Serum tetanus antitoxin levels were determined in 104 World War IIveterans immunized 15 or more years ago while in active militaryservice. The majority had significant antibody. A standard boosterraised the antibody level significantly after seven days. It is feltthat except for grossly contaminated wounds, such a boosterprovides ample protection even though the initialimmunization was done 15 years earlier.

ANTIBODY RESPONSE TO TETANUS TOXOID 15 YEARS

AFTER INITIAL IMMUNIZATION

James R. McCarroll, M.D., F.A.P.H.A.; Irving Abrahams, Ph.D.; and Paul A. Skudder, M.D.

NO PREVENTIVE measure the physiciancan offer his patient promises surer

protection than active immunizationwith tetanus toxoid. Nevertheless, thefailure of our preventive programs toreach many groups of our citizens forcesthe daily use of large amounts of tetanusantitoxin in the treatment of traumaticinjuries. The considerable morbidityassociated with this use of antiserum1results in the paradox of more illnessdue to the prevention of a disease thanwould result from the disease itself.At the present time only two large

groups may be presumed with anydegree of accuracy to have been im-munized against tetanus. These are vet-erans of service in the American armedforces since 1942, and most Americanchildren under the age of 18. Even forthis latter group the presumption cannotbe made with any degree of certaintysince the almost universal use of diph-theria-pertussis-tetanus immunization inchildhood is only now becoming a re-ality. Immunization of armed forcespersonnel, however, has been a uni-v-ersal requirement of military servicesince 1942, so that it has become routine

in most hospital emergency units to ac-cept a history of military service asevidence of previous active immuniza-tion against tetanus. This is of consider-able practical importance since most ofthe 22 million veterans are members ofthe active working population runningthe risk of repeated injuries for whichprophylaxis against tetanus must be of-fered. Until recently, therefore, it hasbeen customary to rely on a singlebooster dose of tetanus toxoid as pro-phylaxis against tetanus in treatingthese injuries. This procedure has of-fered the multiple advantages of certainprotection against the disease, elimina-tion of the considerable hazards andmorbidity associated with antitoxin, andof bolstering the immune status of theindividual.As the length of time since World

War II and discharge from active mili-tary service increases for most veterans,the question of duration of protectionremaining from the initial immunizationbecomes more important. Persistence ofsolid immunity in most persons togetherwith a rapid anamnestic response to abooster dose of toxoid for the remainder

OCTOBER, 19962 1 669

has been well demonstrated for sizablegroups of persons up to ten years afterinitial immunization.2 A few studieshave included scattered cases of personsfollowed for longer periods,3'4 but nosizable group has been carefully studiedfor more than ten years from the timeof initial immunization. To determinethe present immunity status of WorldWar II veterans now 15 or more yearsafter their initial tetanus immunization,and to determine whether a booster doseof tetanus toxoid provides sufficientlyrapid antibody response to provide pro-tection at this time, the present studywas carried out.

Materials and Methods

Population Studied

The study population was composedentirely of veterans of World War IIadmitted as patients to the medical andsurgical services of the New York Vet-erans Administration Hospital. EveryWorld War II veteran admitted to theseservices during a three-month periodwas carefully interviewed to determinehis suitability for inclusion in this study.Only two criteria were used to deter-mine this suitability. First, activeservice in any one of the branches ofthe armed forces during World War IIwas considered evidence of active im-munization against tetanus. Initially itwas planned to use the records of theArmed Forces Records Bureau to deter-mine the exact dates of basic tetanusimmunizations and subsequent boosterdoses for each individual. These rec-ords, however, were not sufficiently com-plete for this purpose. However, allveterans included in this study spentsome period of time on active duty be-tween 1942 and 1946, during whichtime active immunization against teta-nus with one form or another of tetanustoxoid was a universal requirement ofall components of the American armedforces.

The second criterion for inclusion inthe study was absence of any evidenceof either active or passive tetanus immu-nization since discharge from militaryservice. A careful medical history wastaken from each patient and all personswere eliminated who gave any historyof parenteral injection unless the agentadministered was definitely known. Fur-thermore, all patients were eliminatedwho gave a history of any type of injury,burn, or bite for which medical treat-ment was given which might have in-cluded either active or passive tetanusimmunization. To aid in the recollectionof injuries and illnesses which mighthave occurred up to 15 years previously,a second identical interview wasconducted after three or four days.Through this double screening processa study group was derived which in ourjudgment included only individuals whohad received a full immunizing courseof tetanus toxoid at least 15 years pre-viously and had not received anybooster injections after discharge fromservice. Approximately 300 patientswere interviewed to obtain the studysample of 104 persons. No other cri-teria were used for their selection. Pa-tients with all the diagnoses commonlyencountered on general medical andsurgical services were included.The resulting group approximates the

general composition of the World WarII veteran population of New York City.Their ages varied from 32 to 64 years,with an average age of 46.1 years.Four of the group were females, 17were Negroes, and 2 were Orientals.Veterans of service in all branches ofthe armed forces were included and nodistinction among the various compo-nents was made, although basic immu-nizing schedules and agents variedsomewhat among them. If the patientwas deemed suitable for inclusion inthe study, a base line serum was drawnand a standard booster dose of 0.5-mlfluid tetanus toxoid was administered

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ANTIBODY RESPONSE TO TETANUS TOXOID

intramuscularly. Repeat bleedings wereobtained at 4, 7, and 14 days.

Laboratory DeterminationsTitrations for serum antitoxin levels

were made in 20-gram Swiss Webstermale mice from the Camm ResearchInstitute. Tetanus toxin and standardNIH antitoxin were prepared and gen-erously supplied to us by Lt. Colonel A.S. Benenson of the Walter Reed ArmyInstitute of Research. The test methodswere modified slightly from those usedin Benenson's laboratory. Tests for anti-toxin levels were made from 0.0025units per ml to 30 units per ml at two-fold or half-log intervals. Two levels oftoxin test doses were used: 5L+/ ,ooodoses per ml or 5L+/ioo doses per ml.Titrations of standard NIH antitoxinwere made with each test as a controlon the toxin dilution preparation andon other factors of the test. The mix-tures of serum and toxin were kept atroom temperature for one hour, and0.6-ml amounts were injected subcu-taneously at the base of the tail. Theend point was taken as that mixture ofserum and toxin which resulted in sur-vival with symptoms of tetanus at theend of five days after injection. If noend point reactions were observed, the

titer was estimated between the highestdilution of serum permitting survivaland the lowest dilution resulting indeath.

ResultsBase Line Antibody Levels

Of 104 persons from whom base lineserum was obtained, only 8 showed com-plete absence of tetanus antitoxin at thelowest dilution tested, 0.0025 units perml (Figure 1). Forty-five (43.3 percent) had antibody levels of 0.1 unitsor greater, or well within the commonlyaccepted protective level. The remain-ing 46 patients had antitoxin titers be-tween 0.01 units and 0.055 units per ml.If 0.01 units per ml can be accepted asoffering significant protection againsttetanus, then 88 per cent of this groupstill had significant protection remainingfrom immunizations received duringWorld War II. There was no apparentcorrelation between clinical diagnosisand the base line antibody titer.

Antibody Response Four Days afterBooster Injection

Four days after receiving a standardbooster dose of 0.5-ml fluid tetanustoxoid intramuscularly there was no es-

Units ofAntitoxin/ml.

Serum (0.0025 0.0025 0.005 0.01 0.017 0.03 0.055 0.1 0.17 0.3 0.55 1.0 >I

Number ofSubjects 8 2 3 8 16 4 18 3 23 2 13 1 3

l -IIVl l I

13(12.5%)

46(44.2%)

45(43.3%)

IV

91(87.5%)

Figure 1-Serum Tetanus Antitoxin Titers of 104 World War IT Veterans 15 orMore Years after Initial Immunization

OCTOBER, 1962 1671

SERUM TETANUS ANTITOXIN TITERS4 DAYS AFTER BOOSTER DOSE OF TETANUS TOXOID

(0.01 0.01 0.03 0.1, 0.3, 10aover-__ _L *

2

SERUM TETANUS ANTITOXIN TITERS7 DAYS AFTER BOOSTER DOSE OF TETANUS TOXOID

<0 01 0.01 0.03 0 1 00.3 0, 5.0 10.0 aover

4 3 2 9~~V~~~i17irnF13 2

iu 9 6 2

4 4

SERUM TETANUS ANTITOXIN TITERS14 DAYS AFTER BOOSTER DOSE OF TETANUS TOXOID

<0.01 0.01 °0.03I°31,03I 5-0, 10030083over

= SUB-OPTIMA- PPCTEIVE TIT PS

= NC THADsNE FROM PRE-SCESTER T TER

Figure 2

sential antill(odv chaingye in most subjectstestedl (Figure 2).* Only 9 of the 101perso,is tested at 4 days showed any de-tectable rise in antibodv titer. ancd 7

ulljects, hiadl ani actual drop in titer. The

* For clarity the nulmlber of diluitionis listedin Figure 2 hlas been reduiced, each b)ox in-clidin, both serunm5s whose enid poinit titer

O(C111 redl at that diluition, aind those with endpoint titers at the next higher unlisted dilii-tioii, e._., 0.03 includes both 0.03 anid 0.055.Ibis,,combining occasionally miiasks in thetaille a few of the more mll inlol changes citedif] the text.

c(han-es in antibody titer at 4 davs,wN-hether rise or fall. wvere all of lowv de-a-ree. None of the indlividuals showingra fall at 4 days had an eInd point titermoIre thain 1 (ilutioi less thani his base

line level. The rises at 4 days w-ere alsoof slirht de-ree wvith oinly 2 subjectsshowin- a rise of 2 dilutions (a 3-foldrise). There wvas no apparent relationi-ship between the base line antibody titerand a rise or fall at 4 days. Amongpersons showN'ing- anv change in either

VOL. 52, NO. 10, A.J.P.H.

Pre - BoosterSerum

Antitoxin Titerunits/nm I.

< 0.0 1

0.01

0.03

0.

0.3

1.0+

Pre - BoosterSerum

Antitoxin Titeru n t s/mi.

< 0 0 1

0 0 1

0. 03

0.

0. 3

.0+

Pre-BoosterS e r u m

Antitoxin Titerunits/ml.

(0.01

0.01

0.03

0.1

0.3

1 .0+

T o t aS ub je cts

3

23

20

25

6

4

10

T o t aISu bj ec t s

2

8

6

2

4

8 1

T o t a

Subj ects

9

2

3

3

10

2

59

1 672

2

ANTIBODY RESPONSE TO TETANUS TOXOID

direction at 4 days were subjects withall levels of base line antibody titer,from extremely low to very high.

Antibody Response Seven Days afterBooster Injection

After 7 days, 87 per cent (70 of 81)of the subjects tested showed a signifi-cant rise in antibody titer. Only 3 per-sons showed a fall of one dilution inantibody titer at this point. Againthere was no correlation between theinitial antibody level and the responseat 7 days. Of the 8 subjects showingno antibody response at this time, 4 hadno detectable antibody whatever, andthe remaining 4 had base line levels of0.1 units or greater. The majority ofpatients (85 per cent), however, hadserum antitoxin titers of 0.1 units perml, or greater, by 7 days, placing themdefinitely in the "protected range."

Antibody Response 14 Days afterBooster Injection

Fourteen-day bleedings could be ob-tained from only 59 of the originalgroup, but all of these had antibodytiters of 0.1 units or greater regardlessof the base line antibody level. All ofthese, furthermore, represented a markedincrease in antibody titer with a singleexception. This one patient had a highantibody titer (0.55 units per ml) at thetime of his initial bleeding; this titerpersisted unchanged on subsequent de-terminations, although his hospitalchart records the booster dose as given.

The Correlation of Clinical Diagnosiswith Antibody Response

There was no correlation whateverbetween either rapidity or degree ofantibody response and the clinical diag-nosis recorded for these study patients.

The most frequent clinical diagnosis wascirrhosis, often associated with chronicalcoholism and malnutrition, but thesepatients showed as rapid and high anantibody response as the total group.Many types of malignant disease werealso present in the study group, includ-ing malignancies of the lymphopoieticsystem, but all such patients showedantibody responses equal in degree andrapidity of rise to the total group. Therewas also no discernible correlation be-tween the clinical diagnosis and an ini-tial fall in antibody titer at either 4 or7 days. Sex, race, and age also bore norelation to antibody response.

Discussion

The nearly perfect record of tetanustoxoid in protecting immunized individ-uals against active disease is one of themost remarkable in preventive medicine.In degree of protection afforded andduration of immunity provided, it is farsuperior to any other nonliving immu-nizing agent.

Although fewer than 500 cases of tet-anus are reported each year in theUnited States, the occurrence of evenone case of a completely preventable dis-ease is excessive. Since tetanus, fur-thermore, may develop after the mostminor and inconspicuous of injuries, aphysician treating a patient for any typeof trauma must always consider the pos-sibility of the disease and offer sometype of prophylaxis against it. Althoughadequate wound care, including carefuldebridement, is the most effective meas-ure in preventing tetanus, some patientswill still require passive protection withtetanus antitoxin. Since up to half ofall persons tested may show sensitivityto the equine and bovine preparations,1and since human antitoxin is expensiveand not readily available, the paradoxarises of a preventive measure produc-ing more morbidity than is due to thedisease it prevents. There is obviously

OCTOBER. 1962 1 673

no substitute for active immunizationagainst tetanus and every opportunityto accomplish this should be taken. Im-munization with tetanus toxoid shouldalways be started on unimmunized in-dividuals at the time injuries are treated,regardless of whether or not passiveimmunization with antitoxin is also usedto protect the individual at that time.The persistence of significant amounts

of antibody in these World War II vet-erans at least 15 years after their initialimmunization series confirms the im-pression that artificially produced activeimmunity against tetanus persists forlong periods of time. The prompt risein antibody titer in response to a boosterdose of toxoid indicates reliance for pro-tection may .be placed on a booster doseof tetanus toxoid in the case of personsactively immunized up to 15 years pre-viously. In occasional cases in whichmassive contamination of wounds exist,and in which adequate debridementmay be delayed or impossible, it may bedeemed advisable to add the protectionof antitoxin as well as tetanus toxoid.The excellent antibody response of eventhe most debilitated and cachectic pa-tients, and those with chronic and termi-nal illnesses, indicates these conditionsdo not interfere with adequate antibodyresponse.The practical importance of this in-

formation is obvious. Since more than22 million veterans of military serviceand a considerable portion of the 66million children under the age of 20have received a basic course in tetanusimmunization, the hazards of tetanusantitoxin administration can usually beavoided for most members of thesegroups. It is apparent that a singlebooster dose of tetanus toxoid providessufficient antibody response to protectagainst clinical tetanus (excepting, per-haps, in grossly contaminated wounds)in all individuals immunized up to 15years previously and probably forlonger periods.

Summary

Serum tetanus antitoxin levels weredetermined by a standard mouse protec-tion test on 104 World War II veteranswho had been immunized 15 or moreyears previously during active militaryservice. Careful histories were obtainedto eliminate all subjects from the studygroup who might have received tetanustoxoid since discharge from militaryservice. A standard booster dose of fluidtetanus toxoid was administered andserum drawn at 4, 7, and 14 days fortetanus antibody determinations. Only8 of the 104 subjects tested had no de-tectable antibody 15 or more yearsafter immunization; 45 had at least 0.1units of antitoxin per ml.

Eighty-four per cent of the patientstested 4 days after toxoid injectionshowed no change in titer; the remain-der showed slight increases or decreases.Seven days after booster injection 70 of81 patients (86 per cent) showed a sig-nificant rise in antibody level, and allbut one of these had titers of 0.1 unitsper ml or greater. Fourteen days afterbooster injection 58 of 59 patients testedshowed marked increase in titer overbase line levels. The one patient show-ing no increase in antibody titer had ahigh base line level and it is questionedwhether he actually received his boosterinjection.

There was no correlation of eitherrapidity or degree of antibody responsewith any of the various clinical diag-noses recorded on these patients. Pa-tients with advanced liver disease andmalignancies of the hematopoietic sys-tem showed equally good and rapid re-sponses as all other groups of patients.It is considered that except for grosslycontaminated wounds, a single boosterdose of tetanus toxoid provides ampleprotection against clinical tetanus for allpersons immunized at any time up to15 years previously, and probably forconsiderably longer periods.

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ANTIBODY RESPONSE TO TETANUS TOXOID

ACKNOWLEDGMEN¶I[S - The authors grate-fully acknowledge the generous cooperationof Dr. J. James Smith, chief of the medicalservice, New York Veterans AdministrationHospital, and of all the staff of the medicaland surgical services who assisted in thisproject. Blaine A. Braniff, Robert BruceMcFadden, and Richard M. Sallick of theCornell University Medical College conscien-tiously recorded the time-consuming medicalhistories and obtained all necessary serumspecimens. The technical assistance of Mr.Thomas Gilleran in the laboratory is alsoacknowledged with gratitude.

REFERENCES

1. Skudder, P.; McCarroll, J.; Ecker, R.; and Cahow,E. The Incidence of Reactions Following the Admin-istration of Tetanus Antitoxin. J. Trauma 1:41, 1961.

2. Turner, T.; Stafford, E.; and Goldman, L. Studieson the Duration of Protection Afforded by ActiveImmunization Against Tetanus. Bull. Johns HopkinsHosp. 94:204, 1954.

3. Loonery, J.; Edsall, G.; Ipsen, J.; and Chasen, W.Persistence of Antitoxin Levels after Tetanus ToxoidInoculation in Adults, and Effect of a Booster Doseafter Various Intervals. New England J. Med. 254:6(Jan. 5), 1956.

4. Ruegsegger, J. Further Observations on the Per-manence of Tetanus Antitoxin. A.M.A. Arch. Int.Med. 106:410 (Sept.), 1960.

Dr. McCarroll is director, Division of Epidemiologic Research, and assistantprofessor of public health; Dr. Abrahams is assistant professor of microbiologyand immunology; and Dr. Skudder is surgeon to the Emergency Unit of NewYork Hospital-Cornell Medical Center and clinical assistant professor ofsurgery; Cornell University Medical College, New York, N. Y.

This paper was presented before the Epidemiology Section of the AmericanPublic Health Association at the Eighty-Ninth Annual Meeting in Detroit,Mich., November 14, 1961.

This investigation was supported (in part) by a Public Health Service re-search grant (E3369) from the National Institute of Allergy and InfectiousDisease.

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OCTOBER, 1962 1675