CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, Sept. 2003, p. 886–890 Vol. 10, No.

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1071-412X/03/$08.00⫹0 DOI: 10.1128/CDLI.10.5.886–890.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Group A Streptococcal Antibodies in Subjects with or without

Rheumatic Fever in Areas with High or Low Incidences
of Rheumatic Fever
Elia M. Ayoub,1* Beverly Nelson,2 Stanford T. Shulman,3 Douglas J. Barrett,1 J. Douglas Campbell,4
George Armstrong,5 John Lovejoy,6 Gerald H. Angoff,7 and Sol Rockenmacher8
Department of Pediatrics, University of Florida, Gainesville,1 and Department of Pediatrics, University Medical Center,4
Woolfson Children’s Hospital,5 and St. Luke’s Hospital,6 Jacksonville, Florida; Ministry of Health, General Hospital,
St. George’s, Grenada2; Department of Pediatrics, Northwestern University Feinberg School of Medicine,
Children’s Memorial Hospital, Chicago, Illinois3; and New England Heart Institute, Manchester,7 and
Department of Pediatrics, Dartmouth Hitchcock Medical Center, Lebanon,8 New Hampshire
Received 17 March 2003/Returned for modification 5 June 2003/Accepted 23 June 2003

The levels of streptococcal antibody titers in populations with or without rheumatic fever from an area with
a relatively high incidence of rheumatic fever and an area with a low incidence of this disease were compared.
Streptococcal antibody titers were determined for two populations, each of which included children without
rheumatic fever (nonrheumatic children) and rheumatic fever patients. The two populations were derived from
two separate geographic areas, one with a high incidence of rheumatic fever (Grenada) and another with a low
incidence of this disease (central Florida). The results revealed an absence of consistent differences in the
geometric mean antibody titers between the nonrheumatic subjects and the rheumatic fever patients from
Grenada. In the population from Grenada, the mean anti-streptolysin O and anti-DNase B titers were higher
in the nonrheumatic controls (P of 0.085 and 0.029, respectively). However, the mean titer of the antibody to
the group A streptococcal cell wall carbohydrate was higher in the rheumatic fever patients than in the
nonrheumatic controls (P ⴝ 0.047). This finding contrasted with the finding that the means of all three
streptococcal antibody titers in the patients with rheumatic fever were significantly higher than those in the
nonrheumatic subjects from Florida (P ⴝ 0.01–<0.001). The reason for this paradoxical finding became
evident when the streptococcal antibody titers of the nonrheumatic subjects from Grenada and Florida were
compared, revealing significantly higher levels of all three antibodies in the nonrheumatic subjects from
Grenada than in the nonrheumatic subjects from Florida (P < 0.001). These results suggest that nonrheumatic
individuals in an area with a high incidence of rheumatic fever have inordinately elevated levels of strepto-
coccal antibodies in serum. The presence of elevated streptococcal antibody titers in such a population, which
probably reflects a high background prevalence of streptococcal infections, should be taken into consideration
when evaluating the role of the group A streptococcus in nonpurulent complications of infections.

Previous studies of the antibody responses of patients with
acute rheumatic fever compared the antibody levels in such
populations with those in healthy controls, which were defined
as individuals from the same area who had no evidence of
recent group A streptococcal infections or nonpurulent com-
plications of such infections (3, 5, 8, 9, 15, 19). These studies
consistently showed that the levels of the streptococcal anti-
bodies in patients with rheumatic fever, particularly those with
residual rheumatic heart valve disease (1, 8, 15), were signifi-
cantly higher than those of the healthy controls. No studies
have examined the levels of these antibodies in populations
with rheumatic fever and those without rheumatic fever
(termed nonrheumatic) from areas with a high incidence of
rheumatic fever. The present study was undertaken to deter-
mine whether the streptococcal antibody levels in nonrheu-
matic individuals and those with rheumatic fever in an area
with a high incidence of rheumatic fever show the same dif-
ferences as those in populations from areas with a low inci-
dence of this disease.
* Corresponding author. Mailing address: Department of Pediatrics,
P.O. Box 100296, University of Florida, Gainesville, FL 32610. Phone:
(352) 392-2967. Fax: (352) 392-0481. E-mail: ayoubem@peds.ufl.edu.
Streptococcal antibody titers (anti-streptolysin O [ASO],
anti-DNase B, and the antibody to the group A carbohydrate
[anti-A-Cho]) were determined for the sera from individuals
with no history of rheumatic fever and for patients with a
history of rheumatic fever who were from the same population
of the island of Grenada, West Indies, an area where the
incidence of rheumatic fever remains very high (13; P. K.
Noah, R. Kopycinski, B. Nelson, unpublished data; and B.
Nelson and G. F. Armstrong, personal communications). The
levels of the antibodies in these two groups were then com-
pared to those obtained for matched groups of healthy indi-
viduals and patients with a history of rheumatic fever from
Florida, where the incidence of acute rheumatic fever is low
(6).
MATERIALS AND METHODS
Grenada study. The island of Grenada has a population of 99,510, including
about 26,000 in the age range of 5 to 15 years; about 75% of the population is of
African descent (B. Nelson and G. Armstrong, personal communications). The
recent annual incidence of rheumatic fever in Grenada is 52 per 100,000 in
children under the age of 15 years (13; P. K. Noah, R. Kopycinski, and B. Nelson,
unpublished data; B. Nelson and G. F. Armstrong, personal communications).
This incidence contrasts with an incidence of 0.6 per 100,000 in individuals in the
age group of 5 to 19 years in the United States (11). The patients in Grenada are

886
VOL. 10, 2003 GROUP A STREPTOCOCCAL ANTIBODIES AND RHEUMATIC FEVER
monitored through district clinics in their respective parishes to ensure compli-
ance with prophylaxis. Recent data indicate that while the incidence of acute
rheumatic fever has not changed in Grenada, the recurrence rate has declined
significantly following the institution of penicillin prophylaxis in the early 1990s
(13; P. K. Noah, R. Kopycinski, and B. Nelson, unpublished data; B. Nelson and
G. F. Armstrong, personal communications).
(i) Nonrheumatic subjects. Nonrheumatic subjects consisted of 30 children of
African ancestry: 16 were healthy children, 8 children had congenital heart
disease, 4 children had Marfan syndrome, 1 child had congenital rubella, and 1
had congenital hemolytic anemia. These subjects had no history of recent phar-
yngitis and no evidence of active impetigo or complications of group A strepto-
coccal infections. They were seen in the clinic at the same time as the patients
who had rheumatic fever and were matched with these patients for age (range,
4 to 24 years; median age, 14 years).
(ii) Rheumatic fever patients. Patients previously diagnosed as having acute
rheumatic fever were scheduled to return to the clinic, where they were reeval-
uated by one of the physicians involved in this study. The diagnosis of acute
rheumatic fever had been established at the time of the original presentation
based on the updated Jones criteria (7). The history of their illness and their
adherence to prophylaxis were reviewed. The patients received a complete phys-
ical examination. Electrocardiogram and echocardiographic studies were per-
formed on patients with suspected cardiac disease, and the results were reviewed
by a pediatric cardiologist.
Thirty-two patients were included in the Grenada study. Their ages ranged
from 6 to 32 years (median age, 13 years). All patients were of African descent.
None of the patients had acute disease at the time of evaluation. The interval
from the time of acute disease to the present evaluation for the patients ranged
from 0.2 to 20 years, with of a mean of 5.8 years. This interval was ⬍1 year for
3 patients, 1 to 5 years for 16 patients, 6 to 10 years for 7 patients, and 11 to 20
years for 6 patients. Twenty-six of the patients had clinical evidence of chronic
valvular heart disease, and five of these patients had undergone surgical repair of
their mitral valves. The nature of the valvar disease was confirmed by echocar-
diography, which was performed at the time of this evaluation on all but two of
the patients who had undergone mitral valve replacement. Mitral valve abnor-
mality was present in only 15 patients, combined mitral valve and aortic valve
disease was present in 8 patients, and isolated aortic disease was present in 3
patients. Six patients had no clinical or echocardiographic evidence of cardiac
disease; five patients had arthritis and one patient had Sydenham chorea as the
major manifestation of their disease. Twenty-five of the patients were receiving
penicillin prophylaxis: 22 were receiving monthly intramuscular benzathine pen-
icillin G, and 3 were receiving daily oral penicillin.
After the consent of the patients or their parents was obtained, blood was
procured from all Grenada subjects for streptococcal antibody tests.
Gainesville, Fla., study. Antibody titers obtained for the Grenada nonrheu-
matic subjects and the rheumatic fever patients were subsequently compared
with titers obtained for 32 nonrheumatic individuals and 32 patients with rheu-
matic fever from Florida. Only African-American subjects were included.
(i) Nonrheumatic subjects. The nonrheumatic individuals from Florida con-
sisted of healthy school-age children and young adults from whom sera had been
procured for previous studies to serve as healthy controls. These subjects were
screened at the time of blood procurement and were found to have no history of
recent pharyngitis or complications of streptococcal infection. They were
matched for age and race with the rheumatic fever patients. The ages of the
subjects ranged from 5 to 26 years (median age, 13 years).
(ii) Rheumatic fever patients. The rheumatic fever patients included in the
Florida study were age matched (range, 5 to 32 years; median age, 13 years) with
the Grenada patients. They were also matched with respect to cardiac involve-
ment. They included 9 patients without cardiac disease and 23 with valvular
disease: 14 patients had mitral valve disease, 6 had combined mitral and aortic
valve disease, and 3 had isolated aortic valve disease. The interval between the
time of acute disease and procurement of blood samples ranged from 0.2 to 24
years, with a mean interval of 5.9 years. The difference between this interval and
that of the Grenada patients was not significant (P ⫽ 0.49). Data on prophylaxis,
available for 21 patients, revealed that 8 of the patients were receiving monthly
intramuscular benzathine penicillin G prophylaxis and 13 were receiving oral
penicillin prophylaxis.
Streptococcal antibody tests. Serum was separated aseptically from the blood
samples obtained from the Grenada subjects prior to transportation for assay in
our laboratory (University of Florida, Gainesville). All sera were stored at
⫺20°C. Proportional numbers of sera from each of the study groups were assayed
in tandem for the three streptococcal antibodies, namely, ASO, anti-DNase B,
and anti-A-Cho. The assays were performed as previously described (4).
887
Statistical analysis. Analysis of the significance of the difference between
means and geometric means of the antibody titers for the different groups was
assessed by two sample t tests that assume unequal variance provided in the Excel
program in Office 97 (Microsoft Corporation, Redmond, Wash.). Differences
were considered significant if the P value obtained by one-tailed analysis was
⬍0.05.
This study was approved by the University of Florida Medical Center Review
Board for the protection of human subjects.
RESULTS
The distributions of the streptococcal antibody titers ob-
tained for the nonrheumatic subjects and the patients from
Grenada and Florida are illustrated in Fig. 1 and 2, respec-
tively. No marked differences in the distributions of the three
antibodies are noted between the different groups. However, a
comparison of the geometric mean titers obtained for the non-
rheumatic subjects and the rheumatic fever patients from
Grenada and Florida revealed a number of significant differ-
ences.
For the Grenada subjects, the geometric means of the ASO
and anti-DNase B titers were higher in the nonrheumatic pop-
ulation (Table 1). The difference was not significant for the
ASO titers (P ⫽ 0.085), but it was significant for the anti-
DNase B titers (P ⫽ 0.029). In contrast to the higher ASO and
anti-DNase B titers in the nonrheumatic subjects, the mean
anti-A-Cho titer was significantly higher in the rheumatic fever
population than in the nonrheumatic controls (P ⫽ 0.047).
These results contrasted with those for the populations from
Florida where, in keeping with the findings of previous studies
(1, 8, 15), the mean titers of all three antibodies among the
rheumatic fever patients were significantly higher than those
for the nonrheumatic group (P ⫽ 0.01–⬍0.001).
In order to explain the unexpected findings for the strepto-
coccal antibody titers in the nonrheumatic subjects and the
rheumatic fever patients from Grenada, the antibody titers of
the nonrheumatic subjects from Grenada were compared with
those of the nonrheumatic individuals from Florida. As shown
in Table 2, the means of all three antibody titers were signifi-
cantly higher in the Grenada nonrheumatic group than in the
Florida nonrheumatic population (P ⫽ ⬍0.001). A similar
analysis of the geometric means of the antibody titers for the
rheumatic fever groups from Grenada and Florida (Table 1)
revealed no significant differences.
DISCUSSION
Previous studies that established serologic evidence for an
etiologic association between group A streptococcal infection
and rheumatic fever did so by demonstrating significant differ-
ences in the antibody levels of streptococcal antibodies be-
tween healthy controls and patients with rheumatic fever (5, 8,
9). The healthy controls were individuals from the same pop-
ulation or geographic area as the patients with rheumatic fever
but who had no clinical evidence of recent group A strepto-
coccal infection. Because the majority of these studies were
carried out in areas with a low incidence of rheumatic fever (6,
11), the present study was conducted to determine if the strep-
tococcal antibody titers of rheumatic fever patients and non-
rheumatic individuals in populations with higher incidences of
rheumatic fever demonstrate the same differences as those
888 AYOUB ET AL. CLIN. DIAGN. LAB. IMMUNOL.

FIG. 1. Distribution of the streptococcal antibody titers obtained for 30 nonrheumatic individuals and 32 patients with rheumatic fever in
Grenada.

seen in corresponding populations in areas of endemicity
where the prevalence of rheumatic fever is low.
The data obtained in this study confirmed prior observations
regarding the presence of significant differences in the levels of
streptococcal antibodies between healthy individuals and pa-
tients with rheumatic fever in Florida, an area with a low
prevalence of rheumatic fever (6). However, this difference did
not hold when we compared the streptococcal antibody titers
of nonrheumatic individuals to the titers of patients with rheu-
matic fever in Grenada, an area with a high incidence of

FIG. 2. Distribution of the streptococcal antibody titers obtained for 32 nonrheumatic subjects and 32 patients with rheumatic fever in
Florida.
VOL. 10, 2003 GROUP A STREPTOCOCCAL ANTIBODIES AND RHEUMATIC FEVER 889

TABLE 1. Geometric mean streptococcal antibody titers and patients without cardiac sequelae or with transient carditis.
significance of differences between the means for nonrheumatic This finding suggests that in patients with mitral valve disease
individuals and patients with rheumatic fever in
who do not have a history of acute rheumatic fever, the pres-
Grenada and Florida
ence of an elevated anti-A-Cho titer in the presence of normal
Geometric mean titer for indicated ASO and anti-DNase B titers would favor a rheumatic fever
Population source individuals P value
and antibody test etiology as the cause of the mitral valve disease. In areas such
Nonrheumatic With rheumatic fever
as Grenada, where streptococcal infections are highly preva-
Grenada lent, one should approach the interpretation of an elevated
ASO 2.3 2.16 0.085 anti-A-Cho titer with care and should utilize the finding of an
Anti-DNase B 2.68 2.5 0.029
Anti-A-Cho 3.06 3.17 0.047 elevated anti-A-Cho titer as evidence for a rheumatic fever
etiology in a patient with isolated mitral valve insufficiency and
Florida no history of a preceding episode of acute rheumatic fever but
ASO 1.96 2.18 0.01 only if the elevated anti-A-Cho titer is accompanied by normal
Anti-DNase B 2.29 2.57 0.015
Anti-A-Cho 2.71 3.07 ⬍0.001
ASO and/or anti-DNase B titers.
Other than a study done by Urdhal et al. (18), our review of
the published literature failed to reveal studies that evaluated
the levels of streptococcal antibodies in healthy individuals
rheumatic fever (13; P. K. Noah, R. Kopycinski, and B. Nelson, from populations with a high frequency of streptococcal infec-
unpublished data; B. Nelson and G. F. Armstrong, personal tions and/or a high incidence of rheumatic fever. Urdahl and
communications). The reason for this discrepancy became ap- his coworkers determined the serum antistreptokinase titers in
parent when the antibody titers of the nonrheumatic popula- the sera of nonrheumatic adult aboriginals in Australia, a pop-
tions in Grenada and Florida were compared. This revealed ulation with a very high incidence of streptococcal infection
that the streptococcal antibody titers in the nonrheumatic (18). They found that the geometric mean titer of this strep-
group from Grenada were significantly higher than the titers of tococcal antibody was almost 20 times higher than that in
the healthy individuals from Florida (Table 2). nonaboriginal adults. This study, together with data derived
The most likely explanation for this finding is that children in from the present study, indicate that the levels of the strepto-
Grenada are subjected to a high frequency of group A strep- coccal antibodies in healthy populations can vary substantially,
tococcal infections in the form of pharyngitis or impetigo. depending on the frequency of streptococcal infections in
As in other tropical regions, impetigo is reported to be those populations.
highly prevalent in Grenada (B. Nelson, personal communica- Susceptibility to rheumatic fever in certain individuals has
tion). While this may account for the high levels of anti-DNase been ascribed to a number of factors. These include genetic
B in the nonrheumatic population in Grenada, the presence of determinants, for example, HLAs, and the presence of certain
ASO titers in the Grenada nonrheumatic population that are markers such as the B-cell alloantigens (reviewed in reference
significantly higher than those of the healthy population in 2). One of the factors originally considered in rheumatic fever
Florida favors the probability that streptococcal pharyngitis susceptibility is an innate state of immune hyperresponsive-
also contributed to the elevated streptococcal antibodies en- ness, particularly to streptococcal antigens (12, 14, 16, 17).
countered in the nonrheumatic population from Grenada. This While the high frequency of streptococcal infection in a pop-
conclusion is based on previous observations that show that ulation such as the one in Grenada may promote the expres-
patients with impetigo most often show a brisk anti-DNase B sion of acute rheumatic fever, an alternate explanation to con-
response but a feeble ASO response (10). sider is that individuals in such an area develop or possess an
It is of interest that, despite the significantly lower anti- innate immune hyperresponsiveness to streptococcal antigens.
DNase B levels in Grenadian rheumatic fever patients than in Whether the high rate of expression of rheumatic fever in
the local controls, a finding that reflects more recent strepto- certain populations is related to a high frequency of strepto-
coccal infections in the latter group, the anti-A-Cho levels were coccal infections per se or to that in combination with an
significantly higher in the rheumatic fever population. This is innate state of hyperimmune responsiveness to streptococcal
consistent with earlier reports (1, 8, 15) which showed that the antigens remains to be determined.
anti-A-Cho levels remain elevated for several years in patients A number of variables should be taken into consideration
with persistent rheumatic mitral valve insufficiency but not in while interpreting the results of streptococcal antibody titers in
the context of their relationship to the epidemiology of group
A streptococcal infections or the role of such infections in the
TABLE 2. Geometric mean streptococcal antibody titers and
significance of differences, between the means for nonrheumatic
pathogenesis of rheumatic fever. The results of this study point
individuals from Grenada and nonrheumatic to yet another variable, the need to consider the background
individuals from Florida frequency of streptococcal infections in certain populations
when the streptococcal antibody titers of rheumatic fever and
Geometric mean titer for
nonrheumatic subjects from: nonrheumatic populations are being compared. Differences in
Antibody test P value
the distribution of the titers of antibodies between such pop-
Grenada Florida
ulations will become apparent when the titers in nonrheumatic
ASO 2.3 1.96 0.0005 subjects from an area with a high incidence of rheumatic fever
Anti-DNase B 2.68 2.29 0.0004 are compared to those of a similar population from an area
Anti-A-Cho 3.06 2.7 0.0002
with a low incidence of this disease. Had this comparison not
890 AYOUB ET AL.
been done in the present study, it is not inconceivable that one
might have concluded on the basis of the data obtained from
Grenada that group A streptococcal infection does not play a
role in the pathogenesis of rheumatic fever.
ACKNOWLEDGMENTS
The conduct of this study was made possible through the sponsor-
ship of the Children’s Health Organization Relief and Educational
Services (CHORES) and the assistance of the Grenada Ministry of
Health and the personnel at the St. George’s Hospital in Grenada.
We acknowledge the technical assistance of Elaine Harden in per-
forming the antibody assays.
REFERENCES
1. Appleton, R. S., B. E. Victorica, and E. M. Ayoub. 1985. Specificity of
persistence of antibody to the streptococcal group A carbohydrate in rheu-
matic valvular heart disease. J. Lab. Clin. Med. 105:114–119.
2. Ayoub, E. M., M. Kotb, and M. W. Cunningham. 2000. Rheumatic fever
pathogenesis, p. 102–132. In D. L. Stevens and E. L. Kaplan (ed.), Strepto-
coccal infections: clinical aspects, microbiology, and molecular pathogenesis.
Oxford University Press, Inc., New York, N.Y.
3. Ayoub, E. M. 1991. Immune response to group A streptococcal infections.
Pediatr. Infect. Dis. J. 10(Suppl.):S15–S19.
4. Ayoub, E. M., and E. Harden. 1997. Immune response to streptococcal
antigens: diagnostic methods, p. 450–457. In N. R. Rose, E. Conway de
Macario, J. D. Folds, H. C. Lane, and R. M. Nakamura (ed.), Manual of
clinical laboratory immunology, 5th ed. American Society for Microbiology,
Washington, D.C.
5. Ayoub, E. M., and L. W. Wannamaker. 1962. Evaluation of the streptococcal
deoxyribonuclease B and diphosphopyridine nucleotidase antibody tests in
acute rheumatic fever and acute glomerulonephritis. Pediatrics 29:527–538.
6. Children’s Medical Services, Department of Health, State of Florida. 1997.
Rheumatic fever prophylaxis program in Florida. Rheumatic Fever Registry
of the State of Florida, Tallahassee, Fla.
CLIN. DIAGN. LAB. IMMUNOL.
7. Dajani, A. S., E. M. Ayoub, F. Z. Bierman, A. L. Bisno, F. W. Denny, D. T.
Durack, P. Ferrieri, M. Freed, M. Gerber, E. L. Kaplan, A. W. Karchmer, M.
Markowitz, S. H. Rahimtoola, S. T. Shulman, G. Stollerman, T. Masato, A.
Taranta, K. A. Taubert, and W. Wilson. 1992. Guidelines for the diagnosis of
rheumatic fever: Jones Criteria, updated 1992. JAMA 87:302–307.
8. Dudding, B. A., and E. M. Ayoub. 1968. Persistence of streptococcal group A
antibody in patients with rheumatic valvular disease. J. Exp. Med. 128:1081–
1098.
9. Kaplan, E. L., C. D. Rothermel, and D. R. Johnson. 1998. Antistreptolysin O
and anti-deoxyribonuclease B titers: normal values for children ages 2 to 12
in the United States. Pediatrics 101:86–88.
10. Kaplan, E. L., B. F. Anthony, S. S. Chapman, E. M. Ayoub, and L. W.
Wannamaker. 1970. The influence of the site of infection on the immune
response to group A streptococci. J. Clin. Invest. 49:1405–1414.
11. Markowitz, M. 1985. The decline of rheumatic fever: role of medical inter-
vention. The Lewis W. Wannamaker Memorial Lecture. J. Pediatr. 106:545–
550.
12. Meiselas, L. E., S. B. Zinglae, S. L. Lee, et al. 1961. Antibody production in
rheumatic diseases: the effect of brucella antigen. J. Clin. Investig. 40:1872–
1881.
13. Ministry of Health of Grenada. 1998. Data on the incidence of rheumatic
fever in Grenada. Rheumatic Fever Register. Ministry of Health, St.
George’s, Grenada.
14. Rejholec, V. 1957. Incidence of rheumatic fever in relation to immunologic
reactivity. Ann. Rheum. Dis. 16:23–30.
15. Shulman, S. T., E. M. Ayoub, and B. E. Victorica. 1974. Differences in
antibody response to streptococcal antigens in children with rheumatic and
non-rheumatic mitral valve disease. Circulation 50:1244–1251.
16. Stollerman, G. H. 1972. Hypersensitivity and antibody responses in strepto-
coccal disease, p. 501–513. In L. M. Wannamaker and J. M. Matsen (ed.),
Streptococci and streptococcal diseases: recognition, understanding, and
management. Academic Press, New York, N.Y.
17. Swift, H. F. 1929. Rheumatic fever. JAMA 92:2071–2083.
18. Urdahl, K. B., J. D. Matthews, and B. Currie. 2002. Anti-streptokinase
antibodies and streptokinase resistance in an Aboriginal population in north-
ern Australia. Aust. N. Z. J. Med. 26:49–53.
19. Wannamaker, L. W., and E. M. Ayoub. 1960. Antibody titers in acute rheu-
matic fever. Circulation 21:598–614.