Japan. J. Microbiol. Vol. 19(3), 181-185, 1975
Seven-Year Ryukyu Rubella
Follow-Up with
Special
Study
of Rubella
Reference
Hemagglutination
Syndrome
to Persistence
Inhibition
in of
Antibodies
Kohji UEDA,Yukiaki NISHIDA,Kenji OSHIMA,Hiromi YOSHIKAWA, Komei OHASHI, and Saneo NONAKA Kyushu UniversitySchoolof Health Sciences,Fukuoka, Hamanomachi Hospital, Division of Otorhinolaryngology,Fukuoka, Departmentof Ophthalmology,Fukuoka UniversityMedical School,Fukuoka, and Chemo-SeroTherapeutic Research Institute, Kumamoto (Received for publication October 25, 1974)
ABSTRACT
Rubella hemagglutination inhibition (HI) antibodies in 266 children with rubella syndrome born in 1965 in the Ryukyu Islands and their mothers were followed for seven years. Titers of rubella HI antibody in the mothers declined slowly, while those in the children declined rapidly up to 40 months of age. Thereafter decline of titers became extremely slow and only seven cases (three per cent) became seronegative for rubella HI antibody. Rubella HI antibody titers seemed to have no particular correla tion to the severity of clinical manifestations.
It has been known that rubella hemag glutination inhibition (HI) antibody fol lowing congenital rubella infection declines more rapidly than that following postnatal infection [1-3, 5]. However, persistence of antibody following congenital infection has been a target for much controversy [2]. After a rubella epidemic occurring in the Ryukyu Islands in 1965 more than 450 children with rubella syndrome were born [8]. Four hundred and eight cases have so far been registered by the Congenital Rubella Study Group of Kyushu University. This paper presents a follow-up study on rubella HI antibody of 266 cases aged from 6 months to 7 years and of 257 mothers thereof. MATERIALSAND METHODS Studypopulation. Two hundred and sixty six cases with rubella syndrome born in the Ryukyu Islands in 1965 and their 257 Requests for reprints should be addressed to Dr. Kohji Ueda, Kyushu University School of Health Sciences, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812, Japan, 181
mothers were studied. "The cases were classified on the basis of clinical manifesta tions into the following four groups, i. e., cataract group (cataract with congenital heart disease, deafness and/or retinopathy), congenital heart disease (CHD) group (CHD with deafness and/or retinopathy), deafness-1 group (deafness with retinopathy) and deaf ness-2 group (deafness without other clinical manifestations). The diagnosis depended on pediatric examinations, audiometry and oph thalmologic examinations [8]. Each case was examined two to seven times. The mothers of 233 children among a total of 266 cases (88%) had a history of clinical rubella in the first or second trimester of pregnancy. Serum specimens. Serum specimens were obtained four times, i. e., in 1966, 1967, 1968 and 1973. The total number of serum specimens obtained were 449 from the patients and 386 from their mothers. Three or more serum specimens were obtained from 31 cases (12%), two from 116 (44%), and one from 119 (45%). Four hundred and twenty normal children born after 1962
182
K.
Table
a) Age of children with rubella b) Congenital heart disease .
1.
UEDA
Summary
syndrome
ET AL
of specimens
at the
Fig. 1. Rubella HI antibody with rubella syndrome.
in the district served as control. The sera were separated with aseptic care and stored at -20 C until being used (Table 1). Rubella HI test. The antigen used for hemagglutination was prepared in mono layer cultures of BHK-21 cells infected with M-33 strain of rubella virus [7]. Rubella HI antibody was estimated by a microtiter technique according to the method described by the Central Virus Diagnostic Laboratory of the National Institute of Health, Japan [4]. Two-tenths milliliters of a serum specimen was mixed with 0.6 ml of phos phate-buffered saline solution (PBS) and 0.8 ml of a 25% suspension of kaolin made in PBS. Absorption was allowed to proceed
time
collected
of the examination
titers
in 266
.
children
for 20 min at room temperature with frequent shakings. This mixture was centrifuged and the supernatant was removed, to which was added 0.05 ml of a 50% suspension of goose blood cells. Absorption was allowed to proceed for 1 hr at 4 C. The cells were separated by centrifugation. The super natant was considered to represent a 1 : 8 dilution of the original serum. The HI test was performed in a microtiter U plate. Serial twofold dilutions were prepared with a diluent (Veronal-buffered saline solution containing 0.1 % bovine serum albumin and 0.005% gelatin). A 0.025-ml amount of the antigen representing 4 units was added to 0.025 ml of each serum dilution. After
RUBELLA
HI ANTIBODIES
Fig. 2. Rubella HI antibody titers of children with rubella syndrome.
Table 2.
a)
in 257
IN RUBELLA
mothers
183
SYNDROME
Fig. 3. Rubella in 420 normal
HI antibody titers children in 1973.
Relationship between HI antibody titers and clinical manifestations at 7 years (153 cases)
Congenital
b) Geometric
heart
disease
mean
titer .
.
Table 3.
a) Congenital b) Ventricular
Seronegative children (7 cases)
heart disease . septal defect .
incubation at room temperature for 1 hr, 0.05 ml of a 0.2% goose blood cell suspension was added. Reading was done after incuba tion at 4 C for 1 hr followed by additional 30
min standing at room temperature. The titer was expressed by the highest dilution which showed complete inhibition of specific hemagglutination.
184
K. UEDA
RESULTS Rubella
HI Rubella
Antibody
in
Syndrome
and
Persistence
of
children Fig.
6
at
to
rubella
syndrome HI
antibody
from
1 : 32
ranged months 24
months,
and
three
antibody
of
(•ƒ1
: 8) ranged age,
and from
mean
titers
months, and
7
years
is shown
in
titers to
nagative
30
HI
the
titers
in
to
1 : 512.
in
40 for
titers to 1 were
in : 512. sero cases
Geometric aged
30
28.5,
to
positive
those
months,
were
1 : 512
were the
of
: 2048
to
from 1 : 8 four children
24
1
At
1 :8
to
266
1 : 16
and
(GMT)
18
in
age.
children
positive cases At 7 years of negative
from
months
with
antibody
rubella
to
ranged
HI
rubella
12
18
Children Mothers
The
children
at
of
Their
with 1.
the
Sera
6
to
27.0,
40
26.3
to
The
rubella
HI
children
illustrated
in
whose
28.2,
As
to 28.2,
26.1,
in
aged
less
rapid
27.8
and
than
40 in
of
extremely among
a
total
with
, 18 years
7
of
syndrome
seemed
those
to
be
7 years,
of
266
cases
more
Thereafter
children
to
24 old
antibody
rubella
up
to
respectively.
mothers.
in
are mothers
months and
months
titers slow
12
27.0,
their
mothers
of
decline
children
than
decline
6 to months
above,
the
of 257 syndrome
GMT
40
mentioned
titers
sera
2.
were
30
in rubella
Fig.
children
months, were
titers
with
only
became seven
(3%)
eventually
being
seronegative.
Relationship
between
Manifestations Relationship clinical body
in at
the
2.
No
antibody
four
severity
rubella
patients
age
of
7
distinct
with years
of
HI
anti rubella
is shown
difference
was
Clinical Persistence
in
recognized
in
rubella
among
the
groups.
Clinical
manifestations cases
those
seven,
defect
with
are
with
Children
a
of
seven
had
ventricular
Table
another the
history
HI
the in
deafness, and
Distribution
of
presented
one
retinopathy, deafness
the
titer
clinical
negative
and
the
of
Antibody
between
titers
Table
Severity
HI
manifestations
syndrome
HI
the and
sero 3.
deafness
with
five
of
rubella.
Antibody
maternal
in
Sera
Of
septal
remaining
of
younger than 7 years, corresponding to the cases with rubella syndrome, were sero negative. Two out of 46 children aged 7 years had rubella HI antibody, the titers being 1 : 64. It was not known whether these two cases represented congenital or postnatal infection. The incidence of rubella HI antibody among 122 children older than the above rubella syndrome patients was 49 %, the GMT of the seropositive cases being 27.2, and the titers were higher than in the rubella syndrome patients and similar to those in mothers of the patients. DISCUSSION
respectively.
of
Age distribution of rubella HI antibody among 420 normal children in 1973 is shown in Fig. 3. All of 252 normal children
12
months
and
ET AL
only
Normal
Cooper et al [1] examined rubella HI antibody in 270 cases with congenital rubella and their mothers and described that the decline of rubella HI antibody in the cases was more rapid than in mothers, 18.5% of 270 cases became seronegative after 4 years of age. Presence of cases negative for rubella-neutralizing antibody among those with rubella syndrome was reported by Plotkin et al [6] and Weller et al [9] in 1963 and 1964, respectively. Kenrick et al [5] reported that six out of 50 adults with rubella syndrome had no detectable antibody. Hardy et al [3] also noted that rubella HI antibody titers in 50% of 60 cases with congenital rubella declined until a half of those turned to negative by 2 to 4 years. Dudgeon et al [2] reported that, out of 99 cases aged 4 to 16 years with congenital rubella, 4.5% of 44 cases with multiple defects, 5.3% of 38 cases with single defect and 17.6% of 17 cases without defect were negative for rubella HI antibody. In our present study in the Ryukyu Islands, rubella HI antibody titers in the mothers of the children with rubella syndrome declined slowly and the GMT was higher than that of the infected children older than 18 months. The rubella HI antibody titers in children up to 40 months of age showed a rapid decline. However, as far as pre sently studied, the subsequent decline of titers up to 7 years was markedly slow. Only 3% of the 266 children with rubella syndrome were seronegative. Incidence of seronegative
RUBELLA
HI ANTIBODIES
conversion among the children with rubella syndrome in the present investigation was lower than in other reported studies. The possibility of repeated exposure to rubella virus resulting in sustaining the antibody titers was excluded by the control study, in which none of 252 normal children under 6 years had rubella HI antibody. Cooper et al [1] and Dudgeon et al [2] reported that there was no correlation be tween the severity of rubella syndrome and the decline in rubella HI antibody level. Our data so far obtained also indicated that there was no marked difference between rubella HI antibody levels and the severity of clinical manifestations in the patients. Fur ther follow-up studies are required to deter mine the persistence of antibody levels following congenital rubella infection in the Ryukyu Islands. REFERENCES
[ 1]
Cooper, L. Z., Florman, A. L., Ziring, P. R., and Krugman, S. 1971. Loss of rubella hemagglutina tion inhibition antibody in congenital rubella. Amer. J. Dis. Child. 122 : 397-403. [ 2 ] Dudgeon, J.A., Marshall, W. C., and Peckham,
IN RUBELLA
SYNDROME
185
C. S. 1972. Humoral immune responses in con genital rubella. Lancet ii : 480-481. [ 3 ] Hardy, J. B., Sever, J. L., and Gilkeson, M. R. 1969. Declining antibody titers in children with congenital rubella. J. Pediat. 75 : 213-220. [ 4 ] Japan Rubella Vaccine Research Commis sion. March 1971. A study for development of rubella vaccine (I). (in Japanese) [ 5 ] Kenrick, K. G., Slinn, R. F., Dorman, D. C., and Menser, M. A. 1968. Immunoglobulins and rubella-virus antibodies in adults with congenital rubella. Lancet i : 548-551. [ 6 ] Plotkin, S.A., Dudgeon, J. A., and Ramsey, A. M. 1963. Laboratory studies on rubella and the rubella syndrome. British Med. J. 2 : 1296-1299. [ 7 ] Stewart, G.L., Parkman, P. D., Hopps, H. E., Douglas, R. D., Hamilton, J. P., and Meyer, H. M., Jr. 1967. Rubella-virus hemagglutination inhibi tion test. New England J. Med. 276 : 544-557 . [ 8 ] Ueda, K., Nishida, Y., Kano, M., Oshima, K., Takabayashi, K., Kato, H., Nagafuchi, M., Takesue, M., Yoshizawa, Y., and Nagayama, T. 1972. Clinical studies of patients with rubella syndrome occurring in a high incidence in the Ryukyu Islands in 1965 : On the diagnostic sig nificance of clinical manifestations. Acta Pediat . Japan. (oversea eddition) 14: 9-16. [ 9 ] Weller, T. H., Alford, C. A., and Neva, F. A. 1964. Retrospective diagnosis by serologic means of congenitally acquired rubella infection . New England J. Med. 270 : 1039-1041.
Seven-Year Ryukyu Rubella
Follow-Up with
Special
Study
of Rubella
Reference
Hemagglutination
Syndrome
to Persistence
Inhibition
in of
Antibodies
Kohji UEDA,Yukiaki NISHIDA,Kenji OSHIMA,Hiromi YOSHIKAWA, Komei OHASHI, and Saneo NONAKA Kyushu UniversitySchoolof Health Sciences,Fukuoka, Hamanomachi Hospital, Division of Otorhinolaryngology,Fukuoka, Departmentof Ophthalmology,Fukuoka UniversityMedical School,Fukuoka, and Chemo-SeroTherapeutic Research Institute, Kumamoto (Received for publication October 25, 1974)
ABSTRACT
Rubella hemagglutination inhibition (HI) antibodies in 266 children with rubella syndrome born in 1965 in the Ryukyu Islands and their mothers were followed for seven years. Titers of rubella HI antibody in the mothers declined slowly, while those in the children declined rapidly up to 40 months of age. Thereafter decline of titers became extremely slow and only seven cases (three per cent) became seronegative for rubella HI antibody. Rubella HI antibody titers seemed to have no particular correla tion to the severity of clinical manifestations.
It has been known that rubella hemag glutination inhibition (HI) antibody fol lowing congenital rubella infection declines more rapidly than that following postnatal infection [1-3, 5]. However, persistence of antibody following congenital infection has been a target for much controversy [2]. After a rubella epidemic occurring in the Ryukyu Islands in 1965 more than 450 children with rubella syndrome were born [8]. Four hundred and eight cases have so far been registered by the Congenital Rubella Study Group of Kyushu University. This paper presents a follow-up study on rubella HI antibody of 266 cases aged from 6 months to 7 years and of 257 mothers thereof. MATERIALSAND METHODS Studypopulation. Two hundred and sixty six cases with rubella syndrome born in the Ryukyu Islands in 1965 and their 257 Requests for reprints should be addressed to Dr. Kohji Ueda, Kyushu University School of Health Sciences, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812, Japan, 181
mothers were studied. "The cases were classified on the basis of clinical manifesta tions into the following four groups, i. e., cataract group (cataract with congenital heart disease, deafness and/or retinopathy), congenital heart disease (CHD) group (CHD with deafness and/or retinopathy), deafness-1 group (deafness with retinopathy) and deaf ness-2 group (deafness without other clinical manifestations). The diagnosis depended on pediatric examinations, audiometry and oph thalmologic examinations [8]. Each case was examined two to seven times. The mothers of 233 children among a total of 266 cases (88%) had a history of clinical rubella in the first or second trimester of pregnancy. Serum specimens. Serum specimens were obtained four times, i. e., in 1966, 1967, 1968 and 1973. The total number of serum specimens obtained were 449 from the patients and 386 from their mothers. Three or more serum specimens were obtained from 31 cases (12%), two from 116 (44%), and one from 119 (45%). Four hundred and twenty normal children born after 1962
182
K.
Table
a) Age of children with rubella b) Congenital heart disease .
1.
UEDA
Summary
syndrome
ET AL
of specimens
at the
Fig. 1. Rubella HI antibody with rubella syndrome.
in the district served as control. The sera were separated with aseptic care and stored at -20 C until being used (Table 1). Rubella HI test. The antigen used for hemagglutination was prepared in mono layer cultures of BHK-21 cells infected with M-33 strain of rubella virus [7]. Rubella HI antibody was estimated by a microtiter technique according to the method described by the Central Virus Diagnostic Laboratory of the National Institute of Health, Japan [4]. Two-tenths milliliters of a serum specimen was mixed with 0.6 ml of phos phate-buffered saline solution (PBS) and 0.8 ml of a 25% suspension of kaolin made in PBS. Absorption was allowed to proceed
time
collected
of the examination
titers
in 266
.
children
for 20 min at room temperature with frequent shakings. This mixture was centrifuged and the supernatant was removed, to which was added 0.05 ml of a 50% suspension of goose blood cells. Absorption was allowed to proceed for 1 hr at 4 C. The cells were separated by centrifugation. The super natant was considered to represent a 1 : 8 dilution of the original serum. The HI test was performed in a microtiter U plate. Serial twofold dilutions were prepared with a diluent (Veronal-buffered saline solution containing 0.1 % bovine serum albumin and 0.005% gelatin). A 0.025-ml amount of the antigen representing 4 units was added to 0.025 ml of each serum dilution. After
RUBELLA
HI ANTIBODIES
Fig. 2. Rubella HI antibody titers of children with rubella syndrome.
Table 2.
a)
in 257
IN RUBELLA
mothers
183
SYNDROME
Fig. 3. Rubella in 420 normal
HI antibody titers children in 1973.
Relationship between HI antibody titers and clinical manifestations at 7 years (153 cases)
Congenital
b) Geometric
heart
disease
mean
titer .
.
Table 3.
a) Congenital b) Ventricular
Seronegative children (7 cases)
heart disease . septal defect .
incubation at room temperature for 1 hr, 0.05 ml of a 0.2% goose blood cell suspension was added. Reading was done after incuba tion at 4 C for 1 hr followed by additional 30
min standing at room temperature. The titer was expressed by the highest dilution which showed complete inhibition of specific hemagglutination.
184
K. UEDA
RESULTS Rubella
HI Rubella
Antibody
in
Syndrome
and
Persistence
of
children Fig.
6
at
to
rubella
syndrome HI
antibody
from
1 : 32
ranged months 24
months,
and
three
antibody
of
(•ƒ1
: 8) ranged age,
and from
mean
titers
months, and
7
years
is shown
in
titers to
nagative
30
HI
the
titers
in
to
1 : 512.
in
40 for
titers to 1 were
in : 512. sero cases
Geometric aged
30
28.5,
to
positive
those
months,
were
1 : 512
were the
of
: 2048
to
from 1 : 8 four children
24
1
At
1 :8
to
266
1 : 16
and
(GMT)
18
in
age.
children
positive cases At 7 years of negative
from
months
with
antibody
rubella
to
ranged
HI
rubella
12
18
Children Mothers
The
children
at
of
Their
with 1.
the
Sera
6
to
27.0,
40
26.3
to
The
rubella
HI
children
illustrated
in
whose
28.2,
As
to 28.2,
26.1,
in
aged
less
rapid
27.8
and
than
40 in
of
extremely among
a
total
with
, 18 years
7
of
syndrome
seemed
those
to
be
7 years,
of
266
cases
more
Thereafter
children
to
24 old
antibody
rubella
up
to
respectively.
mothers.
in
are mothers
months and
months
titers slow
12
27.0,
their
mothers
of
decline
children
than
decline
6 to months
above,
the
of 257 syndrome
GMT
40
mentioned
titers
sera
2.
were
30
in rubella
Fig.
children
months, were
titers
with
only
became seven
(3%)
eventually
being
seronegative.
Relationship
between
Manifestations Relationship clinical body
in at
the
2.
No
antibody
four
severity
rubella
patients
age
of
7
distinct
with years
of
HI
anti rubella
is shown
difference
was
Clinical Persistence
in
recognized
in
rubella
among
the
groups.
Clinical
manifestations cases
those
seven,
defect
with
are
with
Children
a
of
seven
had
ventricular
Table
another the
history
HI
the in
deafness, and
Distribution
of
presented
one
retinopathy, deafness
the
titer
clinical
negative
and
the
of
Antibody
between
titers
Table
Severity
HI
manifestations
syndrome
HI
the and
sero 3.
deafness
with
five
of
rubella.
Antibody
maternal
in
Sera
Of
septal
remaining
of
younger than 7 years, corresponding to the cases with rubella syndrome, were sero negative. Two out of 46 children aged 7 years had rubella HI antibody, the titers being 1 : 64. It was not known whether these two cases represented congenital or postnatal infection. The incidence of rubella HI antibody among 122 children older than the above rubella syndrome patients was 49 %, the GMT of the seropositive cases being 27.2, and the titers were higher than in the rubella syndrome patients and similar to those in mothers of the patients. DISCUSSION
respectively.
of
Age distribution of rubella HI antibody among 420 normal children in 1973 is shown in Fig. 3. All of 252 normal children
12
months
and
ET AL
only
Normal
Cooper et al [1] examined rubella HI antibody in 270 cases with congenital rubella and their mothers and described that the decline of rubella HI antibody in the cases was more rapid than in mothers, 18.5% of 270 cases became seronegative after 4 years of age. Presence of cases negative for rubella-neutralizing antibody among those with rubella syndrome was reported by Plotkin et al [6] and Weller et al [9] in 1963 and 1964, respectively. Kenrick et al [5] reported that six out of 50 adults with rubella syndrome had no detectable antibody. Hardy et al [3] also noted that rubella HI antibody titers in 50% of 60 cases with congenital rubella declined until a half of those turned to negative by 2 to 4 years. Dudgeon et al [2] reported that, out of 99 cases aged 4 to 16 years with congenital rubella, 4.5% of 44 cases with multiple defects, 5.3% of 38 cases with single defect and 17.6% of 17 cases without defect were negative for rubella HI antibody. In our present study in the Ryukyu Islands, rubella HI antibody titers in the mothers of the children with rubella syndrome declined slowly and the GMT was higher than that of the infected children older than 18 months. The rubella HI antibody titers in children up to 40 months of age showed a rapid decline. However, as far as pre sently studied, the subsequent decline of titers up to 7 years was markedly slow. Only 3% of the 266 children with rubella syndrome were seronegative. Incidence of seronegative
RUBELLA
HI ANTIBODIES
conversion among the children with rubella syndrome in the present investigation was lower than in other reported studies. The possibility of repeated exposure to rubella virus resulting in sustaining the antibody titers was excluded by the control study, in which none of 252 normal children under 6 years had rubella HI antibody. Cooper et al [1] and Dudgeon et al [2] reported that there was no correlation be tween the severity of rubella syndrome and the decline in rubella HI antibody level. Our data so far obtained also indicated that there was no marked difference between rubella HI antibody levels and the severity of clinical manifestations in the patients. Fur ther follow-up studies are required to deter mine the persistence of antibody levels following congenital rubella infection in the Ryukyu Islands. REFERENCES
[ 1]
Cooper, L. Z., Florman, A. L., Ziring, P. R., and Krugman, S. 1971. Loss of rubella hemagglutina tion inhibition antibody in congenital rubella. Amer. J. Dis. Child. 122 : 397-403. [ 2 ] Dudgeon, J.A., Marshall, W. C., and Peckham,
IN RUBELLA
SYNDROME
185
C. S. 1972. Humoral immune responses in con genital rubella. Lancet ii : 480-481. [ 3 ] Hardy, J. B., Sever, J. L., and Gilkeson, M. R. 1969. Declining antibody titers in children with congenital rubella. J. Pediat. 75 : 213-220. [ 4 ] Japan Rubella Vaccine Research Commis sion. March 1971. A study for development of rubella vaccine (I). (in Japanese) [ 5 ] Kenrick, K. G., Slinn, R. F., Dorman, D. C., and Menser, M. A. 1968. Immunoglobulins and rubella-virus antibodies in adults with congenital rubella. Lancet i : 548-551. [ 6 ] Plotkin, S.A., Dudgeon, J. A., and Ramsey, A. M. 1963. Laboratory studies on rubella and the rubella syndrome. British Med. J. 2 : 1296-1299. [ 7 ] Stewart, G.L., Parkman, P. D., Hopps, H. E., Douglas, R. D., Hamilton, J. P., and Meyer, H. M., Jr. 1967. Rubella-virus hemagglutination inhibi tion test. New England J. Med. 276 : 544-557 . [ 8 ] Ueda, K., Nishida, Y., Kano, M., Oshima, K., Takabayashi, K., Kato, H., Nagafuchi, M., Takesue, M., Yoshizawa, Y., and Nagayama, T. 1972. Clinical studies of patients with rubella syndrome occurring in a high incidence in the Ryukyu Islands in 1965 : On the diagnostic sig nificance of clinical manifestations. Acta Pediat . Japan. (oversea eddition) 14: 9-16. [ 9 ] Weller, T. H., Alford, C. A., and Neva, F. A. 1964. Retrospective diagnosis by serologic means of congenitally acquired rubella infection . New England J. Med. 270 : 1039-1041.
