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Tetanus prophylaxis following ocular injuries
The JournaiofEmergency
MeUmne,
Voi 11, pp 677-683.
?rlnted
1993
I-ANUS PROPHYLAXIS
in the USA
CopyrIght
0 ‘993 Per;arx)n
Ore% L!ci
FOLLOW
enson, MD,* Irvin S. Snyder, PhD,j- Valerie and Marian S. Macsai, *Department of Ophthalmology, Medical College Ophthalmology, Microbiology, and Immunology, Reprint Address: William H. Benson, MD, Department
of Virginia, Virginia Commonwealth University, Alchmond, Virginia. Kkpxtmenis o; School of Medicine, West Virginia University Health Sciences Center, kkxgantown of Ophthalmology, Medical College of Virginia, Box 262, Richmond, VA 232904262
been reported in the literature, and none include simple cornea1 abrasion. The 5 case reports lacked details regarding the history and examination so that occult perforation of the cornea, sclera, or skin could not be ruled out (1,9,13,lis). clinical tetanus following an abrasion has not been reported in the li~erat~Ke, and the risk of developing tetanus ~o~~o~~~~~a cornea1 abrasion has not been evaluated by any Kiowa clinical or laboratory investigations B To evaluate whether the present gui prophylaxis against tetanus were appro 17), we developed an animal modei for scalar inoculation of C tetffni in the m signed to determine if tetanus could inoculation of live C tetani 0 toxin through 3 different ro epithelial debridement (abrasio~)~ c~~~~~~ stroma scarification (pe~etratio~)~ and intraca tion (perforation).
0 Abstract-The admIn~st~atioo of prophylaxis against tetanus following a cornea! abrasion is routinely performed in many acute care facilities, despite a lack of support in e literature for its necessity. The risk of developing clinical tetanus from three different types of injuries to the eye was evaluated in an animal model. Clinical tetanus was induced in unimmunized mice by injecting Clostridium tetani organlsms or toxin into the anterior chamber. Immunized mice injected intracamerally did not develop signs of tetanus. Tetanus was not induced by topical inoculation of either live organisms or toxin following cornea1 epithelial t OKsltromal scarification of unimmunized and mice. The results of this study support the administration of prophylaxis against tetanus following perforating ocular injuries. However, our results do not support its routine use following uncomplicated cornea1 abrasions or other types of nonperforating ocular injuries. 3 Keywords - animal model; clostridium tetani; cornea1 ebridement; intracameral; scarification; tetanus
abrasion;
ministration of prophylaxis against tetanus following a cornea1 abrasion is routinely performed in many acute care facilities, despite a lack of support for routine immunization in the literature (1,5). A search of the medical literature from 1847 to 1993 revealed that only 38 cases of clinical tetanus following ocular injury have been reported, of which at least 33 involved perforation through either the cornea or the sclera (5-14). Only 5 cases of clinical tetanus following nonperforating ocular injuries have
~rg~~is~s and Cuiture C tetani A6455 was obtaiae
chamber on prereduced sheep Moo Gram stained to establish purity. plates were prereduced in an a~~e~~~i~
Presented at the Annual Meeting of the Association for Research in Vision and Ophthalmology, Sarasota, May, 1991. RECEIVED: ACCEPTED
1
: 1
1992; FINAL SUBMISSION RECEIVED: y
9 April
1993 677
1993;
678
William H. Benson,
Irvin S. Snyder,
meat broth medium at 35 OC for 24 hours in an anaerobic chamber. The medium was prepared using Bacto-Cooked Meat Medium supplemented to meet the formulation described by Dowell, Lombard, Thompson, and Armfield (18). Agar was not added in order to prepare a broth.
Preparation
of Organisms
Twenty-four hour cultures of C tetani in chopped meat were gently shaken. The meat particles were allowed to settle and the supernatant containing organisms and toxin was removed by decanting. The organisms were sedimented by centrifugation at 12,000 x g for 10 minutes. The supernatant containing toxin was removed and kept cold in ice until needed. The organisms were washed 3 times with a phosphate-gelatin buffer (4 g NaH,PO,-2g gelatin/L, pH 6.2) and resuspended in approximately the original volume. The OD,, was recorded and the number of organisms determined by plating dilutions of the suspension on prereduced sheep blood agar. The cultures were incubated in an anaerobic chamber at 35 OC for 48 hours and the colonies were counted.
Valerie Granus,
J. Vernon Odom, Marian S. Macsai
Toxicity Testing To determine the infectious dose of C tetani for mice, organisms were resuspended in an equal volume of 10% CaCl,. Two-tenths mL were inoculated intramuscularly (IM) into a hind leg of immunized and unimmunized mice (19). The animals were observed for 14 days for signs of clinical tetanus and death cw *
To assess the toxin preparation, 0.2 mL was inoculated IP into immunized and unimmunized mice. The animals were observed for signs of clinical tetanus and death over a 1Cday period. Control mice received the supernatant from uninoculated chopped meat medium.
Ocular Surgical Procedures
Twenty-gram Swiss-Webster mice obtained from Hilltop Lab Animals (Scottdale, PA) were used for the study. The mice were allowed to rest for 24 hours before use and were given food and water ad libitum.
Ocular inoculation with C tetani culture and toxin was performed on unimmunized and immunized mice using each of the debridement, scarification, and intracameral routes. All mice undergoing ocular inoculation were anesthetized for the procedure using a 3 : 1 mixture of ether and chloroform in a closed chamber. Proparacaine was applied topically for local anesthesia. Cornea1 epithelial debridement was performed using a #15 Bard Parker surgical blade (Becton Dickson Co., Lincoln Park, NJ) under an operating microscope. The eyes were then inoculated topically with either C tetani culture or tetanus toxin. Plastic ocular drapes were used to form a funnel around the debrided and sacrificed eyes to allow contact with undilute C tetani culture and tetanus toxin for 3 minutes per eye. Cornea1 stroma scarification was performed using a sharp surgical blade. Eight incisions per cornea were made by placing 1 set of 4 parallel incisions into the stroma at a 90° angle to another set of 4 parallel incisions (Figure 1). Topical application of C tetani culture or tetanus toxin was then performed as described above. The intracameral injections (0.01 mL) were performed using a Hamilton 702 25 ~1 syringe with a 30-gauge needle introduced into the anterior chamber through the limbus.
Immunization
Bacterial Counts and Histopathology
Equine C tetani antitoxin (Sclavo, Inc., Wayne, NJ) was diluted to 200 units/ml in 0.85% NaCl. Mice were passively immunized by intraperitoneal (IP) inoculation of 0.25 mL of the diluted antitoxin (50 units) 24 hours prior to challenge with either organisms or toxin.
Bacterial counts and histopathology were performed on eyes inoculated by the 3 ocular routes with undilute C tetani culture. To perform the bacterial counts, the inoculated eye was removed, placed in 1 mL of phosphate gelatin buffer, and homogenized in a Potter-Elvehjem tissue grinder. One-tenth mL of
Preparation of Toxin The supernate from a 24-hour culture (described above) was sterilized by filtration through a 0.45 pm pore size filter. No growth was obtained on anaerobic culture of the toxin. Dilutions of toxin were prepared in phosphate-gelatin buffer for inoculation.
Mice
679
Tetanus Prophylaxis
tion that develope and injection of organisms the proportion of those dev
analysis of the sample size ne groups to accurate1 tion expected to de (that is, 1.0 in th munized mice). [z/error]*) (22). If a probabilit z = 1.96. Error refers to the ated in one’s estimate. ~xarn~~a~~o~ indicates that as P approaches size required becomes infinitely er, we assumed that P = that a maximum error of 0.11 yielding a minimum sample siz
rn error tolerof the equation
Infection and Toxicity Tedrag Figure
f . cause
eye
foliowin
cornea
stromal
scarification.
serial lo-fold dilutions of homogenized tissue was inoculated onto prereduced sheep blood agar and incubated for 72 hours in an anaerobic chamber at 35 oc. To test the survival of the organisms during grinding and inoculation, a suspension of C tetani containing 3.8 x IO* organisms/ml was carried through the procedure described above. The number recovered O8 organisms/ml, indicating that the procedure did not affect recovery of organisms. For histopathologic study, the inoculated eyes and fixed in 10% formalin. The eyes were embedded in paraffin, sectioned, and stained with periodic acid schiff and hematoxylin and eosin.
Tests ~f~ro~~~tion~ and Determination Sizes- Fewer Analyses
of Sample
We determined that we would compare the proportion of groups receiving ocular surgery that developed tetanus with the proportion of the appropriate control group that developed tetanus. That is, proportions of mice receiving toxin by debridement, scarification, or intracameral injection that developed tetanus would be compared with the proportion of mice receiving II? injections of toxin that developed tetanus (26127 or 96Vo), and with the propor-
ani IM. Inoc~lat~~~ that the cultu
of doubling
supernate contain ~~~~ized mice
were not affected by the toxin.
Clinical lGu&ngs One week following inoculae of d-e. 6 ~~~i~rn~nized mice injected intracame ly with ~~~~l~t~ C tetani manifested signs of clin tetanus r~s~~ti~~ in death. Of the I5 ~nimmuniz ce used for ocular pathology and bacterial counts that received intracamera1 inoculation of undilute C kelei culture, 4 manifested signs of clinical tetanus wit prior to sacrificing on days 4, groups, 6 of the 21 ~nimmu~iz camerally with undilute C te signs of clinical tetanus or de 0.01) (Table 1). Four of the 6 received i~tracameral inoculation of LBE toxin exhibited signs of ical te~a~~~ resulting in death (z = 2.28, P < 0. TRe signs of tetanus exhibited were typical of se ~~od~~e~ in ~r~~~~s mouse models (20-23). They include paralysis of the legs res stable waddling gait, and trismus. Several mice were
William H. Benson,
680
Irvin S. Snyder, Valerie Granus,
Table 1. Clinical Tetanus or Death among Unimmunized Debrided Eyes
Treatment
Scarified Eyes
*** C tetani organisms
lntracameral Injection 6/21 (29%)
***
013
Intramuscular Injection
**
016
et*
C tetani toxin
Marian S. Macsai
Mice (n = 45)
***
016
J. Vernon Odom,
16/l 8 (89%) lntraoeritoneal Ihjection
*
013
416 (67%) 26/27 (96%)
*P < 0.05;~
= 2.19.
*'P
< 0.0002;~
= 3.78.
immobilized while feeding after their jaws became irreversibly clenched to the bar of the cage. None of the unimmunized mice inoculated with undilute C tetani culture or toxin following scarification or debridement demonstrated signs of clinical tetanus or death (Table 1). None of the immunized mice inoculated with undilute C tetani culture or toxin by either intracameral, scarification, or debridement routes exhibited signs of clinical tetanus or death (Table 2). None of the mice in any control group that received ocular inoculation of sterile phosphate-gelatin buffer showed signs of clinical tetanus or death.
***P
5 0.000008:~
=
1 4.0.
due to toxin released from the organisms in vitro during the experimental procedure. Cultures of C tetani were counted at time 0 and after a 2-hour period. The numbers of viable C tetani organisms dropped from 3.8 x 10’ organisms/ml to 3.2 x 10’ organisms/ml, indicating that the organisms survived the length of the experimental procedure. After 2 hours at room temperature (RT), the culture was diluted to approximate the dose injected intracamerally. The culture was filtered and the supernate injected IP into mice to test for released toxin. Clinical tetanus followed inoculation of bacteria-free supernate from cultures kept at room temperature but not from those kept at 4 OC. Cultures of supernate were sterile.
Bacterial Analysis Histopathologic Attempts to isolate C tetani from mice eyes on days 1, 2, and 4 were unsuccessful. In order to determine the survival of C tetani in the mouse eye, the following experiment was performed. Undilute culture (1.6 x lo6 organisms/ml) of C tetani kept at 4 OC was injected intracamerally. The eyes were enucleated for grinding and culture at 0 and 2, 4, 8, 12, and 18 hours following inoculation. At time zero, 5 x lo3 organisms were recovered from the cultured tissue. Organisms decreasing numbers were cultured through 12 hours, and by 18 hours no growth was observed from cultured eyes. We tested the possibility that clinical tetanus was
Organisms were not observed on Gram or BrownHopps stains. Histopathologic examination showed a transient, severe, acute inflammatory reaction in the anterior chamber of unimmunized mice injected intracamerally. The inflammatory response was less severe in immunized mice following intracameral inoculation. By day 3 there was no inflammatory reaction in immunized mice receiving the same injection. The acute infiltration persisted for more than one week in immunized mice and was placed by granulation tissue, as evidenced by a significant proliferation of fibroblasts and endothelial cells.
Table 2. Clinical Tetanus or Death among Immunized Debrided Eyes
Treatment
Findings
Scarified Eyes
Mice (n = 42) lntracameral Injection
Intramuscular Injection
* C terani organisms C tetani
toxin
016
016
o/21
2/9 (22%)
013
013
013
lntraperitoneal Injection o/4
"P < 0.05;~
=
2.18.
Tetanus Prophylaxis
environment Reports of clinical tetanus following ocular injuries are extremely rare. Prior to 1941, almost all cases reported in the medical literature were perforating eye injuries associated with objects in direct contact with horses and the ground, with a mortality rate of 75%. The administration of prophylactic antitoxin was identified for only one of those cases. A review of those cases by Cogan in 1939 (5) and Wetzel in 1942 (9) indicated that prophylactic antitoxin was given in high doses following the onset of clinical tetanus in most of the remaining cases, including many with a fatal outcome. Since 1941, only 3 cases have been reported, with all patients experiencing complete recovery. Only a single case of clinical tetanus following a cornea1 foreign body injury has been reported since 193 I. Despite this rare occurrence, it remains common practice in many acute care facilities to administer prophylaxis against tetanus following routine cornea1 abrasions. We designed an experimental model in the mouse y inoculating C tetani organisms and toxin following 3 types of ocular injuries. The mouse was an appropriate model to evaluate human response to C tetani because, according to known information from previous studies, both mice and humans are among the species most susceptible to toxin (19-20). The various parameters regarding type of injury, type of insculum, and prior immune status were evaluated. Clinical tetanus developed in unimmunized mice following injection of C tetani organisms or toxin into the anterior chamber. Tetanus was not established among any immunized mice, or with topical administration of inoculum following corneal epithelial debridemenl or stromal scarification. We
le 3. Immunization Schedule for Prepared by The Committee
microbiology studies of normal and infects bacterial flora (5-10,13, 24-29). For unimmunized mice inject signs of tetanus preceded death by in 24 hours for those ~~~~~lated I with C tetani organisms. The inc time of death were dependent on ganisms and toxin injected. The la the inoculum, the shorter the ins few as 5 x lo3 organisms were duced enough toxin after intrac produce signs of clinical tetanus son clinical tetanus developed in munized mice injected intracam C tetani organisms is not know the mice with regard to indivi sponses to C tetani organisms or the rratural resistance of the species to C tetaazi may results. These results are similar to t in previous mause studies that use strains of C tetani (19,23). The difficulty with isolation of C te~~.~iorganisms from wounds has been expressed other investigators (8,30-31). Clinical rep5rts animal studies have shown that tetanus is more ely to occur in cases of ~ano~hthalmitis caused by a ~~~5~~~~~ purulent bacterial ~~~taminat~~n and that pure ~~lt~r~s of G tetani inoculated in rabbit eyes cause only a mild iridocyclitis (5,9,12). It has been suggeste tissue necrosis from a purulent ~a~~~bK~~a~rn~~~s may aid in reducing the oxygen co~~~~tra~~~~ in the eye
Tetanus Prophylaxis on Trauma, ~rne~i~a~ Tetanus-Prone Wounds
History Toxoid
of Adsorbed (Doses)
Unknown or fewer 3 or moret
and t
i
Non-Tetanus-Prone Wounds
Tetanus
than
3
Td*
TIG
Td’
TIG
Yes NO-4
Yes
Yes NQ§
No
NO
NO
“For children younger than 7 years of age: DTP (DT, if pertussis vaccine is contraindicated) is preferable to tetanus toxoid alone. For persons 7 years old and older, Td is preferable to tetanus toxoid alone. tlf only 3 doses of fluid toxoid have been received, a 4th dose of toxoid, ~re~er~bl~ an adsorbed toxoid, should be given. $Yes, if more than five years since last dose. (More frequent boosters are not needed and can accentuate side effects.) $Yes, if more than 10 years since last dose. Td = te?anus and diptheria toxoids adsorbed (for adult use). TIG = tetanus immuneglobulin (human).
682
William H. Benson, Table
Clinical
4. Ocular Prone
Wound Wounds
Classification
Irvin S. Snyder, Valerie Granus, for Tetanus-Prone
Tetanus-Prone Wounds
Features
Depth Signs of infection Devitalized tissue Contaminants (dirt,
sufficiently
to allow growth
feces,
soil, saliva)
Corneakcleral perforation Present Present Present
of C tetani organisms
(12).
Specific recommendations for tetanus prophylaxis following ocular injuries are limited. The immunization advisory committee that determines the criteria for prophylaxis against tetanus in wound management does not recommend specific guidelines for the various types of ocular injuries. The decision of when to administer tetanus prophylaxis is subject to interpretation of the present guidelines, which separate all wounds into two categories: “tetanus-prone wounds” and “non-tetanus-prone wounds” (Table 3). The risk factors for a tetanus-prone wound include any one of the following clinical features: Wound age >6 hours, a configuration of stellate shape, avulsion or abrasion, a depth > 1 cm, mechanics of injury due to a missile, crush, burn, or frostbite, or the presence of any signs of infection, devitalized tissue, contaminants (dirt, feces, soil, saliva, and so on), or denervated or ischemic tissue. Current recommendations for tetanus prophylaxis involve a wound with any one of these clinical features. Many of these clinical features are not applicable to most ocular injuries. Therefore, it remains difficult to define precisely which ocular wounds are tetanus-prone. If these criteria are used for a strict interpretation of the present guidelines, then a corneal epithelial abrasion would be considered a “tetanus-prone wound,” because it is an “abrasion.” For example, a cornea1 abrasion would require prophylaxis against tetanus if the patient has had fewer than 3 doses of tetanus toxoid, if it has been more than 5 years since the patient’s last dose, or if the patient’s active immunization status is unknown (15-17). However, cornea1 epithelial abrasions are distinctly different from skin epithelial abrasions and should not qualify as tetanus-prone wounds based solely on the present guidelines. The cornea1 epithelium does not have an underlying blood supply. It receives its nutrients from the aqueous humor inside the eye. The depth of an uncomplicated cornea1 abrasion rarely exceeds 5 cell layers in thickness, and may show substantial healing within 6 hours of injury,
and
J. Vernon Odom,
Marian S. Macsai
Non-Tetanus-
Non-Tetanus Prone Wounds Surface epitheliallabrasion neal scleral penetration Absent Absent Absent
or cor-
with less risk of infection than a comparable skin abrasion. Based on the results of this study and the present fund of knowledge within the literature, we have established a specific wound classification for ocular injuries (Table 4). Any disruption of the cornea1 or conjunctival epithelial surface would be considered an ocular wound. An ocular wound would be classified as “tetanus-prone” in the presence of a cornea1 or scleral perforation, signs of endophthalmitis, devitalized tissue, or contaminants such as dirt, feces, soil, or saliva. Cornea1 or conjunctival epithelial abrasions and penetrating ocular injuries would be classified as “non-tetanus-prone” if signs of perforation, endophthalmitis, devitalized tissue, or wound contaminants were absent. The administration of active and passive tetanus immunization for ocular injuries should follow the current schedule established by the U.S. Public Health Service Advisory Committee on Immunization Practices (USACIP) and the American College of Surgeons Committee on Trauma (15-17,32). The results of this study support the administration of tetanus prophylaxis following perforating eye injuries. However, our results do not support the routine use of tetanus prophylaxis following uncomplicated cornea1 abrasions. Our mouse model indicates that the active immunization status of the patient and type of eye injury are significant factors to be considered with regard to tetanus prophylaxis. An awareness of these risk factors will aid in the appropriate administration of tetanus prophylaxis, and may further reduce unnecessarily frequent tetanus toxoid boosters and health care costs associated with the treatment of cornea1 abrasions. Further clarification of the clinical features of “tetanus-prone” ocular wounds would reduce the existing confusion regarding interpretation of the present guidelines for tetanus prophylaxis.
in part by a grant from Research to Prevent Blindness, Inc.
Acknowledgment-Supported
Tetanus Prophylaxis
68%
REFERENCES 1. Paton D, Goldberg MF. Management of ocular injuries. Philadelphia: WB Saunders; 1976;213. 2. Deutsch TA, Eye and orbit trauma. In: Callaham ML, ed. Current therapy in emergency medicine. Philadelphia: BC Decker; 1987;103-5. 3. Shingleton BJ, Frederick AR Jr, Hutchinson BT. Ocular emergencies. In: Wilkins EW Jr., ed. Emergency medicine. Baltimore: Williams and Wilkins; 1989;876-81. 4. Bouzarth WF, Goldman HW. Trauma to the head. In: Schwartz GR, Safar P, Stone JH, et al, eds. Principles and practice of emergency medicine. Philadelphia: WB Saunders, 1986;1342-3. 5. Cogan DG. Tetanus and the prophylactic use of antitoxin following injuries of the eye. Am J Ophthalmol. 1939;22:1365-7. 6. Sattler R. Penetrating injury limited to the eyeball followed by acute tetanus. Arch Ophthalmol. 1918;47:64-7. 7. Addario la Ferla G. Two cases of tetanus from traumatism of the cornea, one ending fatally. Am J Ophthalmol. 1932; 15:.577. 8. Jayme-Goyaz GG. Cephalic tetanus following injury to the eyeball. Am J Ophthalmol. 1941;24:1281-90. 3. Wetzel JO. Tetanus following eye injury. Am J Ophthalmol. 1942;25:933-44. IO. Wall W. Acute tetanus following a perforating injury of the eye. Eye Ear Nose Throat Monthly. 1948;27:179-82. Il. Tsutsui J. Tetanus infection of cornea. Its treatment with achromycin. Am J Ophthalmol. 1957;43:772-4. 12. Walsh TJ. Clostridial ocular infections. Br J Ophthalmol. 1965;49:472-7.
13. Duke-Elder S, ed. System of ophthalmology, Vol. XIV. Pt. 1: Mechanical iniuries. St. Louis. MO: C.V. Mosbv Co.: 1972:407-410. 14. Czukrasz “I. Unilateral cephalic tetanus from .a conjunctival wound. Klin Mb1 Augenheiik. 1963;143:375-9. 15. American College of Surgeons Committee on Trauma. Prophylaxis against tetanus in wound management. Chicago: American College of Surgeons; 1987. 16. Tetanus-United States, 19821984. MMWR. 1985;34:601-11. 17. American College of Surgeons Committee on Trauma. A guide to prophylaxis against tetanus in wound management. Chicago: American College of Surgeons; 1987.
18. Dowel1 VR Jr, Lombard GL, Thompson FS, Armfield AY. Media for isolation, characterization and identification of obligately anaerobic bacteria. CDC Laboratory Mmual. Atlanta: U.S. Department of Health, Education, and Welfare, Public Health Service, Centers for Disease Control; 1977:44. 19. Dezfulian M. Animal models of botulism and tetanus. In: Botulinum neurotoxin and tetanus toxin. San Diego: Academic Press; 1989;344-6. 20. Pillemer L, Wartma WB. The clinical behavior, incubation period, and pathology of tetanus induced in white Swiss mice by injection of crystalline tetanal toxin. J Immunol. 1947;55: 277-81. 21. Bruning JL, Kintz BL. Computational handbook of statistics. 2nd ed. Glenview, IL: Scott, Foresman; 1977:222-4. 22. Blaisdell EA. Statistics in practice, Fort Worth, Texas: Saunders College Publishing. 1993:355-7. 23. Wright GP. The neurotoxins of CIO$~~~~~M~~ bn!uidn??m and Clostridium tefani. Pharmacol Rev. 1955;7:435-56. 24. Jones DB, Robinson NM. Anaerobic ocular infections. Tram Am Acad Ophthalmol Otolaryngol. 1977;83:309-31. 25. Perry LD, Brinser JH, Kolodner H. Anaerobic cornea1 ulcers. Ophthalmology. 1982;89:636-42. 26. McNatt J, Alien SD, Wilson LA, Dowel1 VR Jr. Anaerobic flora of the normal human conjunctival. sac. Arch Ophthaimol. 1979;11:389-93. 27. Brook I, Pettit TH, Martin WJ, Finegold SM. Anaerobic and aerobic bacteriology of acute conjunctivitis, Ann ~p~~haIrno~. !979;11:389-93. 28. Majekodunmi S, Odugbemi T. Ciostridiurrz wekhii cornea1 ulcer: a case report. Can J Ophthalmol. 1975;!0:290-2. 29. Stern GA, Hodes BL, Stock EL. C~O$~~~~~~~~~ perfingens COTneal ulcer. Arch Ophthalmol. 1979;97:661-3. 30. Znsser H. Microbiology. San M&o, CA: Appleton & Lange; 1988;545-53. 31. Weinstein L, Current concepts: tetanus N Engi J Med. 1973; 289:1293-6. 32. Fraser DW. Preventing tetanus in patients with wounds. Ann Intern Med. 1976;84:9§-7.
MeUmne,
Voi 11, pp 677-683.
?rlnted
1993
I-ANUS PROPHYLAXIS
in the USA
CopyrIght
0 ‘993 Per;arx)n
Ore% L!ci
FOLLOW
enson, MD,* Irvin S. Snyder, PhD,j- Valerie and Marian S. Macsai, *Department of Ophthalmology, Medical College Ophthalmology, Microbiology, and Immunology, Reprint Address: William H. Benson, MD, Department
of Virginia, Virginia Commonwealth University, Alchmond, Virginia. Kkpxtmenis o; School of Medicine, West Virginia University Health Sciences Center, kkxgantown of Ophthalmology, Medical College of Virginia, Box 262, Richmond, VA 232904262
been reported in the literature, and none include simple cornea1 abrasion. The 5 case reports lacked details regarding the history and examination so that occult perforation of the cornea, sclera, or skin could not be ruled out (1,9,13,lis). clinical tetanus following an abrasion has not been reported in the li~erat~Ke, and the risk of developing tetanus ~o~~o~~~~~a cornea1 abrasion has not been evaluated by any Kiowa clinical or laboratory investigations B To evaluate whether the present gui prophylaxis against tetanus were appro 17), we developed an animal modei for scalar inoculation of C tetffni in the m signed to determine if tetanus could inoculation of live C tetani 0 toxin through 3 different ro epithelial debridement (abrasio~)~ c~~~~~~ stroma scarification (pe~etratio~)~ and intraca tion (perforation).
0 Abstract-The admIn~st~atioo of prophylaxis against tetanus following a cornea! abrasion is routinely performed in many acute care facilities, despite a lack of support in e literature for its necessity. The risk of developing clinical tetanus from three different types of injuries to the eye was evaluated in an animal model. Clinical tetanus was induced in unimmunized mice by injecting Clostridium tetani organlsms or toxin into the anterior chamber. Immunized mice injected intracamerally did not develop signs of tetanus. Tetanus was not induced by topical inoculation of either live organisms or toxin following cornea1 epithelial t OKsltromal scarification of unimmunized and mice. The results of this study support the administration of prophylaxis against tetanus following perforating ocular injuries. However, our results do not support its routine use following uncomplicated cornea1 abrasions or other types of nonperforating ocular injuries. 3 Keywords - animal model; clostridium tetani; cornea1 ebridement; intracameral; scarification; tetanus
abrasion;
ministration of prophylaxis against tetanus following a cornea1 abrasion is routinely performed in many acute care facilities, despite a lack of support for routine immunization in the literature (1,5). A search of the medical literature from 1847 to 1993 revealed that only 38 cases of clinical tetanus following ocular injury have been reported, of which at least 33 involved perforation through either the cornea or the sclera (5-14). Only 5 cases of clinical tetanus following nonperforating ocular injuries have
~rg~~is~s and Cuiture C tetani A6455 was obtaiae
chamber on prereduced sheep Moo Gram stained to establish purity. plates were prereduced in an a~~e~~~i~
Presented at the Annual Meeting of the Association for Research in Vision and Ophthalmology, Sarasota, May, 1991. RECEIVED: ACCEPTED
1
: 1
1992; FINAL SUBMISSION RECEIVED: y
9 April
1993 677
1993;
678
William H. Benson,
Irvin S. Snyder,
meat broth medium at 35 OC for 24 hours in an anaerobic chamber. The medium was prepared using Bacto-Cooked Meat Medium supplemented to meet the formulation described by Dowell, Lombard, Thompson, and Armfield (18). Agar was not added in order to prepare a broth.
Preparation
of Organisms
Twenty-four hour cultures of C tetani in chopped meat were gently shaken. The meat particles were allowed to settle and the supernatant containing organisms and toxin was removed by decanting. The organisms were sedimented by centrifugation at 12,000 x g for 10 minutes. The supernatant containing toxin was removed and kept cold in ice until needed. The organisms were washed 3 times with a phosphate-gelatin buffer (4 g NaH,PO,-2g gelatin/L, pH 6.2) and resuspended in approximately the original volume. The OD,, was recorded and the number of organisms determined by plating dilutions of the suspension on prereduced sheep blood agar. The cultures were incubated in an anaerobic chamber at 35 OC for 48 hours and the colonies were counted.
Valerie Granus,
J. Vernon Odom, Marian S. Macsai
Toxicity Testing To determine the infectious dose of C tetani for mice, organisms were resuspended in an equal volume of 10% CaCl,. Two-tenths mL were inoculated intramuscularly (IM) into a hind leg of immunized and unimmunized mice (19). The animals were observed for 14 days for signs of clinical tetanus and death cw *
To assess the toxin preparation, 0.2 mL was inoculated IP into immunized and unimmunized mice. The animals were observed for signs of clinical tetanus and death over a 1Cday period. Control mice received the supernatant from uninoculated chopped meat medium.
Ocular Surgical Procedures
Twenty-gram Swiss-Webster mice obtained from Hilltop Lab Animals (Scottdale, PA) were used for the study. The mice were allowed to rest for 24 hours before use and were given food and water ad libitum.
Ocular inoculation with C tetani culture and toxin was performed on unimmunized and immunized mice using each of the debridement, scarification, and intracameral routes. All mice undergoing ocular inoculation were anesthetized for the procedure using a 3 : 1 mixture of ether and chloroform in a closed chamber. Proparacaine was applied topically for local anesthesia. Cornea1 epithelial debridement was performed using a #15 Bard Parker surgical blade (Becton Dickson Co., Lincoln Park, NJ) under an operating microscope. The eyes were then inoculated topically with either C tetani culture or tetanus toxin. Plastic ocular drapes were used to form a funnel around the debrided and sacrificed eyes to allow contact with undilute C tetani culture and tetanus toxin for 3 minutes per eye. Cornea1 stroma scarification was performed using a sharp surgical blade. Eight incisions per cornea were made by placing 1 set of 4 parallel incisions into the stroma at a 90° angle to another set of 4 parallel incisions (Figure 1). Topical application of C tetani culture or tetanus toxin was then performed as described above. The intracameral injections (0.01 mL) were performed using a Hamilton 702 25 ~1 syringe with a 30-gauge needle introduced into the anterior chamber through the limbus.
Immunization
Bacterial Counts and Histopathology
Equine C tetani antitoxin (Sclavo, Inc., Wayne, NJ) was diluted to 200 units/ml in 0.85% NaCl. Mice were passively immunized by intraperitoneal (IP) inoculation of 0.25 mL of the diluted antitoxin (50 units) 24 hours prior to challenge with either organisms or toxin.
Bacterial counts and histopathology were performed on eyes inoculated by the 3 ocular routes with undilute C tetani culture. To perform the bacterial counts, the inoculated eye was removed, placed in 1 mL of phosphate gelatin buffer, and homogenized in a Potter-Elvehjem tissue grinder. One-tenth mL of
Preparation of Toxin The supernate from a 24-hour culture (described above) was sterilized by filtration through a 0.45 pm pore size filter. No growth was obtained on anaerobic culture of the toxin. Dilutions of toxin were prepared in phosphate-gelatin buffer for inoculation.
Mice
679
Tetanus Prophylaxis
tion that develope and injection of organisms the proportion of those dev
analysis of the sample size ne groups to accurate1 tion expected to de (that is, 1.0 in th munized mice). [z/error]*) (22). If a probabilit z = 1.96. Error refers to the ated in one’s estimate. ~xarn~~a~~o~ indicates that as P approaches size required becomes infinitely er, we assumed that P = that a maximum error of 0.11 yielding a minimum sample siz
rn error tolerof the equation
Infection and Toxicity Tedrag Figure
f . cause
eye
foliowin
cornea
stromal
scarification.
serial lo-fold dilutions of homogenized tissue was inoculated onto prereduced sheep blood agar and incubated for 72 hours in an anaerobic chamber at 35 oc. To test the survival of the organisms during grinding and inoculation, a suspension of C tetani containing 3.8 x IO* organisms/ml was carried through the procedure described above. The number recovered O8 organisms/ml, indicating that the procedure did not affect recovery of organisms. For histopathologic study, the inoculated eyes and fixed in 10% formalin. The eyes were embedded in paraffin, sectioned, and stained with periodic acid schiff and hematoxylin and eosin.
Tests ~f~ro~~~tion~ and Determination Sizes- Fewer Analyses
of Sample
We determined that we would compare the proportion of groups receiving ocular surgery that developed tetanus with the proportion of the appropriate control group that developed tetanus. That is, proportions of mice receiving toxin by debridement, scarification, or intracameral injection that developed tetanus would be compared with the proportion of mice receiving II? injections of toxin that developed tetanus (26127 or 96Vo), and with the propor-
ani IM. Inoc~lat~~~ that the cultu
of doubling
supernate contain ~~~~ized mice
were not affected by the toxin.
Clinical lGu&ngs One week following inoculae of d-e. 6 ~~~i~rn~nized mice injected intracame ly with ~~~~l~t~ C tetani manifested signs of clin tetanus r~s~~ti~~ in death. Of the I5 ~nimmuniz ce used for ocular pathology and bacterial counts that received intracamera1 inoculation of undilute C kelei culture, 4 manifested signs of clinical tetanus wit prior to sacrificing on days 4, groups, 6 of the 21 ~nimmu~iz camerally with undilute C te signs of clinical tetanus or de 0.01) (Table 1). Four of the 6 received i~tracameral inoculation of LBE toxin exhibited signs of ical te~a~~~ resulting in death (z = 2.28, P < 0. TRe signs of tetanus exhibited were typical of se ~~od~~e~ in ~r~~~~s mouse models (20-23). They include paralysis of the legs res stable waddling gait, and trismus. Several mice were
William H. Benson,
680
Irvin S. Snyder, Valerie Granus,
Table 1. Clinical Tetanus or Death among Unimmunized Debrided Eyes
Treatment
Scarified Eyes
*** C tetani organisms
lntracameral Injection 6/21 (29%)
***
013
Intramuscular Injection
**
016
et*
C tetani toxin
Marian S. Macsai
Mice (n = 45)
***
016
J. Vernon Odom,
16/l 8 (89%) lntraoeritoneal Ihjection
*
013
416 (67%) 26/27 (96%)
*P < 0.05;~
= 2.19.
*'P
< 0.0002;~
= 3.78.
immobilized while feeding after their jaws became irreversibly clenched to the bar of the cage. None of the unimmunized mice inoculated with undilute C tetani culture or toxin following scarification or debridement demonstrated signs of clinical tetanus or death (Table 1). None of the immunized mice inoculated with undilute C tetani culture or toxin by either intracameral, scarification, or debridement routes exhibited signs of clinical tetanus or death (Table 2). None of the mice in any control group that received ocular inoculation of sterile phosphate-gelatin buffer showed signs of clinical tetanus or death.
***P
5 0.000008:~
=
1 4.0.
due to toxin released from the organisms in vitro during the experimental procedure. Cultures of C tetani were counted at time 0 and after a 2-hour period. The numbers of viable C tetani organisms dropped from 3.8 x 10’ organisms/ml to 3.2 x 10’ organisms/ml, indicating that the organisms survived the length of the experimental procedure. After 2 hours at room temperature (RT), the culture was diluted to approximate the dose injected intracamerally. The culture was filtered and the supernate injected IP into mice to test for released toxin. Clinical tetanus followed inoculation of bacteria-free supernate from cultures kept at room temperature but not from those kept at 4 OC. Cultures of supernate were sterile.
Bacterial Analysis Histopathologic Attempts to isolate C tetani from mice eyes on days 1, 2, and 4 were unsuccessful. In order to determine the survival of C tetani in the mouse eye, the following experiment was performed. Undilute culture (1.6 x lo6 organisms/ml) of C tetani kept at 4 OC was injected intracamerally. The eyes were enucleated for grinding and culture at 0 and 2, 4, 8, 12, and 18 hours following inoculation. At time zero, 5 x lo3 organisms were recovered from the cultured tissue. Organisms decreasing numbers were cultured through 12 hours, and by 18 hours no growth was observed from cultured eyes. We tested the possibility that clinical tetanus was
Organisms were not observed on Gram or BrownHopps stains. Histopathologic examination showed a transient, severe, acute inflammatory reaction in the anterior chamber of unimmunized mice injected intracamerally. The inflammatory response was less severe in immunized mice following intracameral inoculation. By day 3 there was no inflammatory reaction in immunized mice receiving the same injection. The acute infiltration persisted for more than one week in immunized mice and was placed by granulation tissue, as evidenced by a significant proliferation of fibroblasts and endothelial cells.
Table 2. Clinical Tetanus or Death among Immunized Debrided Eyes
Treatment
Findings
Scarified Eyes
Mice (n = 42) lntracameral Injection
Intramuscular Injection
* C terani organisms C tetani
toxin
016
016
o/21
2/9 (22%)
013
013
013
lntraperitoneal Injection o/4
"P < 0.05;~
=
2.18.
Tetanus Prophylaxis
environment Reports of clinical tetanus following ocular injuries are extremely rare. Prior to 1941, almost all cases reported in the medical literature were perforating eye injuries associated with objects in direct contact with horses and the ground, with a mortality rate of 75%. The administration of prophylactic antitoxin was identified for only one of those cases. A review of those cases by Cogan in 1939 (5) and Wetzel in 1942 (9) indicated that prophylactic antitoxin was given in high doses following the onset of clinical tetanus in most of the remaining cases, including many with a fatal outcome. Since 1941, only 3 cases have been reported, with all patients experiencing complete recovery. Only a single case of clinical tetanus following a cornea1 foreign body injury has been reported since 193 I. Despite this rare occurrence, it remains common practice in many acute care facilities to administer prophylaxis against tetanus following routine cornea1 abrasions. We designed an experimental model in the mouse y inoculating C tetani organisms and toxin following 3 types of ocular injuries. The mouse was an appropriate model to evaluate human response to C tetani because, according to known information from previous studies, both mice and humans are among the species most susceptible to toxin (19-20). The various parameters regarding type of injury, type of insculum, and prior immune status were evaluated. Clinical tetanus developed in unimmunized mice following injection of C tetani organisms or toxin into the anterior chamber. Tetanus was not established among any immunized mice, or with topical administration of inoculum following corneal epithelial debridemenl or stromal scarification. We
le 3. Immunization Schedule for Prepared by The Committee
microbiology studies of normal and infects bacterial flora (5-10,13, 24-29). For unimmunized mice inject signs of tetanus preceded death by in 24 hours for those ~~~~~lated I with C tetani organisms. The inc time of death were dependent on ganisms and toxin injected. The la the inoculum, the shorter the ins few as 5 x lo3 organisms were duced enough toxin after intrac produce signs of clinical tetanus son clinical tetanus developed in munized mice injected intracam C tetani organisms is not know the mice with regard to indivi sponses to C tetani organisms or the rratural resistance of the species to C tetaazi may results. These results are similar to t in previous mause studies that use strains of C tetani (19,23). The difficulty with isolation of C te~~.~iorganisms from wounds has been expressed other investigators (8,30-31). Clinical rep5rts animal studies have shown that tetanus is more ely to occur in cases of ~ano~hthalmitis caused by a ~~~5~~~~~ purulent bacterial ~~~taminat~~n and that pure ~~lt~r~s of G tetani inoculated in rabbit eyes cause only a mild iridocyclitis (5,9,12). It has been suggeste tissue necrosis from a purulent ~a~~~bK~~a~rn~~~s may aid in reducing the oxygen co~~~~tra~~~~ in the eye
Tetanus Prophylaxis on Trauma, ~rne~i~a~ Tetanus-Prone Wounds
History Toxoid
of Adsorbed (Doses)
Unknown or fewer 3 or moret
and t
i
Non-Tetanus-Prone Wounds
Tetanus
than
3
Td*
TIG
Td’
TIG
Yes NO-4
Yes
Yes NQ§
No
NO
NO
“For children younger than 7 years of age: DTP (DT, if pertussis vaccine is contraindicated) is preferable to tetanus toxoid alone. For persons 7 years old and older, Td is preferable to tetanus toxoid alone. tlf only 3 doses of fluid toxoid have been received, a 4th dose of toxoid, ~re~er~bl~ an adsorbed toxoid, should be given. $Yes, if more than five years since last dose. (More frequent boosters are not needed and can accentuate side effects.) $Yes, if more than 10 years since last dose. Td = te?anus and diptheria toxoids adsorbed (for adult use). TIG = tetanus immuneglobulin (human).
682
William H. Benson, Table
Clinical
4. Ocular Prone
Wound Wounds
Classification
Irvin S. Snyder, Valerie Granus, for Tetanus-Prone
Tetanus-Prone Wounds
Features
Depth Signs of infection Devitalized tissue Contaminants (dirt,
sufficiently
to allow growth
feces,
soil, saliva)
Corneakcleral perforation Present Present Present
of C tetani organisms
(12).
Specific recommendations for tetanus prophylaxis following ocular injuries are limited. The immunization advisory committee that determines the criteria for prophylaxis against tetanus in wound management does not recommend specific guidelines for the various types of ocular injuries. The decision of when to administer tetanus prophylaxis is subject to interpretation of the present guidelines, which separate all wounds into two categories: “tetanus-prone wounds” and “non-tetanus-prone wounds” (Table 3). The risk factors for a tetanus-prone wound include any one of the following clinical features: Wound age >6 hours, a configuration of stellate shape, avulsion or abrasion, a depth > 1 cm, mechanics of injury due to a missile, crush, burn, or frostbite, or the presence of any signs of infection, devitalized tissue, contaminants (dirt, feces, soil, saliva, and so on), or denervated or ischemic tissue. Current recommendations for tetanus prophylaxis involve a wound with any one of these clinical features. Many of these clinical features are not applicable to most ocular injuries. Therefore, it remains difficult to define precisely which ocular wounds are tetanus-prone. If these criteria are used for a strict interpretation of the present guidelines, then a corneal epithelial abrasion would be considered a “tetanus-prone wound,” because it is an “abrasion.” For example, a cornea1 abrasion would require prophylaxis against tetanus if the patient has had fewer than 3 doses of tetanus toxoid, if it has been more than 5 years since the patient’s last dose, or if the patient’s active immunization status is unknown (15-17). However, cornea1 epithelial abrasions are distinctly different from skin epithelial abrasions and should not qualify as tetanus-prone wounds based solely on the present guidelines. The cornea1 epithelium does not have an underlying blood supply. It receives its nutrients from the aqueous humor inside the eye. The depth of an uncomplicated cornea1 abrasion rarely exceeds 5 cell layers in thickness, and may show substantial healing within 6 hours of injury,
and
J. Vernon Odom,
Marian S. Macsai
Non-Tetanus-
Non-Tetanus Prone Wounds Surface epitheliallabrasion neal scleral penetration Absent Absent Absent
or cor-
with less risk of infection than a comparable skin abrasion. Based on the results of this study and the present fund of knowledge within the literature, we have established a specific wound classification for ocular injuries (Table 4). Any disruption of the cornea1 or conjunctival epithelial surface would be considered an ocular wound. An ocular wound would be classified as “tetanus-prone” in the presence of a cornea1 or scleral perforation, signs of endophthalmitis, devitalized tissue, or contaminants such as dirt, feces, soil, or saliva. Cornea1 or conjunctival epithelial abrasions and penetrating ocular injuries would be classified as “non-tetanus-prone” if signs of perforation, endophthalmitis, devitalized tissue, or wound contaminants were absent. The administration of active and passive tetanus immunization for ocular injuries should follow the current schedule established by the U.S. Public Health Service Advisory Committee on Immunization Practices (USACIP) and the American College of Surgeons Committee on Trauma (15-17,32). The results of this study support the administration of tetanus prophylaxis following perforating eye injuries. However, our results do not support the routine use of tetanus prophylaxis following uncomplicated cornea1 abrasions. Our mouse model indicates that the active immunization status of the patient and type of eye injury are significant factors to be considered with regard to tetanus prophylaxis. An awareness of these risk factors will aid in the appropriate administration of tetanus prophylaxis, and may further reduce unnecessarily frequent tetanus toxoid boosters and health care costs associated with the treatment of cornea1 abrasions. Further clarification of the clinical features of “tetanus-prone” ocular wounds would reduce the existing confusion regarding interpretation of the present guidelines for tetanus prophylaxis.
in part by a grant from Research to Prevent Blindness, Inc.
Acknowledgment-Supported
Tetanus Prophylaxis
68%
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13. Duke-Elder S, ed. System of ophthalmology, Vol. XIV. Pt. 1: Mechanical iniuries. St. Louis. MO: C.V. Mosbv Co.: 1972:407-410. 14. Czukrasz “I. Unilateral cephalic tetanus from .a conjunctival wound. Klin Mb1 Augenheiik. 1963;143:375-9. 15. American College of Surgeons Committee on Trauma. Prophylaxis against tetanus in wound management. Chicago: American College of Surgeons; 1987. 16. Tetanus-United States, 19821984. MMWR. 1985;34:601-11. 17. American College of Surgeons Committee on Trauma. A guide to prophylaxis against tetanus in wound management. Chicago: American College of Surgeons; 1987.
18. Dowel1 VR Jr, Lombard GL, Thompson FS, Armfield AY. Media for isolation, characterization and identification of obligately anaerobic bacteria. CDC Laboratory Mmual. Atlanta: U.S. Department of Health, Education, and Welfare, Public Health Service, Centers for Disease Control; 1977:44. 19. Dezfulian M. Animal models of botulism and tetanus. In: Botulinum neurotoxin and tetanus toxin. San Diego: Academic Press; 1989;344-6. 20. Pillemer L, Wartma WB. The clinical behavior, incubation period, and pathology of tetanus induced in white Swiss mice by injection of crystalline tetanal toxin. J Immunol. 1947;55: 277-81. 21. Bruning JL, Kintz BL. Computational handbook of statistics. 2nd ed. Glenview, IL: Scott, Foresman; 1977:222-4. 22. Blaisdell EA. Statistics in practice, Fort Worth, Texas: Saunders College Publishing. 1993:355-7. 23. Wright GP. The neurotoxins of CIO$~~~~~M~~ bn!uidn??m and Clostridium tefani. Pharmacol Rev. 1955;7:435-56. 24. Jones DB, Robinson NM. Anaerobic ocular infections. Tram Am Acad Ophthalmol Otolaryngol. 1977;83:309-31. 25. Perry LD, Brinser JH, Kolodner H. Anaerobic cornea1 ulcers. Ophthalmology. 1982;89:636-42. 26. McNatt J, Alien SD, Wilson LA, Dowel1 VR Jr. Anaerobic flora of the normal human conjunctival. sac. Arch Ophthaimol. 1979;11:389-93. 27. Brook I, Pettit TH, Martin WJ, Finegold SM. Anaerobic and aerobic bacteriology of acute conjunctivitis, Ann ~p~~haIrno~. !979;11:389-93. 28. Majekodunmi S, Odugbemi T. Ciostridiurrz wekhii cornea1 ulcer: a case report. Can J Ophthalmol. 1975;!0:290-2. 29. Stern GA, Hodes BL, Stock EL. C~O$~~~~~~~~~ perfingens COTneal ulcer. Arch Ophthalmol. 1979;97:661-3. 30. Znsser H. Microbiology. San M&o, CA: Appleton & Lange; 1988;545-53. 31. Weinstein L, Current concepts: tetanus N Engi J Med. 1973; 289:1293-6. 32. Fraser DW. Preventing tetanus in patients with wounds. Ann Intern Med. 1976;84:9§-7.