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Neonatal tetanus despite protective serum antitoxin concentration
FEMS Microbiology Immunology 76 (1991) 171-176 © 1991 Federation of European Microbiological Societies 0920-8534/91/$03.50 Published by Elsevier ADONIS 092085349100073Y
171
FEMSIM 00162
Neonatal tetanus despite protective serum antitoxin concentration S.Y. Maselle 1, R. M a t r e 3,4, R. Mbise 2 a n d T. H o f s t a d 4 Department of Microbiology and Immunology, and 2 Department of Paediatrics and Child Health, Muhimbili Medical Centre, Dares Salaam, Tanzania, 3 Broegelmann Research Laboratory for Microbiology, and 4 Department of Microbiology and Immunology, The Gade Institute, University of Bergen, Norway Received 12 February 1991 Accepted 15 February 1991
Key words: Pregnancy; Immunization; Neonatal tetanus
1. SUMMARY
2. I N T R O D U C T I O N
Using the ELISA technique to estimate serum antibodies against tetanus toxin, seven neonates with clinical tetanus were found to have antibody levels 4-13 times higher than the presumed minimum protective level of 0.01 I U / m l . All but one of their mothers had been vaccinated with tetanus toxoid in pregnancy. In two other neonates, whose mothers had received multiple booster doses of toxoid during pregnancy, the anti-toxin concentrations were 100- and 400-times the presumed protective level. Therefore the toxin dose may overwhelm the pre-existing anti-toxin level and produce disease. Furthermore, multiple booster injections of tetanus toxoid may not only enhance serum anti-toxin titres, but could also lead to an ineffective immune response.
Many attempts have been made to correlate the amounts of circulating tetanus antitoxin (anti-TT) with the degree of protection in man. However, since human experiments with tetanus are rare, most of the available information is based on animal data which showed that 0.01 international unit (IU) of a n t i - T T / m l of serum was the threshold of protection [1]. This observation was supported by a human experiment in which 2-3 human minimum lethal doses of tetanus toxin failed to produce symptoms of tetanus with pre-existing 0.005-0.01 IU of a n t i - T T / m l of serum [2]. Therefore, it has generally been accepted that an anti-TT level of 0.01 I U / m l is the minimum protective level in man [3,4]. However, clinical tetanus has been reported in adult patients with anti-Tl" concentrations above the presumed minimum protective level at onset of symptoms [5-7]. In Tanzania where neonatal tetanus is an important public health problem, cases have been reported among neonates whose
Correspondence to: R. Matre, Broegelmann Research Laboratory for Microbiology, Armauer Hansen House, N-5021 Bergen, Norway.
172 mothers were vaccinated with tetanus toxoid (TT) in pregnancy [8,9]. Such reports seemed to suggest that there was probably no universal minimum protective level, and that any serum anti-TT concentration could be overwhelmed given a sufficient quantity of toxin. This paper describes the findings in ten newborn babies with clinical tetanus seen in a referral to hospital in Tanzania.
3. M A T E R I A L S A N D M E T H O D S 3. i. Patients studied Full term normal delivery newborns with clinical tetanus admitted to Muhimbili Consultant Hospital in D a r e s Salaam, Tanzania, were studied. Details of their delivery, onset of symptoms, treatment and history of maternal vaccination with tetanus toxoid (TF) in pregnancy were obtained from their records. Patients who had been given therapeutic anti-tetanus serum were excluded from the study. Blood samples were collected on admission, sera separated and stored at - 2 0 ° C until analysis. 3.2. Enzyme immuno-assay Enzyme-linked immunosorbent assay (ELISA) technique was used to determine the anti-TT concentrations in the sera. This technique has been shown to give results comparable with those obtained by in vivo neutralisation test in mice, the conventional method for anti-TT assay [10]. The procedure was essentially similar to that described by Engvall and Perlman [11]. In brief, flat-bottomed polystyrene microtitration plates (Dynatech, Switzerland) were coated with an optimal dilution (see below) of T T ref no. A S / 2 4 / 8 1 - T containing 480 L f / m l (National Institute of Public Health (NIPH), Oslo, Norway), Coating was done at 37°C overnight in 0.05 M bicarbonate buffer, p H 9.6. The plates were kept at 4°C and subsequently used for up to 4 weeks. After washing the plate, phosphate-buffered saline, p H 7.2, containing 1% bovine serum albumin was added to the wells, incubated at 37°C for 1 h and washed. Diluted serum samples were then added, the plate incubated as above and
washed again. Thereafter, swine anti-human IgG phosphatase conjugate (Orion Diagnostic, Espo, Finland) appropriately diluted (see below) was added and incubated as above. After further washing, the substrate, p-nitrophenyl phosphate (Sigma Chem. Co., U.S.A.) was added and the plate kept in the dark at room temperature for 20 min. The enzyme-substrate reaction was stopped with sodium hydroxide solution and optical densities read at 405 nm in a Titertek plate reader (Flow Labs, U.K.). Optimal dilutions of TT (1:500) for coating and conjugate (1:500) for use had earlier been established by checkerboard titration with a positive anti-tetanus toxoid control serum (NIPH). 3.3. Calculation of anti-TT concentrations The optical densities obtained with 14 duplicates of 2-fold dilutions of a positive control serum containing 8 IU of a n t i - T T / m l ( N I P H ) were used to draw a standard curve against corresponding serum dilutions. Each patient's serum had been assayed at three dilutions (1:200, 1:800 and 1:3200) in duplicate and the optical densities obtained were used to determine the anti-TT levels from the standard curve. The final antitoxin concentration for each serum was obtained by averaging any two closest contiguous estimations. A negative control serum from an unvaccinated person and a positive control were included in all assays. One person performed the assays and there were no changes in reagents' lots or equipment.
4. R E S U L T S 4.1. Patients studied Ten neonates with tetanus fulfilled the study criteria. Five of them had been delivered at home and the other five in hospital or maternity homes. Three of the respective mothers were primiparous and the remaining seven were multiparous. All the babies had clinically infected umbilical cord stumps, except one (case no. 10). One patient had been treated with crystalline penicillin and another, who was delivered at home, had local treatment with a mixture of charcoal ash and kerosine. The rest had not received any form of treatment.
173 Table 1 Incubation periods, maternal vaccination status and serum concentrations of tetanus antitoxin (anti-TT) in 10 neonates with tetanus Case no.
Incubation period in days
Maternal "IT vaccination Previous Last pregnancies pregnancy
Serum anti-T]7 IU/ml
1 2 3 4 5 6 7 8 9 10
10 7 6 2 8 8 16 4 4 14
No (p) No (p) 5 doses 3 doses No (p) 11 doses Yes (ns) 3 doses No No
0.130 0.037 0.078 4.220 0.069 1.035 0.124 0.079 0.104 0.002
2 doses No 1 dose 3 doses 1 dose 3 doses 3 doses I dose 2 doses 1 dose
p, primiparous; ns, not specified.
T a b l e 1 shows the i n c u b a t i o n periods, m a t e r n a l T T v a c c i n a t i o n status a n d s e r u m anti-T]" conc e n t r a t i o n s in the ten patients. T h e i n c u b a t i o n p e r i o d s c a l c u l a t e d f r o m the d a y of delivery up to the onset of s y m p t o m s v a r i e d f r o m 2 - 1 6 d a y s with a m e a n of 7.9 days. N i n e m o t h e r s h a d b e e n v a c c i n a t e d with T T in the last p r e g n a n c y , five of w h o m h a d "IT in p r e v i o u s pregnancies. O n e m o t h e r h a d not received a n y T T vaccination. T h e r e was no r e l a t i o n s h i p b e t w e e n the i n c u b a t i o n p e r i o d a n d m a t e r n a l v a c c i n a t i o n status or a n t i - T T levels.
4.2. A n t i - T T levels in the patients' sera Anti-T]" serum levels in the p a t i e n t s shown in T a b l e 1 were all a b o v e 0.01 I U / m l except in o n e p a t i e n t (no. 10) w h o h a d a titre of 0.002 I U / m l . T h e m o t h e r of this p a t i e n t h a d b e e n given a single d o s e of "IT 2 weeks b e f o r e delivery. She h a d n o t b e e n v a c c i n a t e d before. T h e highest a n t i - T T level of 4.220 I U / m l was r e c o r d e d in p a t i e n t no. 4 whose m o t h e r h a d received m u l t i p l e i n o c u l a t i o n s in each of two consecutive p r e g n a n c i e s over a 3-year period. T h e s e c o n d highest level of 1.035 I U / m l was in p a t i e n t no. 6 whose m o t h e r h a d received 14 doses of T T in 5 consecutive pregnancies over a 13-year period. This m o t h e r h a d been given 2 - 3 doses of t o x o i d d u r i n g each o f the 5 pregnancies. O n e p a t i e n t (no. 2) whose m o t h e r
was n o t v a c c i n a t e d h a d a s e r u m a n t i - t o x i n level of 0.037 I U / m l .
4.3. A n t i - T T levels and maternal vaccination T a b l e 2 shows the r e l a t i o n s h i p b e t w e e n m e a n anti-T]" c o n c e n t r a t i o n s a n d the n u m b e r of T T doses given in the last p r e g n a n c y . T h e r e was cons i d e r a b l e i n d i v i d u a l v a r i a t i o n in the a n t i - t o x i n levels as shown b y the wide s t a n d a r d d e v i a t i o n s a n d coefficients of v a r i a t i o n w i t h i n the groups. T h e r e was a general t r e n d for anti-T-l" levels to be higher with i n c r e a s i n g n u m b e r of T T doses. H o w ever, the n u m b e r o f p a t i e n t s s t u d i e d was too small for any statistical analysis.
5. D I S C U S S I O N This s t u d y was u n d e r t a k e n b e c a u s e d e s p i t e an intensive p r o g r a m m e of TI" a d m i n i s t r a t i o n in p r e g n a n c y a n d t r a i n i n g o f b i r t h a t t e n d a n t s in hygienic practices, there are still p r o b l e m s with t e t a n u s n e o n a t o r u m in T a n z a n i a . A l t h o u g h the i n c i d e n c e has fallen f r o m 2% p e r live b i r t h in 1979 [8] to 0.6% in 1989, it is e s t i m a t e d that 40% of the cases occur in n e o n a t e s whose m o t h e r s h a d T T in p r e g n a n c y [8]. This was n o t due to vaccine failure [12], b u t r a t h e r to p r e s u m e d effective levels of a n t i - t o x i n failing to p r e v e n t tetanus. This is the first r e p o r t d o c u m e n t i n g the o c c u r r e n c e of ' p r o tective' levels of a n t i - t o x i n in n e o n a t e s with tetanus. By using the E L I S A t e c h n i q u e to d e t e r m i n e specific I g G a n t i b o d i e s against t e t a n u s toxin, n o n - n e u t r a l i s i n g a n t i b o d i e s could have been inc l u d e d in the e s t i m a t i o n s as it was not p o s s i b l e to
Table 2 Maternal vaccination status and mean tetanus antitoxin (antiT'F) concentrations in patients' sera TF-doses last pregnancy
No. of patients
Mean anti-TT IU/ml
S.D.
S.E.
Coeff. of variation
1 2 3
4 2 3
0.057 0.117 1.793
0.037 0.018 2.151
0 . 0 1 8 64.809 0 . 0 1 3 15.713 1.242 119.946
174 know how much of the absorbency was due to such antibodies. However, previous studies have shown that the technique gives results comparable with those obtained using methods which assay neutralising antibodies [9]. It is likely, therefore, that neonatal tetanus occurred with pre-existing toxin-neutralising antibodies above the presumed m i n i m u m protective level of 0.01 I U / m l . There are previous reports of adult patients with tetanus in w h o m neutralising antibodies assayed using the animal protection model [5-7], were present. In these reports a total of 12 patients had anti-TT concentrations ranging from 4-16-times the protective level. In our study, seven patients had anti-TT concentrations 4 - 1 3 - t i m e s the m i n i m u m protective level. These observations suggest there is no absolute ' m i n i m u m protective level' of tetanus anti-toxin. This m a y be particularly important in neonates who rely on passively acquired immunity; the level of 0.01 I U / m l in maternal serum m a y not provide reliable protection. The toxin dose in relation to the level of neutralising antibodies p r o b a b l y d e t e r m i n e s whether tetanus develops. Two of our patients (nos. 4 and 6) had anti-toxin concentrations 400- and 100-times the m i n i m u m protective level, respectively. These are the highest reported pre-existing anti-toxin levels in patients with tetanus, and an explanation is required. The mothers of the two patients had been given multiple booster doses of TI" in successive pregnancies which might induce p r e d o m i n a n t l y IgG4-restricted response [13]. Antibodies of the I g G 4 subclass are functionally monovalent [14], and m a y be of low affinity in m a n [15]. We did not determine whether the anti-TT in our patients was I g G 4 isotype, but these observations could provide an explanation for the development of tetanus in our patients. If this explanation is correct, repeated use of T T in pregnancy is undesirable because it m a y not be protective as well as frequently causing allergic reactions [12]. The presence of 0.037 I U / m l in patient no. 2, whose mother had not been vaccinated, is more difficult to explain, but could have been due to childhood immunization, although naturally acquired antibodies to tetanus toxin in m a n have been reported [16]. Serum samples are rarely ob-
tained in patients with clinical tetanus, and we have f o u n d only three reports [5-7]. In conclusion, the p r o g r a m m e to prevent tetanus n e o n a t o r u m should avoid hyperimmunisation of women. In addition to the immunisation p r o g r a m m e education and training in hygiene remain vitally important.
ACKNOWLEDGEMENTS This study was supported by grants from the Norwegian Ministry of Development Co-operation and the University of Bergen. We thank the National Institute of Public Health, Oslo for providing us with tetanus toxiod and positive standard sera, and Muhimbili Medical Centre for allowing us to publish these data.
REFERENCES [1] Scheibel, I. (1955) Uses and results of active tetanus immunization. Bull. WHO 13, 381-394. [2] Wolters, K.J. and Dehmek H. (1942) Abschliessende Untersuchungen i~ber die Tetanusprophylaxe durch aktive Immunisierung. Ztschr. fr. Hyg. u. Infektionskr. 124, 326332. [3] Edsall, G. (1959) Specific prophylaxis of tetanus. J. Am. Med. Assoc. 171,417-427. [4] McComb, J.A. (1964) The prophylactic dose of homologous tetanus antitoxin. New Engl. J. Med. 270, 175-178. [5] Goulon, M., Girard, O., Grosbuis, S., Desormeau, J.P. and Capponi, M.F. (1972) Les anticorps antit6tanique: Titrage avant s6roanatoxinotherapie chez 64 t6taniques. Nouv. Presse Med. 1, 3049-3050. [6] Bergener, S.A., Cherubin, C.E., Nelson, S. and Levine, L. (1978). Tetanus despite preexisting antitetanus antibody. J. Am. Med. Assoc. 240, 769-770. [7] Passen, E.L. and Andersen, B.R. (1986) Clinical tetanus despite a "protective" level of toxin neutralizing antibody. J. Am. Med. Assoc. 255, 1171-1173. [8] Ndawi, B.T. (1980) A case control study on the relationship between tetanus toxoid immunization and the development and prognosis of neonatal tetanus at Muhimbili and Bugando Hospitals in Tanzania. A dissertation for Diploma in Public Health of the University of Dares Salaam. [9] Mongi, P.S. (1985) Prognosis of neonatal tetanus with special reference to intrathecal serotherapy. A dissertation for Master of Medicine in Paediatrics and Child Health of the University of Dares Salaam.
175 [10] Melville-Smith, M.E., Seagroatt, V,A. and Watkins, J.T. (1983) A comparison of enzyme-linked immunosorbent assay (ELISA) with the toxin neutralization test in mice as a method for the estimation of tetanus antitoxin in human sera. J. Biol. Stand. 11, 137-144. [11] Engvall, E. and Perlmann, P. (1972) Enzyme-linked immunosorbent assay, ELISA II1. Quantitation of specific antibodies by enzyme-labelled anti-immunoglobulin in antigen-coated wells. J. Immunol. 109, 129-135. [12] Edsall, G., Elliot, M.W., Peebles, T.G., Levine, L. and Eldred, M.C. (1967) Excessive use of tetanus toxoid booster. J, Am, Med. Assoc. 202, 111-113. [13] Aalbersee, R.C., Van der Gaag, R. and Van Leeuwen, J. (1983) Serological aspects of IgG4 antibodies. J. Immunol. 130, 722-726. [14] Van der See, I.S., Van Swieten, P. and Aalbersee, R.C. (1986) Serological aspects of IgG4 antibodies. J. Immunol. 137, 3566-3571. [15] Devey, M.E., Bleasdale, K.M., French, M.A.H. and Harri-
[16]
[17]
[18]
[19]
son, G. (1985) The IgG4 subclass is associated with a low affinity antibody response to tetanus toxoid in man. Immunology 55, 565-567. Veronesi, R., Bizzini, B., Focaccia, R.~ Coscina, A.L., Mazza, C.C., Focaccia, M.T., Carraro, F. and Honningman, M.N. (1983) Naturally acquired antibodies to tetanus toxin in humans and animals from Galhpagos Islands. J. Infect. Dis. 147, 308-3ll. Maselle, S.Y., Mgaya, H., Matre, R. Harthug, S. and Hofstad, T. (1989) Maternal and foetal tetanus toxoid antibody levels following immunization in pregnancy. J. Obst. Gynecol. East. Centr. Afr. 8, 11-14. Rahman, M., Chen, L.C., Chakraborty, J., Yunus, M., Chowdhury, A.I., Sarder, A.M., Bhatia. S. and Curlin, G.T. (1982) Use of tetanus toxoid for the prevention of neonatal tetanus. Bull. WHO 60. 261-267. Leading Article (1983) Prevention of neonatal tetanus. Lancet 1, 1253-1254.
171
FEMSIM 00162
Neonatal tetanus despite protective serum antitoxin concentration S.Y. Maselle 1, R. M a t r e 3,4, R. Mbise 2 a n d T. H o f s t a d 4 Department of Microbiology and Immunology, and 2 Department of Paediatrics and Child Health, Muhimbili Medical Centre, Dares Salaam, Tanzania, 3 Broegelmann Research Laboratory for Microbiology, and 4 Department of Microbiology and Immunology, The Gade Institute, University of Bergen, Norway Received 12 February 1991 Accepted 15 February 1991
Key words: Pregnancy; Immunization; Neonatal tetanus
1. SUMMARY
2. I N T R O D U C T I O N
Using the ELISA technique to estimate serum antibodies against tetanus toxin, seven neonates with clinical tetanus were found to have antibody levels 4-13 times higher than the presumed minimum protective level of 0.01 I U / m l . All but one of their mothers had been vaccinated with tetanus toxoid in pregnancy. In two other neonates, whose mothers had received multiple booster doses of toxoid during pregnancy, the anti-toxin concentrations were 100- and 400-times the presumed protective level. Therefore the toxin dose may overwhelm the pre-existing anti-toxin level and produce disease. Furthermore, multiple booster injections of tetanus toxoid may not only enhance serum anti-toxin titres, but could also lead to an ineffective immune response.
Many attempts have been made to correlate the amounts of circulating tetanus antitoxin (anti-TT) with the degree of protection in man. However, since human experiments with tetanus are rare, most of the available information is based on animal data which showed that 0.01 international unit (IU) of a n t i - T T / m l of serum was the threshold of protection [1]. This observation was supported by a human experiment in which 2-3 human minimum lethal doses of tetanus toxin failed to produce symptoms of tetanus with pre-existing 0.005-0.01 IU of a n t i - T T / m l of serum [2]. Therefore, it has generally been accepted that an anti-TT level of 0.01 I U / m l is the minimum protective level in man [3,4]. However, clinical tetanus has been reported in adult patients with anti-Tl" concentrations above the presumed minimum protective level at onset of symptoms [5-7]. In Tanzania where neonatal tetanus is an important public health problem, cases have been reported among neonates whose
Correspondence to: R. Matre, Broegelmann Research Laboratory for Microbiology, Armauer Hansen House, N-5021 Bergen, Norway.
172 mothers were vaccinated with tetanus toxoid (TT) in pregnancy [8,9]. Such reports seemed to suggest that there was probably no universal minimum protective level, and that any serum anti-TT concentration could be overwhelmed given a sufficient quantity of toxin. This paper describes the findings in ten newborn babies with clinical tetanus seen in a referral to hospital in Tanzania.
3. M A T E R I A L S A N D M E T H O D S 3. i. Patients studied Full term normal delivery newborns with clinical tetanus admitted to Muhimbili Consultant Hospital in D a r e s Salaam, Tanzania, were studied. Details of their delivery, onset of symptoms, treatment and history of maternal vaccination with tetanus toxoid (TF) in pregnancy were obtained from their records. Patients who had been given therapeutic anti-tetanus serum were excluded from the study. Blood samples were collected on admission, sera separated and stored at - 2 0 ° C until analysis. 3.2. Enzyme immuno-assay Enzyme-linked immunosorbent assay (ELISA) technique was used to determine the anti-TT concentrations in the sera. This technique has been shown to give results comparable with those obtained by in vivo neutralisation test in mice, the conventional method for anti-TT assay [10]. The procedure was essentially similar to that described by Engvall and Perlman [11]. In brief, flat-bottomed polystyrene microtitration plates (Dynatech, Switzerland) were coated with an optimal dilution (see below) of T T ref no. A S / 2 4 / 8 1 - T containing 480 L f / m l (National Institute of Public Health (NIPH), Oslo, Norway), Coating was done at 37°C overnight in 0.05 M bicarbonate buffer, p H 9.6. The plates were kept at 4°C and subsequently used for up to 4 weeks. After washing the plate, phosphate-buffered saline, p H 7.2, containing 1% bovine serum albumin was added to the wells, incubated at 37°C for 1 h and washed. Diluted serum samples were then added, the plate incubated as above and
washed again. Thereafter, swine anti-human IgG phosphatase conjugate (Orion Diagnostic, Espo, Finland) appropriately diluted (see below) was added and incubated as above. After further washing, the substrate, p-nitrophenyl phosphate (Sigma Chem. Co., U.S.A.) was added and the plate kept in the dark at room temperature for 20 min. The enzyme-substrate reaction was stopped with sodium hydroxide solution and optical densities read at 405 nm in a Titertek plate reader (Flow Labs, U.K.). Optimal dilutions of TT (1:500) for coating and conjugate (1:500) for use had earlier been established by checkerboard titration with a positive anti-tetanus toxoid control serum (NIPH). 3.3. Calculation of anti-TT concentrations The optical densities obtained with 14 duplicates of 2-fold dilutions of a positive control serum containing 8 IU of a n t i - T T / m l ( N I P H ) were used to draw a standard curve against corresponding serum dilutions. Each patient's serum had been assayed at three dilutions (1:200, 1:800 and 1:3200) in duplicate and the optical densities obtained were used to determine the anti-TT levels from the standard curve. The final antitoxin concentration for each serum was obtained by averaging any two closest contiguous estimations. A negative control serum from an unvaccinated person and a positive control were included in all assays. One person performed the assays and there were no changes in reagents' lots or equipment.
4. R E S U L T S 4.1. Patients studied Ten neonates with tetanus fulfilled the study criteria. Five of them had been delivered at home and the other five in hospital or maternity homes. Three of the respective mothers were primiparous and the remaining seven were multiparous. All the babies had clinically infected umbilical cord stumps, except one (case no. 10). One patient had been treated with crystalline penicillin and another, who was delivered at home, had local treatment with a mixture of charcoal ash and kerosine. The rest had not received any form of treatment.
173 Table 1 Incubation periods, maternal vaccination status and serum concentrations of tetanus antitoxin (anti-TT) in 10 neonates with tetanus Case no.
Incubation period in days
Maternal "IT vaccination Previous Last pregnancies pregnancy
Serum anti-T]7 IU/ml
1 2 3 4 5 6 7 8 9 10
10 7 6 2 8 8 16 4 4 14
No (p) No (p) 5 doses 3 doses No (p) 11 doses Yes (ns) 3 doses No No
0.130 0.037 0.078 4.220 0.069 1.035 0.124 0.079 0.104 0.002
2 doses No 1 dose 3 doses 1 dose 3 doses 3 doses I dose 2 doses 1 dose
p, primiparous; ns, not specified.
T a b l e 1 shows the i n c u b a t i o n periods, m a t e r n a l T T v a c c i n a t i o n status a n d s e r u m anti-T]" conc e n t r a t i o n s in the ten patients. T h e i n c u b a t i o n p e r i o d s c a l c u l a t e d f r o m the d a y of delivery up to the onset of s y m p t o m s v a r i e d f r o m 2 - 1 6 d a y s with a m e a n of 7.9 days. N i n e m o t h e r s h a d b e e n v a c c i n a t e d with T T in the last p r e g n a n c y , five of w h o m h a d "IT in p r e v i o u s pregnancies. O n e m o t h e r h a d not received a n y T T vaccination. T h e r e was no r e l a t i o n s h i p b e t w e e n the i n c u b a t i o n p e r i o d a n d m a t e r n a l v a c c i n a t i o n status or a n t i - T T levels.
4.2. A n t i - T T levels in the patients' sera Anti-T]" serum levels in the p a t i e n t s shown in T a b l e 1 were all a b o v e 0.01 I U / m l except in o n e p a t i e n t (no. 10) w h o h a d a titre of 0.002 I U / m l . T h e m o t h e r of this p a t i e n t h a d b e e n given a single d o s e of "IT 2 weeks b e f o r e delivery. She h a d n o t b e e n v a c c i n a t e d before. T h e highest a n t i - T T level of 4.220 I U / m l was r e c o r d e d in p a t i e n t no. 4 whose m o t h e r h a d received m u l t i p l e i n o c u l a t i o n s in each of two consecutive p r e g n a n c i e s over a 3-year period. T h e s e c o n d highest level of 1.035 I U / m l was in p a t i e n t no. 6 whose m o t h e r h a d received 14 doses of T T in 5 consecutive pregnancies over a 13-year period. This m o t h e r h a d been given 2 - 3 doses of t o x o i d d u r i n g each o f the 5 pregnancies. O n e p a t i e n t (no. 2) whose m o t h e r
was n o t v a c c i n a t e d h a d a s e r u m a n t i - t o x i n level of 0.037 I U / m l .
4.3. A n t i - T T levels and maternal vaccination T a b l e 2 shows the r e l a t i o n s h i p b e t w e e n m e a n anti-T]" c o n c e n t r a t i o n s a n d the n u m b e r of T T doses given in the last p r e g n a n c y . T h e r e was cons i d e r a b l e i n d i v i d u a l v a r i a t i o n in the a n t i - t o x i n levels as shown b y the wide s t a n d a r d d e v i a t i o n s a n d coefficients of v a r i a t i o n w i t h i n the groups. T h e r e was a general t r e n d for anti-T-l" levels to be higher with i n c r e a s i n g n u m b e r of T T doses. H o w ever, the n u m b e r o f p a t i e n t s s t u d i e d was too small for any statistical analysis.
5. D I S C U S S I O N This s t u d y was u n d e r t a k e n b e c a u s e d e s p i t e an intensive p r o g r a m m e of TI" a d m i n i s t r a t i o n in p r e g n a n c y a n d t r a i n i n g o f b i r t h a t t e n d a n t s in hygienic practices, there are still p r o b l e m s with t e t a n u s n e o n a t o r u m in T a n z a n i a . A l t h o u g h the i n c i d e n c e has fallen f r o m 2% p e r live b i r t h in 1979 [8] to 0.6% in 1989, it is e s t i m a t e d that 40% of the cases occur in n e o n a t e s whose m o t h e r s h a d T T in p r e g n a n c y [8]. This was n o t due to vaccine failure [12], b u t r a t h e r to p r e s u m e d effective levels of a n t i - t o x i n failing to p r e v e n t tetanus. This is the first r e p o r t d o c u m e n t i n g the o c c u r r e n c e of ' p r o tective' levels of a n t i - t o x i n in n e o n a t e s with tetanus. By using the E L I S A t e c h n i q u e to d e t e r m i n e specific I g G a n t i b o d i e s against t e t a n u s toxin, n o n - n e u t r a l i s i n g a n t i b o d i e s could have been inc l u d e d in the e s t i m a t i o n s as it was not p o s s i b l e to
Table 2 Maternal vaccination status and mean tetanus antitoxin (antiT'F) concentrations in patients' sera TF-doses last pregnancy
No. of patients
Mean anti-TT IU/ml
S.D.
S.E.
Coeff. of variation
1 2 3
4 2 3
0.057 0.117 1.793
0.037 0.018 2.151
0 . 0 1 8 64.809 0 . 0 1 3 15.713 1.242 119.946
174 know how much of the absorbency was due to such antibodies. However, previous studies have shown that the technique gives results comparable with those obtained using methods which assay neutralising antibodies [9]. It is likely, therefore, that neonatal tetanus occurred with pre-existing toxin-neutralising antibodies above the presumed m i n i m u m protective level of 0.01 I U / m l . There are previous reports of adult patients with tetanus in w h o m neutralising antibodies assayed using the animal protection model [5-7], were present. In these reports a total of 12 patients had anti-TT concentrations ranging from 4-16-times the protective level. In our study, seven patients had anti-TT concentrations 4 - 1 3 - t i m e s the m i n i m u m protective level. These observations suggest there is no absolute ' m i n i m u m protective level' of tetanus anti-toxin. This m a y be particularly important in neonates who rely on passively acquired immunity; the level of 0.01 I U / m l in maternal serum m a y not provide reliable protection. The toxin dose in relation to the level of neutralising antibodies p r o b a b l y d e t e r m i n e s whether tetanus develops. Two of our patients (nos. 4 and 6) had anti-toxin concentrations 400- and 100-times the m i n i m u m protective level, respectively. These are the highest reported pre-existing anti-toxin levels in patients with tetanus, and an explanation is required. The mothers of the two patients had been given multiple booster doses of TI" in successive pregnancies which might induce p r e d o m i n a n t l y IgG4-restricted response [13]. Antibodies of the I g G 4 subclass are functionally monovalent [14], and m a y be of low affinity in m a n [15]. We did not determine whether the anti-TT in our patients was I g G 4 isotype, but these observations could provide an explanation for the development of tetanus in our patients. If this explanation is correct, repeated use of T T in pregnancy is undesirable because it m a y not be protective as well as frequently causing allergic reactions [12]. The presence of 0.037 I U / m l in patient no. 2, whose mother had not been vaccinated, is more difficult to explain, but could have been due to childhood immunization, although naturally acquired antibodies to tetanus toxin in m a n have been reported [16]. Serum samples are rarely ob-
tained in patients with clinical tetanus, and we have f o u n d only three reports [5-7]. In conclusion, the p r o g r a m m e to prevent tetanus n e o n a t o r u m should avoid hyperimmunisation of women. In addition to the immunisation p r o g r a m m e education and training in hygiene remain vitally important.
ACKNOWLEDGEMENTS This study was supported by grants from the Norwegian Ministry of Development Co-operation and the University of Bergen. We thank the National Institute of Public Health, Oslo for providing us with tetanus toxiod and positive standard sera, and Muhimbili Medical Centre for allowing us to publish these data.
REFERENCES [1] Scheibel, I. (1955) Uses and results of active tetanus immunization. Bull. WHO 13, 381-394. [2] Wolters, K.J. and Dehmek H. (1942) Abschliessende Untersuchungen i~ber die Tetanusprophylaxe durch aktive Immunisierung. Ztschr. fr. Hyg. u. Infektionskr. 124, 326332. [3] Edsall, G. (1959) Specific prophylaxis of tetanus. J. Am. Med. Assoc. 171,417-427. [4] McComb, J.A. (1964) The prophylactic dose of homologous tetanus antitoxin. New Engl. J. Med. 270, 175-178. [5] Goulon, M., Girard, O., Grosbuis, S., Desormeau, J.P. and Capponi, M.F. (1972) Les anticorps antit6tanique: Titrage avant s6roanatoxinotherapie chez 64 t6taniques. Nouv. Presse Med. 1, 3049-3050. [6] Bergener, S.A., Cherubin, C.E., Nelson, S. and Levine, L. (1978). Tetanus despite preexisting antitetanus antibody. J. Am. Med. Assoc. 240, 769-770. [7] Passen, E.L. and Andersen, B.R. (1986) Clinical tetanus despite a "protective" level of toxin neutralizing antibody. J. Am. Med. Assoc. 255, 1171-1173. [8] Ndawi, B.T. (1980) A case control study on the relationship between tetanus toxoid immunization and the development and prognosis of neonatal tetanus at Muhimbili and Bugando Hospitals in Tanzania. A dissertation for Diploma in Public Health of the University of Dares Salaam. [9] Mongi, P.S. (1985) Prognosis of neonatal tetanus with special reference to intrathecal serotherapy. A dissertation for Master of Medicine in Paediatrics and Child Health of the University of Dares Salaam.
175 [10] Melville-Smith, M.E., Seagroatt, V,A. and Watkins, J.T. (1983) A comparison of enzyme-linked immunosorbent assay (ELISA) with the toxin neutralization test in mice as a method for the estimation of tetanus antitoxin in human sera. J. Biol. Stand. 11, 137-144. [11] Engvall, E. and Perlmann, P. (1972) Enzyme-linked immunosorbent assay, ELISA II1. Quantitation of specific antibodies by enzyme-labelled anti-immunoglobulin in antigen-coated wells. J. Immunol. 109, 129-135. [12] Edsall, G., Elliot, M.W., Peebles, T.G., Levine, L. and Eldred, M.C. (1967) Excessive use of tetanus toxoid booster. J, Am, Med. Assoc. 202, 111-113. [13] Aalbersee, R.C., Van der Gaag, R. and Van Leeuwen, J. (1983) Serological aspects of IgG4 antibodies. J. Immunol. 130, 722-726. [14] Van der See, I.S., Van Swieten, P. and Aalbersee, R.C. (1986) Serological aspects of IgG4 antibodies. J. Immunol. 137, 3566-3571. [15] Devey, M.E., Bleasdale, K.M., French, M.A.H. and Harri-
[16]
[17]
[18]
[19]
son, G. (1985) The IgG4 subclass is associated with a low affinity antibody response to tetanus toxoid in man. Immunology 55, 565-567. Veronesi, R., Bizzini, B., Focaccia, R.~ Coscina, A.L., Mazza, C.C., Focaccia, M.T., Carraro, F. and Honningman, M.N. (1983) Naturally acquired antibodies to tetanus toxin in humans and animals from Galhpagos Islands. J. Infect. Dis. 147, 308-3ll. Maselle, S.Y., Mgaya, H., Matre, R. Harthug, S. and Hofstad, T. (1989) Maternal and foetal tetanus toxoid antibody levels following immunization in pregnancy. J. Obst. Gynecol. East. Centr. Afr. 8, 11-14. Rahman, M., Chen, L.C., Chakraborty, J., Yunus, M., Chowdhury, A.I., Sarder, A.M., Bhatia. S. and Curlin, G.T. (1982) Use of tetanus toxoid for the prevention of neonatal tetanus. Bull. WHO 60. 261-267. Leading Article (1983) Prevention of neonatal tetanus. Lancet 1, 1253-1254.