Diphtheria-neutralizing antibody levels in healthy adults from Rio de Janeiro, Brazil.
ABSTRACT In Brazil, until 2004, the immunization policy against diphtheria involved childhood vaccination with no official routine booster dose administered after 15 years of age. This study assessed functional antibody levels against diphtheria among blood donors. A total of 140 blood samples were collected, and diphtheria antitoxin levels were evaluated by Vero cell neutralization test. The mean age of the population was 34 years old (range: 18-61 years); 37.8% females and 62.2% males. Overall, 30.7% (95%, CI: 23.4-38.7) individuals presented neutralizing antitoxin antibody titers < 0.01 IU/ml; 42.1% (95%, CI: 34.1-50.4) showed values between 0.01-0.09 IU/ml and, 27.1% (95%, CI: 20.2-34.9) had (3) 0.1 IU/ml. In the subgroup of individuals with history of diphtheria immunization during childhood (85%), a number of 28.5% showed unprotective levels of circulating neutralizing antibody (< 0.01 IU/ml). Despite the continuous progress of immunization programs directed to Brazilian population, currently healthy adults remain susceptible to diphtheria.
- SourceAvailable from: Renata Stavracakis Peixoto[Show abstract] [Hide abstract]
ABSTRACT: Subinhibitory concentrations (Sub-MICs) of antibiotics may alter bacterial surface properties and change microbial physiology. This study aimed to investigate the effect of Sub-MIC (1/8 MIC) of penicillin (PEN) and erythromycin (ERY) on bacterial morphology, haemagglutinating activity, cell surface hydrophobicity, biofilm formation on glass and polystyrene surfaces, as well as the distribution of cell surface acidic anionic residues of Corynebacterium diphtheriae strains (HC01 tox- strain; CDC-E8392 and 241 tox+ strains). All microorganisms tested were susceptible to PEN and ERY. The growth in the presence of PEN induced bacterial filamentation, whereas Sub-MIC of ERY caused cell size reduction of 241 and CDC E-8392 strains. Adherence to human erythrocytes was reduced after growth in the presence of ERY, while cell surface hydrophobicity was increased by Sub-MIC of both antibiotics in BATH assays. Conversely, antibiotic-inhibition of biofilm formation was not observed. All strains enhanced biofilm formation on glass after treatment with ERY, while only 241 strain increased glass adherence after cultivation in the presence of PEN. Biofilm production on polystyrene surfaces was improved by 1/8 MIC of ERY. After growth in the presence of both antimicrobial agents, 241 and CDC-E8392 strains exhibited anionic surface charges with focal distribution. In conclusion, Sub-MIC of PEN and ERY modified bacterial surface properties and enhanced not only biofilm formation but also cell surface hidrophobicity. Antibiotic-induced biofilm formation may contribute to the inconsistent success of antimicrobial therapy for C. diphtheriae infections.Journal of Medical Microbiology 02/2013; · 2.30 Impact Factor
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ABSTRACT: Serologic data on diseases that are preventable by vaccines are necessary to evaluate the success of immunization programs and to identify susceptible subgroups. In the present study, we determined serum IgG levels against diphtheria toxin of military and civilian blood donors (N = 75; 69.3% males and 30.7% females) aged 18-64 years, from the Brazilian Army Biology Institute, Rio de Janeiro, using a commercial diphtheria kit (Diphtheria IgG ELISA; IBL, Germany). Most (63%) unprotected military donors were from the older age group of 41 to 64 years. In contrast, the majority (71%) of young military donors (18 to 30 years) were fully protected. About half of the military donors aged 31 to 40 years were protected against diphtheria. Among the civilians, about 50% of persons aged 18 to 30 years and 31 to 40 years had protective antibody levels against diphtheria as also did 64% of individuals aged 41 to 64 years. All civilians had a similar antibody response (geometric mean = 0.55 IU/mL) independent of age group. Military donors aged 18-30 years had higher IgG levels (geometric mean = 0.82 IU/mL) than military donors of 41-64 years (geometric mean = 0.51 IU/mL; P > 0.05). In conclusion, the existence of a considerable proportion of susceptible adults supports the position that reliable data on the immune status of the population should be maintained routinely and emphasizes the importance of adequate immunization during adulthood.Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas / Sociedade Brasileira de Biofisica ... [et al.] 12/2009; 43(1):120-3. · 1.08 Impact Factor
459Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 101(4): 459-462, June 2006
Diphtheria-neutralizing antibody levels in healthy adults from
Rio de Janeiro, Brazil
Fabricia Pires Pimenta, Paulo Vieira Damasco/*, José Cerbino Neto**,
Guilherme Santoro Lopes***, Raphael Hirata Jr, Lucimar Gonçalves Milagres,
Ana Luíza Mattos-Guaraldi/+
Faculdade de Ciências Médicas, Universidade do Estado do Rio de Janeiro, Av. 28 de Setembro 87, Fundos, 3o andar, 20551-030
Rio de Janeiro, RJ, Brasil *Escola de Medicina e Cirurgia, Universidade Federal do Estado do Rio de Janeiro, Rio de Janeiro, RJ,
Brasil **Setor de Doenças Imunopreveníveis, Secretaria Estadual de Saúde, Rio de Janeiro, RJ, Brasil ***Departamento de
Medicina Preventiva, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil
In Brazil, until 2004, the immunization policy against diphtheria involved childhood vaccination with no
official routine booster dose administered after 15 years of age. This study assessed functional antibody levels
against diphtheria among blood donors. A total of 140 blood samples were collected, and diphtheria antitoxin
levels were evaluated by Vero cell neutralization test. The mean age of the population was 34 years old (range: 18-
61 years); 37.8% females and 62.2% males. Overall, 30.7% (95%, CI: 23.4-38.7) individuals presented neutralizing
antitoxin antibody titers < 0.01 IU/ml; 42.1% (95%, CI: 34.1-50.4) showed values between 0.01-0.09 IU/ml and,
27.1% (95%, CI: 20.2-34.9) had ≥ 0.1 IU/ml. In the subgroup of individuals with history of diphtheria immunization
during childhood (85%), a number of 28.5% showed unprotective levels of circulating neutralizing antibody (<
0.01 IU/ml). Despite the continuous progress of immunization programs directed to Brazilian population, currently
healthy adults remain susceptible to diphtheria.
Key words: diphtheria - diphtheria antitoxin - Vero cells assay - Rio de Janeiro - Brazil
The vast territory and unfavorable economic condi-
tions presented by most regions of our country make dif-
ficult the notification of diphtheria cases to Public Health
authorities and remittance of suspect isolates for bacte-
riological confirmation and toxigenicity tests to reference
laboratories. Local outbreaks have been reported indicat-
ing gaps or failure in vaccine coverage (Mattos-Guaraldi
et al. 2003).
Western Europe epidemics illustrated the potential
susceptibility of adults to diphtheria in the vaccine era.
Although the World Health Organization recommends
evaluation of vaccine-induced immunity among both chil-
dren and adults, a very few serosurveys have been done
outside of North American and Western Europe conti-
nents (Damasco et al. 2005). Nationwide seroepidemio-
logical studies are necessary to elaborate effective strat-
egies to maintain immunity against diphtheria in adults,
including periodic booster doses and immunization of
selected age groups (Galazka & Robertson 1996, Galazka
In Brazil, the National Immunization Program was es-
tablished in the early 1970s. Although diphtheria control
was only achieved in the 1990s, 640 diphtheria cases were
notified with an incidence coefficient (IC) of 0.45/100,000
Financial support: Capes, MCT/CNPq, Pronex, Faperj, SR-2/
+Corresponding author: firstname.lastname@example.org,
Received 16 January 2006
Accepted 5 May 2006
inhabitants at the end of decade. During 2004, 19 cases of
diphtheria were reported in the country with an IC of 0.01/
100,000. Since 1970s, the vaccine coverage to tetanus-
diphtheria toxoid (Td) increased from 66 to 94%. Since
2004, Brazilian immunization policy against diphtheria rec-
ommends childhood vaccination at 2, 4, and 6 months of
age and booster doses including in adolescents and adults
(Brasil 2004). However, there is not much information
about regular immunization coverage and immunity of
Brazilian adult population (Funasa 2002). In 2002, the Public
Health Agency of Rio de Janeiro (SES-RJ 2002) stated a
shifting in the age distribution of cases of diphtheria to
persons over 15 years of age. A change in epidemiologi-
cal aspects of diphtheria has been also recently observed
in São Paulo (Casagrande et al. 2005). Current information
also indicated the circulation of Corynebacterium
diphtheriae in our population, including cancer patients
(Mattos-Guaraldi et al. 2001a) and healthy vaccinated
adults (Mattos-Guaraldi et al. 2001b). Recent investiga-
tions on the prevalence of IgG anti-diphtheria toxin levels
in blood donors from Rio de Janeiro showed that only
30% of adults were fully protected. However, investiga-
tion on the circulating neutralizing toxin antibody levels
in the vaccinated population remains necessary, especially
for individuals with titers < 0.1 IU/ml of specific IgG
(Damasco et al. 2005). The aim of this study was to assess
functional antibody levels against diphtheria toxin among
healthy blood donors living in Rio de Janeiro.
The survey analyzed 140 sera from a random sample
of 240 blood donors of Hospital Universitário Pedro
Ernesto, Universidade do Estado do Rio de Janeiro (Hupe/
Uerj) collected from July to October 2002. The study pro-
tocol was approved by The Institutional Review Board of
460 Diphtheria-neutralizing antibody in adults • Fabricia Pires Pimenta et al.
the University Hospital, and blood donors were included
in the study after signing a written informed consent.
Healthy adults included individuals from 18 to 61 years
old, stratified by age and sex: median age of 34 years; 53
(37.8%) female, and 87 (62.2%) male. A total of 119 (85.5%)
subjects reported basic childhood diphtheria immuniza-
tion. Serum samples were frozen and stored at –70o C until
performance of diphtheria toxin antibody test.
Serum samples were tested for specific anti-diphtheria
toxin neutralizing antibodies using a microtitre plate as
previously described (Mills et al. 2003). Briefly, Vero cells
were grown in modified Eagle’s medium (MEM) supple-
mented with 10% fetal calf serum. A volume of 50 µl of
serial twofold dilutions of undiluted test sera or standard
serum for diphtheria toxin, initially diluted at 1/200 (equine
antiserum from Instituto Vital Brazil, RJ, Brazil; 1000 IU/ml
of antibody to diphtheria toxin) were added to the plates.
Following incubation of plates with diphtheria toxin (80
pg/50 µl) at 37oC for 1h, 100 µl of Vero cells (2.5 x 105 cells/
ml) were added to each well. The neutralizing effects of
antibodies were evaluated by growth of Vero cells after 4
days of incubation at 37oC. Neutralizing antibody titer
was defined as the highest dilution of serum neutralizing
toxin killing of 50% Vero cells (CD50). Viability of cells was
determined, by the MTT assay (Efstratiou et al. 1998).
Neutralizing antibody levels were expressed as IU/ml cal-
culated from the standard serum and were categorized
according to internationally accepted ranges: < 0.01 IU/
ml (non-protective), between 0.01 to 0.09 IU/ml (basic pro-
tection), ≥ 0.1 IU/ml (full protection). The lowest protec-
tive level of serum neutralizing antibody to diphtheria is
considered as > 0.01 IU/ml (Galazka 1993).
Data analysis was carried out using the Epi Info™
software program version 6.03 developed by the Centers
for Disease Control and Prevention (CDC, Atlanta, GA,
Diphtheria antitoxin production, primarily of IgG type,
can be induced by absorption of native toxin during clini-
cal infection or in the carrier state, or by immunization
with diphtheria toxoid (Walory et al. 2000). Protection
against diphtheria is mainly due to the development of
neutralizing toxin antibodies. It is believed that a circulat-
ing diphtheria antitoxin level of 0.01 IU/ml, as determined
by the neutralization test in animals or in cell culture, pro-
vides clinical immunity against disease. The outcome of
revaccination of adults depends on several factors, in-
cluding the immunization schedule, potency and time since
the last dose of toxoid (Galaska 1993).
In developing countries where diphtheria is endemic,
the process of maintaining immunity usually operates
through natural mechanisms, including frequent skin in-
fections caused by C. diphtheriae. Nowadays, adults
might become susceptible to diphtheria due to reduced
opportunities of sub clinical infections. Since diphtheria
infection may also occur among previously vaccinated
persons (Mattos-Guaraldi et al. 2001b), the immunity gap
observed among adults should be closed by regular diph-
theria boosters (Ohuabunwo et al. 2005).
Preliminary studies on IgG diphtheria antitoxin levels
determined by means of an ELISA showed basic immu-
nity in 66.7 and 90.9% of children (0-14 years) and teenag-
ers (15-20 years), respectively (Filardy et al. 2001). The
lack of information on the immunity status against diph-
theria in Brazilian adults prompted us to analyse IgG lev-
els anti-diphtheria toxoid in healthy individuals aged 18-
61 years. The results indicated that only 30.7% of indi-
viduals were fully protected (Damasco et al. 2005). Herein,
data presented in the Table illustrated the distribution by
age of antitoxin neutralizing antibody levels among Rio
de Janeiro citizens blood donors. Of note, only 27.1% of
individuals showed antibody levels ≥ 0.1 IU/ml consid-
ered to be protective against diphtheria. Most individu-
als (42.1%) had antibody levels between 0.01-0.09 IU/ml
and are considered partially protected against the dis-
ease. However, a great proportion (30.7%) of individuals
remains vulnerable to disease due to a lack of specific
antibody (< 0.01 IU/ml).
Among unprotected individuals, 26.6% were from the
age group of 18-30 years. The high percentage of indi-
viduals (72.8%) presenting diphtheria antitoxin levels <
0.1 IU/ml suggests a significantly higher risk of diphthe-
ria among adults in our community. Nevertheless, the cur-
rent strategy of mass immunization during childhood may
indeed be sufficient to prevent major outbreaks due to
generation of memory cells. Despite this hypothesis, epi-
demic diphtheria has re-emerged on a massive scale in the
Newly Independent States (NIS) of the former Soviet
Union, beginning in 1990 and affecting 15 countries by
the end of 1994, with more than 175,000 reported cases
and 5000 deaths before immunization campaigns con-
trolled the epidemic. Diphtheria epidemics also spreaded
to neighbouring countries in Europe, the Middle East and
Neutralizing-antibody levels to diphtheria toxin in blood donors stratified by age
< 0.010.01-0.09> 0.1
Age groups (years)
18 - 30 (n = 60)
31 - 40 (n = 45)
41 - 50 (n = 24)
51 - 61 (n = 11)
Total (n = 140)4330.723.4-38.75942.134.1-50.43827.120.2-34.9
461Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 101(4), June 2006
Asia. According to literature, control of diphtheria epi-
demics in vaccine era requires high levels of immunity
among all age groups of the population (Galazka 2000a,b,
Ohuabunwo et al. 2005).
The heterogeneous selection criteria for the partici-
pants and serological methods used for determining im-
munity to diphtheria make difficult to establish reliable
comparisons of data from different epidemiological stud-
ies (Corbeira et al. 1999). However, data achieved in other
countries in which antitoxin antibody concentrations were
measured by neutralization test (Kjedlsen et al. 1988,
Rappuoli et al. 1993, Mathei et al. 1997, Edmunds et al.
2000, Marlovits et al. 2000, McQuillan et al. 2002), demon-
strated a low percentage of immunity against diphtheria
in healthy adults, as observed in this study.
The present evaluation of neutralizing antibodies
showed that the highest percentage (41.6%) of subjects
susceptible to diphtheria was in the age group of 41-50
years old. During epidemic in Eastern Europe the highest
mortality rate (62%) was also detected in this age group
(Rakhmanova et al. 1996, Brennan et al. 2000, Ohuabunwo
et al. 2005).
Questionnaires answered by blood donors revealed
that 119 (85%) had history of basic diphtheria immuniza-
tion during childhood. In this group, 28.5% and 42%
showed diphtheria neutralizing antibody titers < 0.01 IU/
ml and between 0.01-0.09 IU/ml, respectively. Only 29.4%
of subjects showed protective levels of neutralizing anti-
bodies to diphtheria (≥ 0.1 IU/ml).
We believe that the vast territory and unfavorable eco-
nomic conditions presented by most regions of our coun-
try turn difficult the notification of cases to Public Health
authorities and remittance of suspect isolates for bacte-
riological confirmation and toxigenicity tests to reference
laboratories. Local outbreaks have been reported indicat-
ing gaps or failure in vaccine coverage (Mattos-Guaraldi
et al. 2001b).
In general, diphtheria has become rare in immunized
populations despite the lack of protective antibody titers
in large proportion of the adult population. Some authors
questioned the validity of accepted cut-off values for diph-
theria protective titers. They believe antibodies wane with
time after complete basic immunization while immunologi-
cal memory persists, discarding the need for adult
boostering (Mathias 1985). Nevertheless, during the diph-
theria resurgence in parts of Eastern Europe, 60-70% of
reported cases were among persons aged 15 years and
older (Galazka & Robertson 1996). Therefore, if immuno-
logical memory generated by vaccination in childhood is
enough to protect adult individuals, without additional
booster doses, needs further investigations.
This seroepidemiologic survey indicates a low preva-
lence of diphtheria neutralizing antibody levels in healthy
adults from Rio de Janeiro, Brazil, with 30.7% (95%, CI:
23.4-38.7) of individuals presenting titers < 0.01 IU/ml.
The existence of susceptible adults creates an epidemic
potential in our community and reinforces the need of
introduction and surveillance of Td vaccination for adults
in order to ensure adequate diphtheria neutralizing anti-
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