). The NBI is a function of the number of surviving bacteria after the incubation period: number surviving in patient's test average number surviving in control tests Hence, higher numbers reflect poorer phagocyte bactericidal activity. Duplicate tests were performed using E. coli 075 as the test organism. Chemotaxis of neutrophils in vitro was studied only in the first 12 patients utilizing complement-derived chemotaxins as previously described.15 Radial immunodiffusion17 was used to measure levels of C3(B), IgG, properdin and factor B using nondialyzed frozen sera. All measurements were compared to the control pooled normal human serum (PNHS). Concentrations of C3(B), properdin and factor B in the control PNHS were determined by comparison with standard sera.* Antisera to IgG, C3(f3Ic/,8Ia), and factor B were obtained commercially from Behring Diag*
Provided by Dr. Douglas Fearon.
Ann. Surg. * December 1978
nostics, Somerville, N.J. 08876. Antisera to C3(B) (the B antigenic determinant of C3) were prepared from anti-,3lc/,3la by the method of Molenaar.18 Properdin was prepared by the method of Ogle et al. ,21 and antisera to properdin were prepared in goats. Albumin levels were determined routinely as part of an SMA-12 group of tests. The ability of patient sera to support opsonization was performed by a modification of the method of Hirsch and Strauss12 using 4% concentration of patient serum which was previously dialyzed to remove any antibiotics potentially present. Control PNHS was similarly dialyzed and treated. E. coli 075 was used as the test organism. We have found that this strain has a requirement for factor B and properdin for opsonization, but that opsonization proceeds normally in IgGdepleted serum. The ability to opsonize organisms obtained from positive blood cultures was similarly studied using the patient's sera obtained before, after, and nearest the time of the event. The results were expressed as an opsonic index which is: log10 kill with patient serum log10 kill with control serum The response of lymphocytes in whole blood to PHA was determined by the method of Eskola et al.8 For studying the effects of patient sera on PHA responses with normal lymphocytes, lymphocytes were purified by centrifugation on a Ficoll-hypaque density gradient, washed with Eagle's minimum essential medium (MEM), resuspended in MEM containing penicillin, streptomycin and 5% PNHS (or patient serum) and dispensed (200,ul) into sterile microculture plates (Falcon, Oxnard, California). PHA (0.5 ug) was added to each well. After 48 hours of incubation at 370 in 5% C02, 1 mCi of 3H-thymidine (20 mCi/nmole, New England Nuclear, Boston, Massachusetts) was added to each well. After an additional 18-24 hours incubation, the lymphocytes were harvested on glass fiber filters using a MASH III (Microbiological Associates, Bethesda, Maryland) and the incorporation of 3H-thymidine into DNA determined by liquid scintillation spectroscopy. All determinations were performed in sets of six for each patient and included nonstimulated controls.
Normal Controls Identical tests were performed on twenty normal individuals to provide a basis for comparison to a normal population. Statistical Analysis of Data Data are presented as mean + standard error of the mean. Levels of significance were determined by standard Student's t-tests.
Vol. 188 . No. 6
811
SEVERE THERMAL INJURY
Results
20 19
Patient Groupings
18
Thirteen of the 20 patients, hereafter referred to as infected, had 19 episodes of positive blood cultures at some time during the period of observation. The average burn size for infected patients was 62% total! 44% 30 (BI = 53) compared to 54% totalI46% 30 (BI = 50) for noninfected.
.
Is 17 16 15 14
13 E.
12
. II
,j
Tests of Neutrophil Function
_
Infected patients had significantly worse neutrophil function (p < 0.01) against S. aureus 502A overall than did noninfected patients (Fig. 1). When all patients were divided into two groups according to burn size, those with burns of 45% 30 or greater had an average NBI of 6.0 + 0.7 compared to 4.9 0.4 for patients with 44% 30 or less (p > 0.1). The antibacterial function of neutrophils against S. aureus correlated poorly with the antibacterial function of the same neutrophils against E. coli (Fig. 2), and there was no correlation of function against E. coli with the development of infection, either when examined individually or as a group (infected versus non-
10 9 08
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0
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1
1
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2 3 4 5 6 7 8 9 10 11 12 13 14
15
16 17
18
NBI E. coli FIG. 2. Scattergram illustrating the lack of correlation between neutrophil function determined using S. aureus and E. coli as the test organisms. Tests were performed in parallel.
infected). The NBI (E. coli) for the infected group 3.8 + 0.6 compared to 2.8 + 0.7 for the noninfected group (p = not significant). Chemotaxis of neutrophils also correlated poorly with infection although chemotaxis was frequently abnormal. There was no correlation between the chemotactic index and the NBI using the same samples with tests performed at the same time (Fig. 3). The functional chemotactic index for the infected patients was 100 + 17, as compared to 76 11 for the noninfected patients. Sequential studies on the same patients showed no relationship to size of burn or ultimate outwas
10 9
8
7
WECTED
6~~~~~~~~
±
5
come.
4
Tests of Opsonization
1-4
5-8
912 Q3-6
17-20 21-24 25-e8 29-36 3744 P B D
FIG. 1. Sequential neutrophil function (NBI) in burn patients. Values shown are the mean + S.E.; normal values are 2.1 + 0.4. The broken line represents patients who were never infected (bacteremic) during the period of study, while the solid line is pooled data from patients who were infected (bacteremic) at any time during the study. Neutrophil function was significantly worse in infected patients (p < 0.01).
The opsonic index was depressed in all but one patient studied during the first five postburn days, returning to nearly normal values by the fourth to the fourteenth postburn day (Fig. 4). There was no difference between infected and noninfected patients. Similarly, there was no difference in the time-course relationship of the opsonic indices to extent of burn. Of 13 available organisms isolated from blood cultures, there was no evidence of an organism-specific defect in opsonization in any instance when the infecting organisms were tested comparing PNHS with sequential sera from the infected patient before, during and after the event.
812
ALEXANDER AND OTHERS 0
10
0
Ann.
Surg. 9 December 1978
the second week, with only insignificant differences between infected and noninfected groups (Fig. 9). (Data for individual patients are discussed below).
0 0
9
Tests of Lymphocyte Function The responsiveness of isolated lymphocytes to PHA was measured in only seven patients. In no instances were the lymphocytes abnormal in their ability to be stimulated. However, marked depressions in reactivity were present when responsiveness was measured in the patient's whole blood (stimulation index = 5.3 + 0.2 compared to normal controls = 22 + 3). Both dialyzed and nondialyzed serum from the patients inhibited the responsiveness of normal lymphocytes to PHA. There were no differences between infected and noninfected
0 0
8 0
7 0
6
0
0
0
0
0
0
5
0
4
0
0
.0
t0I
3
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0
06
2
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0
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.
patients (Fig. 10).
0
* 0
-
0* 0
00-
20
40
0
*-
0
60
80
100 120
40
160 180
FUNCTIONAL CHEMOTACTIC INDEX
Analysis of Sequential Measurements in Infected Patients Nineteen episodes of bacteremia occurred in the 13 patients in the infected group (Table 1). One patient had three episodes, and two episodes occurred in each
FIG. 3. Scattergram illustrating the lack of correlation between neutrophil function (using S. aureus) and functional chemotaxic index (FCI). Tests were performed in parallel. 1.2
Serum Concentrations of IgG, Albumin, Factor B, and C3 Serum albumin levels were initially low in both infected and noninfected patients, rising from an average of 2.8 gm/dl on days 1-15 to 3.2 gm/dl by days 36-45 (Fig. 5). However, there was no difference between the infected and noninfected patients. Serum concentrations of IgG were significantly depressed during the early resuscitative period, rising rapidly between the eighth and twentieth postburn days to high normal levels in noninfected patients and low normal levels in infected patients (Fig. 6). The values for infected patients were significantly lower than for noninfected patients after the thirteenth postburn day (p < 0.001), even though they were within normal values. Properdin levels, while initially low, remained within the normal range after the ninth postburn day (Fig. 7). There were no significant differences between infected and noninfected patients. Serum concentrations of factor B rose rapidly during the first three weeks after injury to more than double normal levels in many patients (Fig. 8). Infected and noninfected groups were comparable. C3 concentrations, measured by the B antigenic determinant, rose to higher than normal values during
1.0
0.9
0.8
0.7 0.6
0.5
0.4 0.3
0.2 0.1
1-4
5B
9"2
316 17-20 21-24 25-28 27-36 37-44 PBD
FIG. 4. Sequential analysis of the ability of patient serum to support opsonophagocytosis of E. coli 075. Values shown are the mean ± S.E.; normal values are 0189 ± 0.03. A depression in opsonic index is seen the first week following the burn, but returns to low normal by three weeks:
VOl. 188.* NO. 6
SEVERE THERMAL INJURY
of four patients. Three of the infected patients died, one on PBD 12 from smoke inhalation and pneumonia (59-year-old woman, 63% totalI13% 30), one from a cardiac arrhythmia associated with staphylococcal pericarditis on PBD 28 (12-year-old girl, 55% total/45% 30, and one from septicemia on PBD 34 (55-year-old man, 80% total/33% 3°). All other patients survived and have been discharged. Eleven of the septic episodes were caused by S. aureus, two by Enterobacter cloacae, one each by Klebsiella pneumonia, Candida albicans, Pseudomonas aeruginosa, Streptococcus viridans and a Bacillus sp. and one episode by multiple organisms: S. aureus with E. cloacae. Fourteen of the nineteen episodes of bacteremia were associated with a significant preceding abnormality of neutrophil function against S. aureus 502A (NBI 2 4) (Table 1). The average NBI immediately preceding all bacteremia episodes was 7.6 ± 1.2. The average NBI was also 7.6 immediately preceding the three infections caused by gram negative bacilli. Two other instances were associated with mild neutrophil dysfunction (NBI 2-4), and neutrophil function was normal ( 4) at the time of five of these seven episodes. Circulating levels of C3(B) increased in the serum of patients associated with 12 episodes of bacteremia and
45
40
4.0
r 35
30 3.0 F
25
20 2.0 P Non-Infeced Patient
-
Infected Patient
1.0
I0 5
1-5
6-10
1
16-20
21-25 26 30 31-35 36
40
41-45
DAYS POST BURN
FIG. 5. Sequential analysis of serum albumin in burn patients. Values shown are the mean ± S.E.; the normal range of albumin is 3.5-5.0 gm/dl. No consistent, significant differences are seen between noninfected and infected patients.
*4
5-6
9e
6
r?-0
0214 25-es 29-a6 37-44
P BD
FIG. 7. Sequential analysis of properdin in burn patients. Values shown are the mean + S.E.; normal values are 28 + 1 ,ug/ml.
814
l~ ~F
ALEXANDER AND OTHERS
7001
600
i'
A lllf 4 . X,.~I - /1 500
)
/
300 200
*
100
INFECTED
W N---ECTED
-4
5-8
942 13*6 17-20 21-24 25-28 29-36 37-44 PBD
FIG. 8. Sequential analysis of factor B in burn patients. Values shown are the mean + S.E.; normal values are 261 + 15 ,ug/ml. Note the rapid rise of factor B, suggesting that this component is an acute phase protein.
fell during six episodes. In all of these last six episodes, there was also a fall in the opsonic index, but the opsonic index fell in six other patients without reduction in C3(B). Factor B rose during nine episodes of infection, fell during eight, remained unchanged in one, and was not determined in one. Properdin levels increased during ten episodes, fell during five episodes
241
14001
F4
8-
942 5MS rT-2 a-04 z~a -a 37-44
F16. 9. Sequential analysis of C3(B) levels in burn patients. Values shown are the mean ± S.E.; normal values are 1030 ± 23 ,.tgIml. Initial levels are low, but rise to elevated values by the second week.
Ann. Surg. * December 1978
and remained unchanged during one episode. Sufficient data were not available for three episodes. Strong evidence for consumption (a drop of -50% in C3B and opsonic index) occurred in three patients with suggestive evidence (drops of 25-49%) in two others. In one instance properdin and factor B seemed to be involved in a consumptive process (patient 8-2).
Discussion It is well known that the antibacterial function of neutrophils, the opsonic activity of serum, lymphocyte function and function of the reticuloendothelial system (RES) all contribute to resistance to bacterial infections in experimental animals and man. All of these immunologic functions have been shown to be abnormal following burn injury.1 However, no previous study has investigated the significance of isolated immunologic deficiencies relative to other variables of host defense against infection. We have attempted to examine those variables which were potentially of critical importance, except for function of the RES which was excluded from consideration because adequate tests for sequential evaluation in man are not currently available. The results of this study strongly suggest that acquired abnormalities of neutrophil function, measured by the ability to kill S. aureus 502A, are of major and primary importance in the development of bacteremia following major thermal injury. Increased susceptibility to infection associated with abnormalities of neutrophil function against S. aureus are in 100 _
90
_
80
70-
JL 30
-
~
~
lii
\
20
"
Non-infected
/infected
1=
10 0
1-4
5-8
l
I
9-12 13-16 17-20 21-24 25-28 29-36 PBD
oIG. 10. Sequential analysis of the inhibition of lymphocyte PHA responses by patient serum. Values shown are the mean ± S.E.; normal serum always inhibited PHA responses less than 5%. No consistent, significant differences in serum inhibitory activity between infected and noninfected patients is apparent.
Vol. 188 * No. 6
815
SEVERE THERMAL INJURY TABLE 1. Relationship of Immunologic Variables to Bacteremia Preinfection*
Patient and Episode
Organism
1-1 2-1 3-1
S. aureus S. aureus E. cloacae
3-2
S. aureus &
4-1 4-2 5-1 6-1 7-1 7-2 8-1 8-2 9-1 10-1 10-2
10-3 11-1 12-1 13-1
E. cloacae S. aureus S. aureus S. aureus K. pneumoniae E. cloacae S. aureus S. aureus Candida S. aureus
Bacillus S. S. P. S.
aureus aureus aerug. aureus
Strep.
NBI 5.0 9.9 16.7
Opsonic Index 0.31 0.89 0.56
Postinfection
C3(B)
Factor B
Properdin
IgG
jtg/ml
tg/ml
jg/mi
mg/dl
NBI
1404 1697 1638
960 480 375
N.D.t 27 45
1635 1369 960
N.D. N.D. 12.6
420
38
1200
No
450 360 420 450 420 840 900 630 570 270 420 294 420 420
27 N.D. 35 26 27 32 27 8 27 8 27 24 20 28
1023
Mild
870
No Yes No No Mild No Yes No No No No No Yes
jg/ml
jg/ml
0.94 0.46
2691 2340 1872
870 495 450
1.02
2574
0.67 0.78 20.0 3.9 7.7 2.5 2.0 6.2 4.3 4.4 3.5
14 25 25
No No No
Index
0.96
22 27
1635 1495 624
Properdin
0.88 0.42 0.85 0.56 0.95 1.05 0.56 1.05 0.73
A MAJOR THERMAL BURN is the most severe form of
A injury that man can sustain and survive. Infection
has always been the leading cause of death following major burn injuries, and multiple lines of both clinical and laboratory evidence have indicated that an increased susceptibility to infection exists over and above that simply associated with loss of the skin barrier. Indeed, virtually every variable of immunologic resistance to infection has been studied and reported to be abnormal following severe thermal injury.' However, these studies have examined only isolated variables, and most were performed before the current practice of intensive nutritional therapy was available. This study was designed to examine the spectrum of immunologic resistance to infection to determine which variables are of primary importance. Supported by USPHS Grants AI-R936-01A1 and 5P01-GM15428-09. Submitted for publication: April 4, 1978.
From the Surgical Immunobiology Laboratory of the Department of Surgery, the Cincinnati Shriners Burns Institute, and the Paul I. Hoxworth Blood Center, University of Cincinnati Medical Center, Cincinnati, Ohio
Methods
Patient Material and Analysis
Twenty consenting patients with burn injuries involving -45% total body surface area were studied over a period of 18 months. Thirteen were children treated at the Shriners Burns Institute (SBI) and seven were adults treated at the Cincinnati General Hospital (CGH). The average burn size was 59.5% total with 44% 30 (burn index = 52). The patients were admitted to study up to 21 days following burn (the average was 13 days, half of the patients were admitted within four days). Informed consent was obtained before each patient was entered. Therapy of the patients was not altered by their participation in this study. All patients received routine topical therapy, initially with silver sulfadiazine with alterations as indicated by surface cultures. Systemic antibiotics were administered only on specific indications, primarily bacteremia. The caloric requirements to reflect basal plus anticipated hypermetabolic demands were estimated for each of the patients, depending on the size of the burn, and an attempt was made to provide these calories by continuous nasogastric instilfation in patients unable to voluntarily ingest enough to meet their requirements. In most patients (SBI), daily measurements were made of weight and caloric intake. In 14 patients for whom adequate data were available for the calculation, the average maximum weight loss from preburn weight was 12%. In each patient, there was routine monitoring for evidence of infection, and blood cultures were obtained whenever such existed. Blood was taken for study three times per week for six weeks or until closure of the wound or death of the patient. At each study interval, the following tests
0003-4932/78/1200/0809 $00.90 © J. B. Lippincott Company
809
810
ALEXANDER AND OTHERS
were done: measurement of the antibacterial function of neutrophils using S. aureus and E. coli; chemotaxis of neutrophils; serum levels of albumin, C3(B), IgG, properdin and factor B; measurement of the opsonic activity of serum; lymphocyte response to phytohemagglutinin (PHA); and measurement of inhibition of PHA response of normal lymphocytes in the presence of patient serum. Tests of cellular function were performed on the day of collection, and serum related tests were performed in groups. Patient serum was procured within two hours of collection. Half was divided into aliquots and quickly frozen (-90°) and the other half was dialyzed at 40 for 24 hours against normal saline before division and freezing (-90°). A single large pool of normal human serum (PNHS) from ten normal adults was similarly processed at the beginning of the study to serve as standards and controls throughout the study. Patients were divided into two groups, infected and noninfected, based upon the presence or absence of a positive blood culture during the period of study. For each test, pooled data were then analyzed comparing infected versus noninfected groups. All of the data available were plotted individually for each of the patients, including major clinical events such as surgery or anesthesia, the administration of blood and blood products, weight, temperature, caloric intake, positive blood cultures and systemic antibiotics. Tests Performed
The antibacterial function of neutrophils was measured by the technique of Alexander and Meakins3 using S. aureus 502A as the test organism. Results were recorded as a neutrophil bactericidal index (NBI). The NBI is a function of the number of surviving bacteria after the incubation period: number surviving in patient's test average number surviving in control tests Hence, higher numbers reflect poorer phagocyte bactericidal activity. Duplicate tests were performed using E. coli 075 as the test organism. Chemotaxis of neutrophils in vitro was studied only in the first 12 patients utilizing complement-derived chemotaxins as previously described.15 Radial immunodiffusion17 was used to measure levels of C3(B), IgG, properdin and factor B using nondialyzed frozen sera. All measurements were compared to the control pooled normal human serum (PNHS). Concentrations of C3(B), properdin and factor B in the control PNHS were determined by comparison with standard sera.* Antisera to IgG, C3(f3Ic/,8Ia), and factor B were obtained commercially from Behring Diag*
Provided by Dr. Douglas Fearon.
Ann. Surg. * December 1978
nostics, Somerville, N.J. 08876. Antisera to C3(B) (the B antigenic determinant of C3) were prepared from anti-,3lc/,3la by the method of Molenaar.18 Properdin was prepared by the method of Ogle et al. ,21 and antisera to properdin were prepared in goats. Albumin levels were determined routinely as part of an SMA-12 group of tests. The ability of patient sera to support opsonization was performed by a modification of the method of Hirsch and Strauss12 using 4% concentration of patient serum which was previously dialyzed to remove any antibiotics potentially present. Control PNHS was similarly dialyzed and treated. E. coli 075 was used as the test organism. We have found that this strain has a requirement for factor B and properdin for opsonization, but that opsonization proceeds normally in IgGdepleted serum. The ability to opsonize organisms obtained from positive blood cultures was similarly studied using the patient's sera obtained before, after, and nearest the time of the event. The results were expressed as an opsonic index which is: log10 kill with patient serum log10 kill with control serum The response of lymphocytes in whole blood to PHA was determined by the method of Eskola et al.8 For studying the effects of patient sera on PHA responses with normal lymphocytes, lymphocytes were purified by centrifugation on a Ficoll-hypaque density gradient, washed with Eagle's minimum essential medium (MEM), resuspended in MEM containing penicillin, streptomycin and 5% PNHS (or patient serum) and dispensed (200,ul) into sterile microculture plates (Falcon, Oxnard, California). PHA (0.5 ug) was added to each well. After 48 hours of incubation at 370 in 5% C02, 1 mCi of 3H-thymidine (20 mCi/nmole, New England Nuclear, Boston, Massachusetts) was added to each well. After an additional 18-24 hours incubation, the lymphocytes were harvested on glass fiber filters using a MASH III (Microbiological Associates, Bethesda, Maryland) and the incorporation of 3H-thymidine into DNA determined by liquid scintillation spectroscopy. All determinations were performed in sets of six for each patient and included nonstimulated controls.
Normal Controls Identical tests were performed on twenty normal individuals to provide a basis for comparison to a normal population. Statistical Analysis of Data Data are presented as mean + standard error of the mean. Levels of significance were determined by standard Student's t-tests.
Vol. 188 . No. 6
811
SEVERE THERMAL INJURY
Results
20 19
Patient Groupings
18
Thirteen of the 20 patients, hereafter referred to as infected, had 19 episodes of positive blood cultures at some time during the period of observation. The average burn size for infected patients was 62% total! 44% 30 (BI = 53) compared to 54% totalI46% 30 (BI = 50) for noninfected.
.
Is 17 16 15 14
13 E.
12
. II
,j
Tests of Neutrophil Function
_
Infected patients had significantly worse neutrophil function (p < 0.01) against S. aureus 502A overall than did noninfected patients (Fig. 1). When all patients were divided into two groups according to burn size, those with burns of 45% 30 or greater had an average NBI of 6.0 + 0.7 compared to 4.9 0.4 for patients with 44% 30 or less (p > 0.1). The antibacterial function of neutrophils against S. aureus correlated poorly with the antibacterial function of the same neutrophils against E. coli (Fig. 2), and there was no correlation of function against E. coli with the development of infection, either when examined individually or as a group (infected versus non-
10 9 08
0
0
0~~~~~ 7
6 5 4
*
* r
0
0
0
0
3
0
0
2 01 %
1
1
1
1
2 3 4 5 6 7 8 9 10 11 12 13 14
15
16 17
18
NBI E. coli FIG. 2. Scattergram illustrating the lack of correlation between neutrophil function determined using S. aureus and E. coli as the test organisms. Tests were performed in parallel.
infected). The NBI (E. coli) for the infected group 3.8 + 0.6 compared to 2.8 + 0.7 for the noninfected group (p = not significant). Chemotaxis of neutrophils also correlated poorly with infection although chemotaxis was frequently abnormal. There was no correlation between the chemotactic index and the NBI using the same samples with tests performed at the same time (Fig. 3). The functional chemotactic index for the infected patients was 100 + 17, as compared to 76 11 for the noninfected patients. Sequential studies on the same patients showed no relationship to size of burn or ultimate outwas
10 9
8
7
WECTED
6~~~~~~~~
±
5
come.
4
Tests of Opsonization
1-4
5-8
912 Q3-6
17-20 21-24 25-e8 29-36 3744 P B D
FIG. 1. Sequential neutrophil function (NBI) in burn patients. Values shown are the mean + S.E.; normal values are 2.1 + 0.4. The broken line represents patients who were never infected (bacteremic) during the period of study, while the solid line is pooled data from patients who were infected (bacteremic) at any time during the study. Neutrophil function was significantly worse in infected patients (p < 0.01).
The opsonic index was depressed in all but one patient studied during the first five postburn days, returning to nearly normal values by the fourth to the fourteenth postburn day (Fig. 4). There was no difference between infected and noninfected patients. Similarly, there was no difference in the time-course relationship of the opsonic indices to extent of burn. Of 13 available organisms isolated from blood cultures, there was no evidence of an organism-specific defect in opsonization in any instance when the infecting organisms were tested comparing PNHS with sequential sera from the infected patient before, during and after the event.
812
ALEXANDER AND OTHERS 0
10
0
Ann.
Surg. 9 December 1978
the second week, with only insignificant differences between infected and noninfected groups (Fig. 9). (Data for individual patients are discussed below).
0 0
9
Tests of Lymphocyte Function The responsiveness of isolated lymphocytes to PHA was measured in only seven patients. In no instances were the lymphocytes abnormal in their ability to be stimulated. However, marked depressions in reactivity were present when responsiveness was measured in the patient's whole blood (stimulation index = 5.3 + 0.2 compared to normal controls = 22 + 3). Both dialyzed and nondialyzed serum from the patients inhibited the responsiveness of normal lymphocytes to PHA. There were no differences between infected and noninfected
0 0
8 0
7 0
6
0
0
0
0
0
0
5
0
4
0
0
.0
t0I
3
I
0
0
06
2
0
0
0
0
.
patients (Fig. 10).
0
* 0
-
0* 0
00-
20
40
0
*-
0
60
80
100 120
40
160 180
FUNCTIONAL CHEMOTACTIC INDEX
Analysis of Sequential Measurements in Infected Patients Nineteen episodes of bacteremia occurred in the 13 patients in the infected group (Table 1). One patient had three episodes, and two episodes occurred in each
FIG. 3. Scattergram illustrating the lack of correlation between neutrophil function (using S. aureus) and functional chemotaxic index (FCI). Tests were performed in parallel. 1.2
Serum Concentrations of IgG, Albumin, Factor B, and C3 Serum albumin levels were initially low in both infected and noninfected patients, rising from an average of 2.8 gm/dl on days 1-15 to 3.2 gm/dl by days 36-45 (Fig. 5). However, there was no difference between the infected and noninfected patients. Serum concentrations of IgG were significantly depressed during the early resuscitative period, rising rapidly between the eighth and twentieth postburn days to high normal levels in noninfected patients and low normal levels in infected patients (Fig. 6). The values for infected patients were significantly lower than for noninfected patients after the thirteenth postburn day (p < 0.001), even though they were within normal values. Properdin levels, while initially low, remained within the normal range after the ninth postburn day (Fig. 7). There were no significant differences between infected and noninfected patients. Serum concentrations of factor B rose rapidly during the first three weeks after injury to more than double normal levels in many patients (Fig. 8). Infected and noninfected groups were comparable. C3 concentrations, measured by the B antigenic determinant, rose to higher than normal values during
1.0
0.9
0.8
0.7 0.6
0.5
0.4 0.3
0.2 0.1
1-4
5B
9"2
316 17-20 21-24 25-28 27-36 37-44 PBD
FIG. 4. Sequential analysis of the ability of patient serum to support opsonophagocytosis of E. coli 075. Values shown are the mean ± S.E.; normal values are 0189 ± 0.03. A depression in opsonic index is seen the first week following the burn, but returns to low normal by three weeks:
VOl. 188.* NO. 6
SEVERE THERMAL INJURY
of four patients. Three of the infected patients died, one on PBD 12 from smoke inhalation and pneumonia (59-year-old woman, 63% totalI13% 30), one from a cardiac arrhythmia associated with staphylococcal pericarditis on PBD 28 (12-year-old girl, 55% total/45% 30, and one from septicemia on PBD 34 (55-year-old man, 80% total/33% 3°). All other patients survived and have been discharged. Eleven of the septic episodes were caused by S. aureus, two by Enterobacter cloacae, one each by Klebsiella pneumonia, Candida albicans, Pseudomonas aeruginosa, Streptococcus viridans and a Bacillus sp. and one episode by multiple organisms: S. aureus with E. cloacae. Fourteen of the nineteen episodes of bacteremia were associated with a significant preceding abnormality of neutrophil function against S. aureus 502A (NBI 2 4) (Table 1). The average NBI immediately preceding all bacteremia episodes was 7.6 ± 1.2. The average NBI was also 7.6 immediately preceding the three infections caused by gram negative bacilli. Two other instances were associated with mild neutrophil dysfunction (NBI 2-4), and neutrophil function was normal ( 4) at the time of five of these seven episodes. Circulating levels of C3(B) increased in the serum of patients associated with 12 episodes of bacteremia and
45
40
4.0
r 35
30 3.0 F
25
20 2.0 P Non-Infeced Patient
-
Infected Patient
1.0
I0 5
1-5
6-10
1
16-20
21-25 26 30 31-35 36
40
41-45
DAYS POST BURN
FIG. 5. Sequential analysis of serum albumin in burn patients. Values shown are the mean ± S.E.; the normal range of albumin is 3.5-5.0 gm/dl. No consistent, significant differences are seen between noninfected and infected patients.
*4
5-6
9e
6
r?-0
0214 25-es 29-a6 37-44
P BD
FIG. 7. Sequential analysis of properdin in burn patients. Values shown are the mean + S.E.; normal values are 28 + 1 ,ug/ml.
814
l~ ~F
ALEXANDER AND OTHERS
7001
600
i'
A lllf 4 . X,.~I - /1 500
)
/
300 200
*
100
INFECTED
W N---ECTED
-4
5-8
942 13*6 17-20 21-24 25-28 29-36 37-44 PBD
FIG. 8. Sequential analysis of factor B in burn patients. Values shown are the mean + S.E.; normal values are 261 + 15 ,ug/ml. Note the rapid rise of factor B, suggesting that this component is an acute phase protein.
fell during six episodes. In all of these last six episodes, there was also a fall in the opsonic index, but the opsonic index fell in six other patients without reduction in C3(B). Factor B rose during nine episodes of infection, fell during eight, remained unchanged in one, and was not determined in one. Properdin levels increased during ten episodes, fell during five episodes
241
14001
F4
8-
942 5MS rT-2 a-04 z~a -a 37-44
F16. 9. Sequential analysis of C3(B) levels in burn patients. Values shown are the mean ± S.E.; normal values are 1030 ± 23 ,.tgIml. Initial levels are low, but rise to elevated values by the second week.
Ann. Surg. * December 1978
and remained unchanged during one episode. Sufficient data were not available for three episodes. Strong evidence for consumption (a drop of -50% in C3B and opsonic index) occurred in three patients with suggestive evidence (drops of 25-49%) in two others. In one instance properdin and factor B seemed to be involved in a consumptive process (patient 8-2).
Discussion It is well known that the antibacterial function of neutrophils, the opsonic activity of serum, lymphocyte function and function of the reticuloendothelial system (RES) all contribute to resistance to bacterial infections in experimental animals and man. All of these immunologic functions have been shown to be abnormal following burn injury.1 However, no previous study has investigated the significance of isolated immunologic deficiencies relative to other variables of host defense against infection. We have attempted to examine those variables which were potentially of critical importance, except for function of the RES which was excluded from consideration because adequate tests for sequential evaluation in man are not currently available. The results of this study strongly suggest that acquired abnormalities of neutrophil function, measured by the ability to kill S. aureus 502A, are of major and primary importance in the development of bacteremia following major thermal injury. Increased susceptibility to infection associated with abnormalities of neutrophil function against S. aureus are in 100 _
90
_
80
70-
JL 30
-
~
~
lii
\
20
"
Non-infected
/infected
1=
10 0
1-4
5-8
l
I
9-12 13-16 17-20 21-24 25-28 29-36 PBD
oIG. 10. Sequential analysis of the inhibition of lymphocyte PHA responses by patient serum. Values shown are the mean ± S.E.; normal serum always inhibited PHA responses less than 5%. No consistent, significant differences in serum inhibitory activity between infected and noninfected patients is apparent.
Vol. 188 * No. 6
815
SEVERE THERMAL INJURY TABLE 1. Relationship of Immunologic Variables to Bacteremia Preinfection*
Patient and Episode
Organism
1-1 2-1 3-1
S. aureus S. aureus E. cloacae
3-2
S. aureus &
4-1 4-2 5-1 6-1 7-1 7-2 8-1 8-2 9-1 10-1 10-2
10-3 11-1 12-1 13-1
E. cloacae S. aureus S. aureus S. aureus K. pneumoniae E. cloacae S. aureus S. aureus Candida S. aureus
Bacillus S. S. P. S.
aureus aureus aerug. aureus
Strep.
NBI 5.0 9.9 16.7
Opsonic Index 0.31 0.89 0.56
Postinfection
C3(B)
Factor B
Properdin
IgG
jtg/ml
tg/ml
jg/mi
mg/dl
NBI
1404 1697 1638
960 480 375
N.D.t 27 45
1635 1369 960
N.D. N.D. 12.6
420
38
1200
No
450 360 420 450 420 840 900 630 570 270 420 294 420 420
27 N.D. 35 26 27 32 27 8 27 8 27 24 20 28
1023
Mild
870
No Yes No No Mild No Yes No No No No No Yes
jg/ml
jg/ml
0.94 0.46
2691 2340 1872
870 495 450
1.02
2574
0.67 0.78 20.0 3.9 7.7 2.5 2.0 6.2 4.3 4.4 3.5
14 25 25
No No No
Index
0.96
22 27
1635 1495 624
Properdin
0.88 0.42 0.85 0.56 0.95 1.05 0.56 1.05 0.73
