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The protective effect of pneumococcal vaccination following partial splenectomy
JOURNAL
OF SURGICAL
RESEARCH
45, 56-59 (1988)
The Protective Effect of Pneumococcal Vaccination following Partial Splenectomy’ RANDALL W. POBELL,M.D., *,* WILLIAM E. BLAYLOCK, B.S., CHARLES J. HOFF, PH.D.,? ANDSTEPHEN A. CHARTRAND, M.D.$ Department of *Surgery, Medical Genetics,t and *Pediatrics, University of South Alabama College of Medicine, Mobile, Alabama 36617 Presented at the Annual Meeting of the Association for Academic Surgery, Orlando, Florida, November l-4, 1987 Weanling CD-l male rats were subjected to 100, 75, and 50% splenectomy. One week following splenectomy, animals received either a sham immunization with 0.1 ml NS or pneumococcal immunization with 0.1 ml of a polyvalent (23) vaccine. Eight weeks following surgery, all animals received an intraperitoneal innoculation with 1O6Streptococcus pneumoniae Type 3 organisms and were observed for mortality. Significant differences in mortality were seen between sham and immunized animals undergoing 100 or 75% splenectomy, while in the 50% group a difference was noted which did not reach statistical significance (Mantel-Cox log rank test). Patients undergoing greater than 50% splenectomy may be afforded greater protection against overwhelming pneumococcal infections by immunization with pneumococcai vachne. 0 1988 Academic Ress, Inc.
INTRODUCTION
Overwhelming postsplenectomy infection (OPSI), a syndrome predicted by Morris and Bullock in 19 19 [I] and confirmed by King and Schumaker [2] and Singer [3] in 1952 and 1973, prompted many surgeons to practice splenic salvage [4, 51. Interest in partial splenectomy for other splenic problems such as cysts [6], metabolic disease [7], and staging procedures [8] should result in increasing numbers of patients undergoing such procedures. Pneumococcal vaccination has been shown to decrease mortality from pneumococcal sepsis in both human and animal studies [9, lo]. The purpose of this study was to evaluate the possible protective effects of pneumococcal vaccine in an animal model of varying degrees of splenectomy. ’ This study was supported by an Intramural Grant Award from the College of Medicine, University of South Alabama. *To whom reprint requests should be addressed at Division of Pediatric Surgery, USAMC, 245 1 Fillingim St., Mobile, AL 366 17. 0022-4804188 $1.50 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
MATERIALS
Experimental
AND METHODS
Protocol
One hundred-sixty weanling male CD-l rats (60- 125 g in weight) underwent varying degrees of splenectomy via a midline celiotomy under general anesthesia induced by intraperitoneal (ip) injection of a mixture of ketamine (40 mg/kg) and xyalcine (5 mg/kg) with intramuscular (im) supplementation as necessary. All procedures were performed under clean surgical conditions. Total splenectomy was accomplished by electrocautery of all blood vessels to the spleen. Partial splenectomy (50 or 75%) was accomplished by electrocautery of vessels supplying 50 or 75% of the spleen with the devascularized spleen removed by bipolar electrocoagulation through splenic tissue. Any persistent bleeding was controlled by electrocautery or application of microfibrillar collagen hemostat (Avitene-Alcon, Inc.) One week following the surgical procedure, animal groups received either a sham immunization with 0.1 ml normal saline im or pneumococcal vaccination with 0.1 ml of a polyvalent pneumo56
POWELL TABLE
ET AL.:
PNEUMOCOCCAL
Statistical Methods
1
EXPERIMENTAL GROUPS AND RESULTS Group 123456
% Splenectomy Immunization N Mortality N %
57
VACCINE
100 -
100 +
50 -
50 +
75 -
75 +
40
40
20
20
20
18
38 95
30 75
18 90
15
20 100
13 72
75
coccal vaccine (Pneumovac-23, Merck Sharp & Dohme). Eight weeks following the surgical procedure, all animals received an ip injection of lo6 Streptococcus pneumoniae Type 3. Animals were observed at 8-hr intervals following the ip innoculation for mortality for a total of 6 days. The protocol resulted in six groups as depicted in Table 1. This protocol was reviewed and approved by the Institutional Animal Use and Care Committee of the University of South Alabama College of Medicine (Animal Care and Use Assurance Number A 3288-01)
Microbiologic Protocol S. pneumoniae Type 3 (American
Type Culture Collection) was suspended in an equal volume of 15% mucin/4% hemoglobin and then 1 ml was injected ip in a 20-g CF- 1 mouse. Six hours after the ip injection, heart blood was subcultured on blood agar plates and incubated overnight (35°C room air). Isolated colonies were then suspended in Muellar Hinton broth with 25% fetal calf serum and grown to log phase (3-4 hr). The organisms were then pelleted (12508 X 15 min) and resuspended in normal saline to a final inoculum density of 106- 106.5 colonyforming units/ml (optical density method). The final suspension was administered to each experimental animal by ip injection.
The survival rates and variables were analyzed by x2 likelihood ratio. The survival curves based on S-hr survival rates over 6 days were analyzed by the Mantel-Cox log rank analysis. RESULTS
The mortality rates by group are depicted in Table 1. When variables were analyzed by x2 likelihood ratio, the significant variable was whether or not the animal was immunized with a relative risk value of 2.46; i.e., immunized animals had a likelihood of survival 2.46 times greater than that of shamimmunized animals. When survival curves were analyzed, significant differences were noted in the group pairings depicted in Table 2. DISCUSSION
Overwhelming postsplenectomy infection remains a concern in the patient undergoing
TABLE
2
PAIRWISE COMPARISONSOF KAPLAN-MEIER SURVIVAL CURVES BY MANTEL-C• X LOG-RANK TEST Group pairing 1:2 1:3 1:4 1:5 1:6 2:3 2:4 2~5 2:6 3:4 3:5 316 4:5 4:6 5:6
Stat N
P value
18.134 2.032 8.144 0.651 10.489 4.496 0.131 0.198 0.214 1.948 0.525 3.956 3.936 0.340 7.289
0.000 1 N.S. 0.0043
N.S. 0.0012 0.0340
N.S. 0.0024
N.S. N.S. N.S. 0.0467 0.0473
N.S. 0.0069
Note. Stat N = Mantel-Cox log-rank statistic. Group pairings are experimental groups identified in Table 1.
58
JOURNAL
OF SURGICAL
RESEARCH: VOL. 45, NO. 1, JULY
splenectomy and has resulted in efforts to conserve the spleen. Frequently, splenorrhaphy may require resection of devitalized or severely injured portions of the spleen. Goldthorn et al. demonstrated an increased LDso for rats undergoing partial splenectomy (75%) when challenged with intravenous pneumococci [ 1 I]. Church et al. demonstrated normal immunologic response to sheep red blood cell infusion in rats undergoing 50% splenectomy, but a decreased response in animals undergoing 75% splenectomy [ 121. Haque et al. demonstrated increased survival in an animal model with 50% of the spleen removed when compared to total splenectomy and splenic reimplantation [ 131. Van Wyck et al. utilizing graded splenic resection in rats demonstrated that greater than one-third of the splenic mass was necessary to maintain host resistance against intravenous pneumococcal challenge [ 141. These studies lead to the conclusion that in animal models 50% of the spleen seems to be necessary to maintain bacterial clearance and humoral functions of the spleen at normal levels. The pneumococcal vaccine, now in a polyvalent vaccine covering 23 types of the organism, is widely used for patients rendered asplenic or hyposplenic by surgery or disease. Ammann et al. demonstrated the efficacy of the vaccine in patients with sicklecell disease and patients undergoing splenectomy [9]. Many animal studies have also documented the efficacy of the vaccine [ 10, 15, 161. In our experimental model, a severe bacterial challenge resulted in high mortalities in all groups. When the two variables, i.e., percentage splenectomy and immunization, were analyzed by x2 likelihood ratios, the degree of splenectomy was not significant, but animals receiving the pneumococcal vaccine had a likelihood of survival 2.46 times greater than that of sham-immunized animals. When the different groups were compared utilizing the Mantel-Cox log rank test (Table 2), significant differences occurred be-
1988
tween sham and immunized groups in all pairings except between groups 3 and 4 (50% splenectomy). The most significant differences were between the animals undergoing 100 and 75% splenectomy with immunized animals experiencing 20 and 28% decreases in mortality, respectively. In the 50% splenectomy groups, sham animals sustained a 90% mortality versus 75% in the immunized animals, but the difference did not reach statistical significance. As splenic salvage becomes more popular and widely utilized and other indications for partial splenic resection increase, more patients will undergo partial splenectomy. Our experimental model indicates that patients undergoing removal of 50% or greater of their splenic mass may be afforded increased protection from severe pneumococcal infections by pneumococcal vaccination. REFERENCES 1. Morris, D. H., and Bullock, F. D. The importance of the spleen in resistance to infection. Ann. Surg. 70: 513, 1919. 2. Ring, H., and Schumacker, H. B., Jr. Splenic studies. I. Susceptibility to infection after splenectomy in infancy. Ann. Surg. 136: 239, 1952. 3. Singer, D. B. Postsplenectomy sepsis. In H. S. Rosenberg, and R. P. Bolands (Eds.), Perspectives in Pediatric Pathology. Chicago: Year Book Med. Pub. 1973. Pp. 285-3 11. 4. Buntain, W. L., and Lynn, H. B. Splenorrhaphy: Changing concepts for the traumatized spleen. Surgery 86: 748, 1979. 5. Sherman, R. Management of trauma to the spleen. Adv. Surg. 17: 37, 1984. 6. Morgenstem, L., and Shapiro, S. J. Partial splenectomy for nonparasitic splenic cysts. Amer. J. Surg. 139: 278, 1980. I. Rubin, M., Yampolski, I., Lambrozo, R., Zaizov, R., and Dintsman, M. Partial splenectomy in Gaucher’s disease. J. Pediatr. Surg. 21: 125, 1986. 8. Tubbs, R. R., Thomas, F., Norris, D., and Firor, H. V. Is hemisplenectomy a satisfactory option to total splenectomy in abdominal staging of Hodgkin’s disease?J. Pediatr. Surg. 22: 727, 1987. 9. Ammann, A. J., Addiego, J., Wara, D. W., Lubin, B., Smith, W. B., and Mentzer, W. C. Polyvalent pneumococcal-polysaccharide immunization of patients with sickle-cell anemia and patients with splenectomy. N. Engl. J. Med. 297: 897, 1977.
POWELL
ET AL.: PNEUMOCOCCAL
10. Dawes, L. G., Malangoni, M. A., Spiegel, C. A., and Schiffman, G. Response to immunization after partial and total splenectomy. J. Surg. Rex 39: 53, 1985. 11. Goldthorn, J. F., Schwartz, A. D., Swift, A. J., and Winkelstein, J. A. Protective effects of residual splenic tissue after subtotal splenectomy. J. Pediatr. Surg. 13: 587, 1978. 12. Church, J. A., Mahour, G. H., and Lipsey, A. I. Antibody responses after splenectomy and splenic autoimplantation in rats. J. Surg. Res. 31: 343, 1981. 13. Haque, A. U., Hudson, P., Wood, G., and Lindsey, N. J. Splenic autotransplant and residual partial spleen: Prevention of septicemia. Japan. J. Surg. 14: 407, 1984.
VACCINE
59
14. Van Wyck, D. B., Witte, M. H., Witte, C. L., and Thies, A. C., Jr. Critical splenic mass for survival from experimental pneumococcemia. J. Surg. Res. 28: 14, 1980. 15. Cooney, D. R., Dearth, J. C., Swanson, S. E., Dewanjee, M. K., and Telander, R. L. Relative merits of partial splenectomy, splenic reimplantation, and immunization in preventing postsplenectomy infection. Surgery 86: 56 1, 1979. 16. Scher, K. S., Wroczynski, A. F., and Jones, C. W. Protection from postsplenectomy sepsis: Effect of prophylactic penicillin and pneumococcal vaccine on clearance of type 3 Pneumococcus. Surgery 93: 792, 1983.
OF SURGICAL
RESEARCH
45, 56-59 (1988)
The Protective Effect of Pneumococcal Vaccination following Partial Splenectomy’ RANDALL W. POBELL,M.D., *,* WILLIAM E. BLAYLOCK, B.S., CHARLES J. HOFF, PH.D.,? ANDSTEPHEN A. CHARTRAND, M.D.$ Department of *Surgery, Medical Genetics,t and *Pediatrics, University of South Alabama College of Medicine, Mobile, Alabama 36617 Presented at the Annual Meeting of the Association for Academic Surgery, Orlando, Florida, November l-4, 1987 Weanling CD-l male rats were subjected to 100, 75, and 50% splenectomy. One week following splenectomy, animals received either a sham immunization with 0.1 ml NS or pneumococcal immunization with 0.1 ml of a polyvalent (23) vaccine. Eight weeks following surgery, all animals received an intraperitoneal innoculation with 1O6Streptococcus pneumoniae Type 3 organisms and were observed for mortality. Significant differences in mortality were seen between sham and immunized animals undergoing 100 or 75% splenectomy, while in the 50% group a difference was noted which did not reach statistical significance (Mantel-Cox log rank test). Patients undergoing greater than 50% splenectomy may be afforded greater protection against overwhelming pneumococcal infections by immunization with pneumococcai vachne. 0 1988 Academic Ress, Inc.
INTRODUCTION
Overwhelming postsplenectomy infection (OPSI), a syndrome predicted by Morris and Bullock in 19 19 [I] and confirmed by King and Schumaker [2] and Singer [3] in 1952 and 1973, prompted many surgeons to practice splenic salvage [4, 51. Interest in partial splenectomy for other splenic problems such as cysts [6], metabolic disease [7], and staging procedures [8] should result in increasing numbers of patients undergoing such procedures. Pneumococcal vaccination has been shown to decrease mortality from pneumococcal sepsis in both human and animal studies [9, lo]. The purpose of this study was to evaluate the possible protective effects of pneumococcal vaccine in an animal model of varying degrees of splenectomy. ’ This study was supported by an Intramural Grant Award from the College of Medicine, University of South Alabama. *To whom reprint requests should be addressed at Division of Pediatric Surgery, USAMC, 245 1 Fillingim St., Mobile, AL 366 17. 0022-4804188 $1.50 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
MATERIALS
Experimental
AND METHODS
Protocol
One hundred-sixty weanling male CD-l rats (60- 125 g in weight) underwent varying degrees of splenectomy via a midline celiotomy under general anesthesia induced by intraperitoneal (ip) injection of a mixture of ketamine (40 mg/kg) and xyalcine (5 mg/kg) with intramuscular (im) supplementation as necessary. All procedures were performed under clean surgical conditions. Total splenectomy was accomplished by electrocautery of all blood vessels to the spleen. Partial splenectomy (50 or 75%) was accomplished by electrocautery of vessels supplying 50 or 75% of the spleen with the devascularized spleen removed by bipolar electrocoagulation through splenic tissue. Any persistent bleeding was controlled by electrocautery or application of microfibrillar collagen hemostat (Avitene-Alcon, Inc.) One week following the surgical procedure, animal groups received either a sham immunization with 0.1 ml normal saline im or pneumococcal vaccination with 0.1 ml of a polyvalent pneumo56
POWELL TABLE
ET AL.:
PNEUMOCOCCAL
Statistical Methods
1
EXPERIMENTAL GROUPS AND RESULTS Group 123456
% Splenectomy Immunization N Mortality N %
57
VACCINE
100 -
100 +
50 -
50 +
75 -
75 +
40
40
20
20
20
18
38 95
30 75
18 90
15
20 100
13 72
75
coccal vaccine (Pneumovac-23, Merck Sharp & Dohme). Eight weeks following the surgical procedure, all animals received an ip injection of lo6 Streptococcus pneumoniae Type 3. Animals were observed at 8-hr intervals following the ip innoculation for mortality for a total of 6 days. The protocol resulted in six groups as depicted in Table 1. This protocol was reviewed and approved by the Institutional Animal Use and Care Committee of the University of South Alabama College of Medicine (Animal Care and Use Assurance Number A 3288-01)
Microbiologic Protocol S. pneumoniae Type 3 (American
Type Culture Collection) was suspended in an equal volume of 15% mucin/4% hemoglobin and then 1 ml was injected ip in a 20-g CF- 1 mouse. Six hours after the ip injection, heart blood was subcultured on blood agar plates and incubated overnight (35°C room air). Isolated colonies were then suspended in Muellar Hinton broth with 25% fetal calf serum and grown to log phase (3-4 hr). The organisms were then pelleted (12508 X 15 min) and resuspended in normal saline to a final inoculum density of 106- 106.5 colonyforming units/ml (optical density method). The final suspension was administered to each experimental animal by ip injection.
The survival rates and variables were analyzed by x2 likelihood ratio. The survival curves based on S-hr survival rates over 6 days were analyzed by the Mantel-Cox log rank analysis. RESULTS
The mortality rates by group are depicted in Table 1. When variables were analyzed by x2 likelihood ratio, the significant variable was whether or not the animal was immunized with a relative risk value of 2.46; i.e., immunized animals had a likelihood of survival 2.46 times greater than that of shamimmunized animals. When survival curves were analyzed, significant differences were noted in the group pairings depicted in Table 2. DISCUSSION
Overwhelming postsplenectomy infection remains a concern in the patient undergoing
TABLE
2
PAIRWISE COMPARISONSOF KAPLAN-MEIER SURVIVAL CURVES BY MANTEL-C• X LOG-RANK TEST Group pairing 1:2 1:3 1:4 1:5 1:6 2:3 2:4 2~5 2:6 3:4 3:5 316 4:5 4:6 5:6
Stat N
P value
18.134 2.032 8.144 0.651 10.489 4.496 0.131 0.198 0.214 1.948 0.525 3.956 3.936 0.340 7.289
0.000 1 N.S. 0.0043
N.S. 0.0012 0.0340
N.S. 0.0024
N.S. N.S. N.S. 0.0467 0.0473
N.S. 0.0069
Note. Stat N = Mantel-Cox log-rank statistic. Group pairings are experimental groups identified in Table 1.
58
JOURNAL
OF SURGICAL
RESEARCH: VOL. 45, NO. 1, JULY
splenectomy and has resulted in efforts to conserve the spleen. Frequently, splenorrhaphy may require resection of devitalized or severely injured portions of the spleen. Goldthorn et al. demonstrated an increased LDso for rats undergoing partial splenectomy (75%) when challenged with intravenous pneumococci [ 1 I]. Church et al. demonstrated normal immunologic response to sheep red blood cell infusion in rats undergoing 50% splenectomy, but a decreased response in animals undergoing 75% splenectomy [ 121. Haque et al. demonstrated increased survival in an animal model with 50% of the spleen removed when compared to total splenectomy and splenic reimplantation [ 131. Van Wyck et al. utilizing graded splenic resection in rats demonstrated that greater than one-third of the splenic mass was necessary to maintain host resistance against intravenous pneumococcal challenge [ 141. These studies lead to the conclusion that in animal models 50% of the spleen seems to be necessary to maintain bacterial clearance and humoral functions of the spleen at normal levels. The pneumococcal vaccine, now in a polyvalent vaccine covering 23 types of the organism, is widely used for patients rendered asplenic or hyposplenic by surgery or disease. Ammann et al. demonstrated the efficacy of the vaccine in patients with sicklecell disease and patients undergoing splenectomy [9]. Many animal studies have also documented the efficacy of the vaccine [ 10, 15, 161. In our experimental model, a severe bacterial challenge resulted in high mortalities in all groups. When the two variables, i.e., percentage splenectomy and immunization, were analyzed by x2 likelihood ratios, the degree of splenectomy was not significant, but animals receiving the pneumococcal vaccine had a likelihood of survival 2.46 times greater than that of sham-immunized animals. When the different groups were compared utilizing the Mantel-Cox log rank test (Table 2), significant differences occurred be-
1988
tween sham and immunized groups in all pairings except between groups 3 and 4 (50% splenectomy). The most significant differences were between the animals undergoing 100 and 75% splenectomy with immunized animals experiencing 20 and 28% decreases in mortality, respectively. In the 50% splenectomy groups, sham animals sustained a 90% mortality versus 75% in the immunized animals, but the difference did not reach statistical significance. As splenic salvage becomes more popular and widely utilized and other indications for partial splenic resection increase, more patients will undergo partial splenectomy. Our experimental model indicates that patients undergoing removal of 50% or greater of their splenic mass may be afforded increased protection from severe pneumococcal infections by pneumococcal vaccination. REFERENCES 1. Morris, D. H., and Bullock, F. D. The importance of the spleen in resistance to infection. Ann. Surg. 70: 513, 1919. 2. Ring, H., and Schumacker, H. B., Jr. Splenic studies. I. Susceptibility to infection after splenectomy in infancy. Ann. Surg. 136: 239, 1952. 3. Singer, D. B. Postsplenectomy sepsis. In H. S. Rosenberg, and R. P. Bolands (Eds.), Perspectives in Pediatric Pathology. Chicago: Year Book Med. Pub. 1973. Pp. 285-3 11. 4. Buntain, W. L., and Lynn, H. B. Splenorrhaphy: Changing concepts for the traumatized spleen. Surgery 86: 748, 1979. 5. Sherman, R. Management of trauma to the spleen. Adv. Surg. 17: 37, 1984. 6. Morgenstem, L., and Shapiro, S. J. Partial splenectomy for nonparasitic splenic cysts. Amer. J. Surg. 139: 278, 1980. I. Rubin, M., Yampolski, I., Lambrozo, R., Zaizov, R., and Dintsman, M. Partial splenectomy in Gaucher’s disease. J. Pediatr. Surg. 21: 125, 1986. 8. Tubbs, R. R., Thomas, F., Norris, D., and Firor, H. V. Is hemisplenectomy a satisfactory option to total splenectomy in abdominal staging of Hodgkin’s disease?J. Pediatr. Surg. 22: 727, 1987. 9. Ammann, A. J., Addiego, J., Wara, D. W., Lubin, B., Smith, W. B., and Mentzer, W. C. Polyvalent pneumococcal-polysaccharide immunization of patients with sickle-cell anemia and patients with splenectomy. N. Engl. J. Med. 297: 897, 1977.
POWELL
ET AL.: PNEUMOCOCCAL
10. Dawes, L. G., Malangoni, M. A., Spiegel, C. A., and Schiffman, G. Response to immunization after partial and total splenectomy. J. Surg. Rex 39: 53, 1985. 11. Goldthorn, J. F., Schwartz, A. D., Swift, A. J., and Winkelstein, J. A. Protective effects of residual splenic tissue after subtotal splenectomy. J. Pediatr. Surg. 13: 587, 1978. 12. Church, J. A., Mahour, G. H., and Lipsey, A. I. Antibody responses after splenectomy and splenic autoimplantation in rats. J. Surg. Res. 31: 343, 1981. 13. Haque, A. U., Hudson, P., Wood, G., and Lindsey, N. J. Splenic autotransplant and residual partial spleen: Prevention of septicemia. Japan. J. Surg. 14: 407, 1984.
VACCINE
59
14. Van Wyck, D. B., Witte, M. H., Witte, C. L., and Thies, A. C., Jr. Critical splenic mass for survival from experimental pneumococcemia. J. Surg. Res. 28: 14, 1980. 15. Cooney, D. R., Dearth, J. C., Swanson, S. E., Dewanjee, M. K., and Telander, R. L. Relative merits of partial splenectomy, splenic reimplantation, and immunization in preventing postsplenectomy infection. Surgery 86: 56 1, 1979. 16. Scher, K. S., Wroczynski, A. F., and Jones, C. W. Protection from postsplenectomy sepsis: Effect of prophylactic penicillin and pneumococcal vaccine on clearance of type 3 Pneumococcus. Surgery 93: 792, 1983.