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Abstract
Tetanus and diphtheria (Td) antibodies were studied in HIV-1-infected women during puerperium. HIV group (n = 61) was compared
with Control group (n = 101). Twenty-one women from HIV and 13 from Control group who had antibody levels lower than 0.1 IU/mL
received a booster with Td vaccine. Antibodies were assessed by double antigen ELISA. Mean tetanus and diphtheria antibody levels from
HIV group were lower than those from Control group. Multiple linear regression analysis showed that tetanus and diphtheria antibody
levels were decreased by HIV-1-infection, and that was independent of the reduction due to the time interval between last booster and
antibody assessment. After a booster dose, both groups had an increase in mean tetanus and diphtheria antibody levels, but in Control
group the levels were higher than in HIV group.
© 2004 Elsevier Ltd. All rights reserved.
Keywords: Tetanus; Diphtheria; HIV infection
0264-410X/$ – see front matter © 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.vaccine.2004.03.023
3708 T.C.S. Bonetti et al. / Vaccine 22 (2004) 3707–3712
women whose children were regulary followed up at For diphtheria antibodies, the same method was applied
the Pediatric AIDS Clinic; Control group, with healthy with some modifications: 0.05 g/mL of diphtheria toxoid
HIV-seronegative women who had given birth at the Ma- (Butantan Institute, Brazil), diphtheria reference serum (in
ternity Hospital of the Federal University of São Paulo. house standard calibrated against “diphtheria antitoxin hu-
HIV-1-infection was defined using the CDC criteria [13]. man serum 91/534”—NIBSC reagent) and biotin-labeled
Women from HIV group who had recent available CD4+ T diphtheria toxoid were used as substitutes for the correspon-
cells counts were divided into three groups: CD4+ T lym- dent tetanus reagents.
phocyte count ≥500, <500 and ≥200 and <200 cells/L. Tetanus and diphtheria antibodies were expressed in In-
For each participant a form was completed with informa- ternational Units per millilitre (IU/mL) using the curve com-
tion about age, previous immunization, antenatal care and parison method to transform optical density in concentration
most recent CD4+ T lymphocyte count. Women who re- units.
ferred at least one dose of tetanus and diphtheria vaccine in
the last 10 years before this study were selected for antibody 2.5. Protective antibody levels
analysis.
Patients were classified according to internationally
2.2. Blood sample collection accepted criteria of protective antibody levels [15–19].
Tetanus and diphtheria antibody concentrations lower than
Venous blood samples were collected from all participants 0.01 IU/mL were considered without protection; levels be-
at study entry. For individuals who received a booster dose tween 0.01 and 0.09 IU/mL, basic immunity; and levels
of Td, blood was also drawn at two other occasions: up to greater than 0.1 IU/mL, full protection.
90 days before vaccination and 25–90 days after.
Samples were centrifuged at 800 × g for 10 min. Serum
was separated and stored at −70 ◦ C until tested. 2.6. Statistical analysis
Table 1 Table 2
Epidemiologic characteristics of the study population Distribution of women from Control and HIV groups in categories of
immunity for tetanus and diphtheria
Group Control HIV P
Control group HIV group
Mean age, in years 27 (16–43) 30 (19–44) 0.002a
(range) Tetanusa (IU/mL)
Antenatal care during 97.5% 96.7% 0.063b <0.01 2% (2/101) 11% (7/61)
pregnancy ≥0.01 and <0.1 7% (7/101) 18% (11/61)
Women born in the 54.9% 55.0% 0.995c ≥ 0.1 91% (92/101) 71% (43/61)
State of São Paulo
Diphtheriab (IU/mL)
Mean time interval, in 1.5 (0.01–8.0) 0.7 (0.1–8.0) 0.617a
<0.01 1% (1/101) 3% (2/61)
yearsd (range)
≥0.01 and <0.1 5% (5/101) 21% (13/61)
a Student’s t-test. ≥0.1 94% (95/101) 76% (46/61)
b Fisher exact test.
c Chi-square test. a The proportion of women in Control group with tetanus antibody
d Time interval, in years, between last booster and antibody assessment. levels ≥0.1 IU/mL (91%) was higher than that of women in HIV group
(71%) (χ2 : P = 0.001). The proportion of women in Control group
with tetanus antibody levels <0.01 IU/mL (2%) was smaller than that of
HIV group: 0.7 years (range, 0.1–8.0; Student’s t-test, P = women in HIV group (11%) (Fisher exact test: P = 0.027).
b The proportion of women in Control group with diphtheria antibody
0.617). The epidemiologic characteristics of the individuals
levels ≥0.1 IU/mL (94%) was higher than that of women in HIV group
enrolled in the study are summarized in Table 1. (76%) (χ2 : P = 0.001). The proportion of women in Control group
with diphtheria antibody levels <0.01 IU/mL (1%) was similar to that of
3.2. Seroprevalence of tetanus and diphtheria antibodies women in HIV group (3%) (Fisher exact test: P = 0.557).
Mean tetanus antibody levels were significantly lower act test, P = 0.557). However, for tetanus, the propor-
in HIV group (0.22 IU/mL) than in Control group tion of women in Control group with antibodies lower than
(1.34 IU/mL), (Student’s t-test, P < 0.001) (Fig. 1A). The 0.01 IU/mL (2%) was smaller than that of women in HIV
same was observed for diphtheria antibodies, with mean group (11%) (Fisher exact test, P = 0.027).
levels in HIV group (0.41 IU/mL) significantly lower than
those from Control group (1.83 IU/mL) (Student’s t-test, 3.3. Tetanus and diphtheria antibodies according to
P < 0.001) (Fig. 1B). CD4+ T lymphocyte counts
The percentage of individuals with full protection against
tetanus in HIV group (71%) was lower than in Control group Mean tetanus antibody levels in HIV-1-infected individ-
(91%) (χ2 , P = 0.001). For diphtheria, the percentage of uals subdivided according to CD4+ T lymphocyte counts
individuals with antibody levels ≥0.1 IU/mL was also lower were significantly lower in two subgroups (CD4+ ≥500 and
in HIV group (76%) than in Control group (94%) (χ2 , P = <500 and ≥200) when compared with Control group (Tukey
0.001) (Table 2). No statistical difference was noticed be- comparison, P = 0.01 and 0.01, respectively) (Fig. 2A).
tween HIV and Control groups when women were divided For diphtheria antibodies, the same situation was observed:
using 0.01 IU/mL antibody levels for diphtheria (Fisher ex- HIV subgroups CD4+ ≥ 500 and CD4+ < 500 and ≥ 200
12.2
12.2
1.0 1.0
0.08 0.08
0.007 0.007
Fig. 1. (A) Tetanus antibody levels in Control and HIV groups. (B) Diphtheria antibody levels in Control and HIV groups. Geometric mean (GM) antibody
levels and number of individuals in each group are shown over graphs for both tetanus and diphtheria. Statistical analysis employed Student’s t-test.
3710 T.C.S. Bonetti et al. / Vaccine 22 (2004) 3707–3712
p = 0.01 p = 0.03
p = 0.01 p = 0.01
GM (IU/mL) 1.34 0.17 0.24 0.41 GM (IU/mL) 1.83 0.48 0.35 1.06
Diphtheria antibody levels (IU/mL)
Tetanus antibody levels (IU/mL)
12.2 12.2
1.0 1.0
0.08 0.08
0.007 0.007
Control CD4 < 500 Control CD4 < 500 CD4 < 200
CD4 >= 500 CD4 < 200 CD4 >= 500
(A) group and >= 200 (B) group and >= 200
HIV HIV
group group
Fig. 2. Tetanus and diphtheria antibody levels in Control and HIV groups. HIV group was subdivided according to CD4+ T lymphocyte counts (cells/L).
Geometric mean (GM) antibody levels and number of individuals in each group are shown over graphs for both tetanus and diphtheria. Statistical analysis
employed ANOVA, with differences between groups assessed by Tukey comparison.
3.5. Tetanus and diphtheria antibodies after one Td dose mean diphtheria antibody levels after booster (0.55 IU/mL)
than Control group (2.94 IU/mL), with P value close to
We compared the response to one Td vaccine dose in 21 significance level (Student’s t-test, P = 0.069) (Fig. 3).
women from HIV and 13 women from Control group. Be- Due to the small number of women who were assessed
fore booster, women from both groups had tetanus and/or for the response to a booster Td dose, we were not able to
diphtheria antibodies lower than 0.1 IU/mL. Mean antibody subdivide the HIV group according to CD4+ T lymphocyte
levels did not differ either: 0.04 and 0.04 IU/mL for tetanus levels.
(Student’s t-test, P = 0.43) and 0.02 and 0.04 IU/mL for
diphtheria (Student’s t-test, P = 0.085) in HIV and Control
groups, respectively. 4. Discussion
After a booster dose, both groups had an increase in
mean tetanus and diphtheria antibody levels. Mean tetanus In this study, we have tested tetanus and diphtheria anti-
post-booster antibody levels were significantly lower in HIV bodies in Brazilian HIV-1-infected women of childbearing
group (2.94 IU/mL) than in Control group (7.87 IU/mL) age. We chose this population because, apart from assessing
(Student’s t-test, P = 0.025). HIV group had also lower specific immunity to these diseases in the women, we would
T.C.S. Bonetti et al. / Vaccine 22 (2004) 3707–3712 3711
P = 0.025 P = 0.069
GM (IU/mL) 0.04 7.87 0.04 2.94 GM(IU/mL) 0.04 2.94 0.02 0.55
148.4
Diphtheria antibody levels (IU/mL)
148.4
Tetanus antibody levels (IU/mL)
20.1 20.1
2.72 2.72
0.37 0.37
0.05 0.05
0.007 0.007
0.001 0.001
pre-boosterp ost-boosterp re-booster post-booster pre-booster post-booster pre-booster post-booster
Fig. 3. Tetanus and diphtheria antibody levels in Control and HIV groups before and after one Td dose. Geometric mean (GM) antibody levels and
number of individuals in each group are shown over graphs for both tetanus and diphtheria. Statistical analysis employed paired Student’s t-test to
assess differences between pre- and post-booster antibody levels within each group; Student’s t-test was used to assess differences between post-booster
antibody levels in Control and HIV groups for both tetanus and diphtheria.
be indirectly evaluating their capacity to transmit tetanus into immunologic categories for HIV infection. However, no
passive immunity to their offspring in the event of a future difference among HIV subgroups was observed. Although
pregnancy. this finding may indicate a qualitative derangement in early
Women from HIV and Control groups had similar epi- phases of HIV disease, it may also reflect a small sample
demiologic characteristics, except for age (Table 1). As size in different subgroups.
tetanus and diphtheria antibodies tend to decrease with Kroon et al. [10] did not find statistically lower diph-
time [16,20], we were aware that difference in age between theria antibodies in HIV-infected individuals, and found
groups might introduce a bias in the study. However, results lower tetanus antibodies only in those with CD4+ T cells
from multiple regression analysis proved that other variables lower than 100 cells/L when compared with Control
were independently associated with antibody levels. HIV-negative subjects.
Both tetanus and diphtheria antibodies were lower in We have then performed multiple regression analyses to
HIV group when compared with Control group (tetanus, assess the effect of each separate variable on tetanus and
1.34 IU/mL versus 0.22 IU/mL; diphtheria, 1.83 IU/mL ver- diphtheria antibodies. We have shown that HIV infection
sus 0.41 IU/mL) (Fig. 1A and B). Previous studies have was associated with a strong reduction in both tetanus and
not found differences in tetanus [11] or diphtheria antibod- diphtheria mean antibodies and that was independent of the
ies [10] when HIV and healthy controls were compared. effect of time interval between last booster and antibody as-
This lack of difference might be due to different laboratory sessment, which was also associated with lower antibodies.
techniques to assess antibodies with different degrees of ac- Interestingly, older age was associated with a mean decrease
curacy [11]. Also, some researchers had small sample sizes in tetanus but not diphtheria antibodies. Other authors have
[10], what might have interfered with their results. On the found that susceptibility to both diseases increased with age
other hand, as will be discussed later, studying a group of [18,21–24].
HIV-infected individuals with a less altered immune system Higher mean diphtheria antibodies were associated with
might lead to a reduced difference with healthy subjects. women who were born in Brazilian States other than the
The distribution of both groups in categories for tetanus State of São Paulo. While diphtheria is controlled in the
and diphtheria (Table 2) also showed a lower proportion of latter, cases of the disease still occur in other Brazilian re-
HIV-infected women fully immune to tetanus and diphtheria gions [25]. We then hypothesize that more frequent natural
(≥0.1 IU/mL). We have also observed a higher proportion boosters might have contributed to the association observed
of women from HIV group who were susceptible to tetanus through multiple regression analysis.
(<0.01 IU/mL). The large percentage of HIV-infected Lastly, we assessed the effect of a booster Td dose in
women in the intermediate immune category for both dis- women from both groups who had shown low tetanus and/or
eases probably reflects the effect of a faster antibody decay diphtheria antibodies (lower than 0.1 IU/mL). They were in-
and a lower response to booster doses in that population [6]. dividuals who really needed the intervention and pre-booster
We expected to find a progressive reduction in mean an- antibody assessment was similar in both groups. Our results
tibody levels when women from HIV group were divided have shown that women from HIV and Control groups are
3712 T.C.S. Bonetti et al. / Vaccine 22 (2004) 3707–3712
able to respond to the vaccination. However, the magnitude differentiation of B lymphocytes from asymptomatic human
of the response is larger for Control group, whose individu- immunodeficiency virus—seropositive male homosexuals. J Immunol
als have reached higher antibody levels for both tetanus and 1987;139:2929–35.
[8] Janoff EN, Hardy WD, Smith PD, Wahl SM. Humoral recall response
diphtheria (Fig. 3A and B). in HIV infection. Levels, specificity, and affinity of antigen-specific
Other groups have already shown similar results to a IgG. J Immunol 1991;147:2130–5.
Td dose [10,12]. That reinforces the necessity of keeping [9] Kjeldsen K, Heron I, Skinhoj P. Immunity against diphtheria and
HIV-infected individuals up-to-date with their vaccination. tetanus in human immunodeficiency virus-infected Danish men born
Moreover, women of childbearing age, apart from being 1950–1959. Acta Pathol Microbiol Scand 1992;100:803–8.
[10] Kroon FP, van Dissel JT, Labadie J, van Loon AM, van Furth R.
protected, can also transfer tetanus antibodies to their off- Antibody response to diphtheria, tetanus, and poliomyelitis vaccines
spring in the event of future pregnancy. And because placen- in relation to the number of CD4+ T lymphocytes in adults infected
tal transfer is reduced in that population [11], antibody lev- with human immunodeficiency virus. Clin Infect Dis 1995;21:1197–
els must be higher than those found in a non-HIV-infected 203.
population. [11] de Moraes-Pinto MI, Almeida ACM, Kenj G, Filgueiras TE, Tobias
W, Santos AM, et al. Placental transfer and maternally acquired
Finally, the presence of a pool of susceptible individuals neonatal IgG immunity in human immunodeficiency virus infection.
in any population can contribute to the triggering of a diph- J Infect Dis 1996;173:1077–84.
theria epidemic. That has already been proved in a recent [12] Dieye TN, Sow PS, Simonart T, Gueye-Ndiaye A, Popper SJ,
past [4] and must be avoided. Delforge ML, et al. Immunological and virologic response after
tetanus toxoid booster among HIV-1 and HIV-2 infected Senegales
individuals. Vaccine 2002;20:905–13.
[13] Centers for Diseases and Control (CDC). Appendix: revised
Acknowledgements surveillance case definition for HIV infection. Morb Mortal Wkly
Rep 1999;48:29–31.
We thank the staff from the Department of Preventive [14] Kristiansen M, Aggerbeck H, Heron I. Improved ELISA for
Medicine, Federal University of São Paulo, Brazil, for as- determination of anti-diphtheria and/or anti-tetanus antitoxin
antibodies in sera. Acta Pathol Microbiol Scand 1997;105:843–53.
sistence in statistical analyses. We thank Dr. Abês Mahmed
[15] Ipsen J. Circulating antitoxin at the onset of diphtheria in 425 patients.
Amed, Department of Obstetrics of the Federal University J Immunol 1946;54:325–47.
of São Paulo, Brazil, for assistance in data collection. We [16] Galazka AM. The immunological basis for immunization
thank Dr. Reinaldo Salomão, Division of Infectious Diseases series. Module 2: Diphtheria. World Health Organization; 1993.
of the Federal University of São Paulo, Brasil, for review- WHO/EPI/GEN/93.12.
[17] Gupta RK, Griffin Jr P, Xu J, Rivera R, Thompson C, Siber GR.
ing the manuscript. This work was supported by FAPESP,
Diphtheria antitoxin levels in US blood and plasma donors. J Infect
Brazil—protocol number: 00/01332-7 and 97/06118-9. Dis 1996;173:1493–7.
[18] Stark K, Schonfeld C, Barg J, Molz B, Vornwald A, Bienzle
U. Seroprevalence and determinants of diphtheria, tetanus and
References poliomyelitis antibodies among adults in Berlin, Germany. Vaccine
1999;17:844–50.
[1] Centers for Diseases and Control (CDC). Recommended childhood [19] von Hunolstein C, Aggerbeck H, Andrews N, Berbers G,
and adolescent immunization schedule. Morb Mortal Wkly Rep Fievet-Groyne F, Maple PAC, et al. European sero-epidemiology
2003;52:Q1–Q10. network: standardisation of the results of diphtheria antitoxin assays.
[2] Galazka AM. The changing epidemiology of diphtheria in the vaccine Vaccine 2000;18:3287–96.
era. J Infect Dis 2000;181:S2–9. [20] Galazka AM. The immunological basis for immunization series.
[3] World Health Organization (WHO). Tetanus toxoid vaccine. Geneve: Module 3: Tetanus. World Health Organization; 1993. WHO/EPI/
WHO-Vaccines, Immunization and Biologicals; 2003. Available at: GEN/93.13.
http://www.who.int/vaccines/en/tetanus.shtml#eliminating. Accessed [21] Crossley K, Irvine P, Warren B, Lee BK, Mead K. Tetanus and
30 April 2003. diphtheria immunity in urban Minnesota adults. J Am Med Assoc
[4] Vitek CR, Wharton M. Diphtheria in the former Soviet Union: 1979;242:2298–300.
reemergence of a pandemic disease. Emerg Infect Dis 1998;4:1–17. [22] Kjeldsen K, Simonsen O, Heron I. Immunity against diphtheria and
[5] Zolopa AR, Kemper CA, Shiboski S, Hamilton JR, Moss AR, tetanus in age group 30–70 years. J Infect Dis 1988;20:177–85.
Deresinski SC. Progressive immunodeficiency due to infection with [23] Gergen PJ, McQuillan GM, Kiely M, Ezzati-Rice TM, Sutter RW,
human immunodeficiency virus does not lead to waning immunity to Virella G. A population-based serologic survey of immunity to
measles in a cohort of homosexual men. Clin Infect Dis 1994;18:636– tetanus in the United States. N Engl J Med 1995;332:761–6.
8. [24] Yuan L, Lau W, Thipphawong J, Kasenda M, Xie F, Bevilacqua J.
[6] World Health Organization (WHO). EPI vaccines in HIV-infected Diphtheria and tetanus immunity among blood donors in Toronto.
individuals. Geneva: WHO-Vaccines, immunization and biologicals; Can Med Assoc J 1997;156:985–90.
2001. Available at: http://www.who.int/vaccines-diseases/diseases/ [25] Boletim Eletrônico Epidemiológico. Ano 2, no 2. Ministério da
HIV.shtml. Accessed 30 April 2003. Saúde—Fundação Nacional de Saúde; 2002. Available at: http://
[7] Teeuwsen UJP, Logtenberg T, Siebelink KHJ, Lange JM, Goudsmit www.funasa.gov.br/pub/boletim eletronico epi/boletim eletronico
J, Uytdehaag FG, et al. Analysis of the antigen- and mitogen-induced epi 0202.pdf. Accessed 30 April 2003.