JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1990, p. 413-415 Vol. 28, No.

3
0095-1137/90/030413-03$02.00/0
Copyright C 1990, American Society for Microbiology

Evaluation of a New Latex Agglutination Test for Detection of

Streptolysin O Antibodies
MICHAEL A. GERBER,* LOLITA S. CAPARAS, AND MARTIN F. RANDOLPH
Department of Pediatrics, University of Connecticut Health Center, Farmington, Connecticut 06032
Received 25 August 1989/Accepted 28 November 1989

Acute- and convalescent-phase serum specimens were collected from 50 patients with group A streptococcal
pharyngitis. The anti-streptolysin O (ASO) titer for each serum specimen was determined by using both the

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standard neutralization assay and the latex agglutination (LA) test (Rheumagen ASO; Biokit Inc., New Britain,
Conn.). When the ASO titers derived by the two methods were compared, the correlation coefficient was 0.93.
When the ability of the LA test to demonstrate a significant ASO titer rise (.2 dilutions) was compared with
that of the standard neutralization assay, the LA test had a sensitivity of 91%, a specificity of 86%, a positive
predictive value of 83%, and a negative predictive value of 92%. Triplicate LA test determinations were
performed on a subset of 31 serum specimens, and for 29 (94%), the repeated ASO titers were all within 1
dilution of each other; the width of the 95% confidence interval for the triplicate measurements of each serum
specimen was ±32.8 IU. We found the Rheumagen ASO to be a simple, rapid LA procedure for measuring
ASO titers that produces results that are highly reproducible, show little lot-to-lot variability, and are
comparable to the ASO titers obtained with the standard neutralization assay.

The measurement of antibodies to streptococcal extracel-
lular antigens has been used by clinicians to help support the
diagnosis of one of the nonsuppurative complications of
group A beta-hemolytic streptococcal (GABHS) infections:
acute rheumatic fever or acute poststreptococcal glomerulo-
nephritis. Measurement of these antibodies has also been
used by investigators studying the epidemiology of GABHS
infections, as well as the epidemiology and pathogenesis of
their nonsuppurative sequelae. Since the original description
of the anti-streptolysin O (ASO) procedure by Todd in 1932
(18), this test has been the most widely used for determina-
tion of antibodies to streptococcal extracellular antigens. In
many clinical laboratories, it is the only streptococcal anti-
body test available. However, the neutralization assay that
is most often used to determine ASO titers is a relatively
complicated procedure requiring a well-equipped laboratory.
The neutralization assay also requires the use of rabbit
erythrocytes that are unstable on storage.
The purpose of this investigation was to compare a new,
simple latex agglutination (LA) method for measuring ASO
titers with the standard neutralization procedure.
MATERIALS AND METHODS
During the winter and spring of 1984 and 1985, children
seen in a private pediatric office (M.F.R.) with clinical
findings suggestive of GABHS pharyngitis were enrolled in
an investigation of this disease after informed written con-
sent had been obtained. The details of the study protocol are
presented elsewhere (5). Briefly, individuals had throat
cultures performed, and serum specimens were obtained at
the first visit and again at a follow-up visit approximately 4
weeks later. All patients with a positive throat culture for
GABHS were treated with an appropriate course of antibi-
otics. Sera were stored at -70°C, and both acute- and
convalescent-phase serum specimens from each subject
were tested simultaneously for ASO antibodies by using the
microdilution procedure for the neutralization assay (4) with
*
Corresponding author.
ASO reference control serum and streptolysin O reagent
obtained from Difco Laboratories (Detroit, Mich.). Titers
were reported in Todd units.
The ASO titer for each serum specimen was also deter-
mined by using a new LA test (Rheumagen ASO; Biokit
Inc., New Britain, Conn.). In place of the dilution scheme
suggested by the manufacturer (1:200, 1:400, 1:800, etc.), the
following dilutions of serum with normal saline were made in
an attempt to approximate the neutralization ASO dilution
scheme: 1:50, 1:100, 1:150, 1:200, etc. A drop (0.050 ml) of
each dilution was placed in one section of a disposable slide.
A drop of the latex reagent was placed next to the drop of
diluted serum, and the drops were then mixed. The slide was
gently rotated manually for 3 min, and the presence or
absence of agglutination was then determined. The acute-
and convalescent-phase serum specimens from each patient
were tested simultaneously, along with appropriate positive
and negative controls. Titers were reported in international
units. One international unit equals 1.04 Todd units (16). A
significant rise in antibody titer for either ASO assay was
defined as a rise of 2 dilution increments or more from acute-
to convalescent-phase serum specimens. The dilution
scheme for the neutralization ASO is constructed so that a
significant rise (2 dilutions or greater) in antibody titer
represents exactly a .0.2-log rise, while for the LA test, by
using the dilution scheme derived for this study, a significant
rise (2 dilutions or greater) in antibody titer represents
approximately a .0.2-log rise.
For a subset of 31 serum specimens, the LA test was
performed on three different occasions and the ASO titers
derived were compared as a measure of the reproducibility
of the assay. For another subset of 25 serum specimens,
each specimen was simultaneously tested with three dif-
ferent lots of Rheumagen reagent (1-4588, H-3689, and
C-3089) and the ASO titers derived were compared as a
measure of lot-to-lot variability.
The data were analyzed by using Student's paired t test,
linear regression analysis, and Hoyt's method (7), which
uses an analysis of variance approach to assess reliability.

413
414 GERBER ET AL.
1000
' 800
É 600 e e
ab.
s (ASO) can be used to confirm the diagnosis of a GABHS
400
z
200
t.*
s eeX
,s. . .
ea
0 patients with various streptococcal infections by neutralizing
0 200 400 600 800 1000
Rh.umagen LA Test (NU)
FIG. 1. Correlation between ASO titers obtained with Rheuma-
gen LA test and standard neutralization assay.
RESULTS
Acute- and convalescent-phase serum specimens were
available from 50 individuals (mean age, 9.9 years; range, 2
to 20 years), all of whom had acute pharyngitis and GABHS
isolated from their upper respiratory tracts. When the ASO
titers of these 100 serum specimens as determined by the LA
test were compared with the corresponding ASO titers as
determined by the neutralization assay, the correlation co-
efficient was 0.93 (Fig. 1) and Student's paired t test showed
no significant difference (P > 0.05). For only 11 of the 100
(11%) serum specimens was there more than a 1 dilution
discrepancy between the ASO titer as determined by the LA
test and the ASO titer as determined by the neutralization
assay.
By using the titers as determined by the neutralization
assay as the "gold standard," the sensitivity, specificity, and
predictive values of the LA test in demonstrating a signifi-
cant rise in ASO titers were determined (Table 1). The LA
test had a sensitivity of 91%, a specificity of 86%, a positive
predictive value of 83%, and a negative predictive value of
92%. Of the four false-positive LA results, three did have a
rise in neutralization assay titers but of only 1 dilution in
magnitude. In addition, both of the patients with false-
negative LA results did have a rise in LA titers but of only 1
dilution in magnitude.
For 29 of the 31 (94%) serum specimens that were run in
triplicate by using the LA test, the ASO titers were all within
1 dilution (50 IU) of each other. In addition, for these
triplicate measurements of each serum specimen, Hoyt's r =
0.98 and the width of the 95% confidence interval was ±32.8
IU, which is less than 1 dilution. For 21 of the 25 (84%)
serum specimens that were tested simultaneously with three
different lots ofRheumagen reagent, the ASO titers obtained
were identical. For 4 of the 25 (16%) serum specimens, the
ASO titer obtained with one lot (not always the same lot)
was 1 dilution higher than the titer obtained with the other
two lots.
TABLE 1. Significant rise in ASO titer
Rise in No. with rise by standard
Rheumagen neutralization assay
LA test Yes
Yes 20
No 2 24
J. CLIN. MICROBIOL.
DISCUSSION
Streptolysin O (SO) is an oxygen-labile hemolysin that is
one of a variety of extracellular products elaborated by
group A streptococci. SO elicits an antibody response in the
host during a GABHS infection. This antibody response
infection or the diagnosis of one of its nonsuppurative
sequelae.
In the initial report of the ASO procedure, Todd (18)
demonstrated the presence of ASO antibodies in the sera of
SO with serial amounts of sera of the patients. The excess,
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unneutralized SO was then revealed by adding erythrocytes
to the system as an indicator. The endpoint was the highest
dilution of serum having no hemolysis, with the ASO titer
expressed in Todd units, which are equivalent to the recip-
rocal of the dilution. This technique was subsequently mod-
ified by Rantz and Randall (12), and later a microtitration
procedure developed by Edwards (4) was introduced.
Approximately 80% of patients who have had a group A
streptococcal infection have a demonstrable antibody re-
sponse to a single streptococcal extracellular antigen. If one
looks for antibody responses to multiple streptococcal extra-
cellular antigens, the percentage approaches 95% (1). How-
ever, most laboratories are unable to perform multiple
streptococcal antibody assays and rely solely on the ASO
determination. Unfortunately, the standard neutralization
assay used to determine ASO titers is a relatively expensive,
time-consuming, and complicated procedure requiring a
well-equipped laboratory. In fact, the American Heart As-
sociation's revised Jones criteria of 1965 for the diagnosis of
acute rheumatic fever (17), which required evidence of a
prior streptococcal infection, were not initially accepted by
the WHO Expert Committee on Prevention of Rheumatic
Fever because there were often insufficient laboratory re-
sources in developing countries to provide the necessary
serologic tests (21).
Over the years a number of attempts have been made to
develop simplified, rapid tests for measuring either multiple
or single streptococcal antibody titers. The Streptozyme test
(Wampole Laboratories, Cranbury, N.J.) is a hemagglutina-
tion procedure that is said to detect antibodies to five
different streptococcal extracellular enzymes. However, a
number of studies have demonstrated problems with the use
of the Streptozyme test (6, 10, 11). The WHO has concluded
that this test is insufficiently reliable for assaying streptococ-
cal antibodies and recommends that it no longer be used (20).
In 1978, Ricci and co-workers (15) introduced a simplified
neutralization assay for measuring ASO antibodies in whole
blood rather than serum, using the patient's own erythro-
cytes as the indicator. This procedure was based on the fact
that the capacity of SO to hemolyze erythrocytes, but not to
bind specific antibodies, is lost when sulfhydryl groups are
oxidized. The results with this procedure showed a good
correlation with the results when the standard neutralization
assay was used, but, despite the fact that this technique was
later modified to allow for automation (14), the procedure
has never gained wide acceptance. In 1986, Reitano and
co-workers (13) reported on the use of an enzyme-linked
immunosorbent assay for measuring ASO antibodies, and in
No 1988, Umeda and co-workers (19) described a neutralization
4 assay that used carboxyfluorescein-entrapped liposomes in-
stead of rabbit erythrocytes. While both of these procedures
showed a good correlation with the standard method, neither
VOL. 28, 1990 LATEX AGGLUTINATION TEST FOR STREPTOLYSIN O ANTIBODIES
represented a major advance with respect to simplification of
the ASO procedure.
There have been several earlier attempts at developing a
LA test for measuring ASO antibodies. The Leap Strep
(Organon Teknika, Malvern, Pa.) is a liposome-enhanced
LA test which measures not only ASO but anti-DNase B
antibodies as well. However, when compared with the
standard neutralization assay for ASO antibodies, the Leap
Strep was judged to have insufficient sensitivity and could
not be recommended as a useful test for screening for ASO
antibodies (8, 9). The Check-Spectra (Diagniostic Technol-
ogy, Hauppauge, N.Y.) is a LA test for measuring ASO
antibodies that was also found to be too insensitive to be
useful in screening serum specimens (9). The Mercia ASL
Latex Kit (Mercia Diagnostic Ltd, Great Britain) is another
LA test for determining ASO antibody titers (2). When used
to screen serum specimens with a cutoff of .200 IU for
positivity, this test had a sensitivity of 100% and a specificity
of 74% when compared with the standard neutralization
assay. However, when specific titers, as determined by the
two methods, were compared, the correlation was poor.
Finally, the Rapi Tex ASL (Behring, Hounslow, England) is
another new LA test for measuring ASO antibodies. The
data comparing the Rapi Tex ASL with the standard neu-
tralization assay look promising but are, at this time, very
limited (3).
We have examined the Rheumagen ASO LA test for the
measurement of ASO antibodies and have found it to be a
simple procedure that requires no special equipment or
expertise. The Rheumagen ASO can be used as either a
qualitative or quantitative test, takes only several minutes to
complete, and has a shelf life of up to one year. In addition,
with as little as 225 ,ul of serum, titers ranging from 50 to 900
IU can be determined. Fingerstick samples would, there-
fore, be adequate for the performance of this test. We found
that the ASO titers measured by the Rheumagen ASO were
highly reproducible, showed little lot-to-lot variability, and
correlated very closely with the ASO titers measured by the
standard neutralization procedure. We also found that the
ability of the Rheumagen ASO to demonstrate a significant
rise in ASO titer was comparable to that of the standard
neutralization procedure. Much of the discrepancy between
the two procedures in this regard appeared to be attributable
to differences in the dilution schemes. If these findings are
corroborated by additional investigations, the Rheumagen
ASO LA test could replace the neutralization assay as the
procedure of choice for the determination of ASO antibod-
ies. Ideally, LA tests for other streptococcal antibodies
would be developed so that in the future one could simulta-
neously perform LA tests for multiple streptococcal anti-
body assays.
ACKNOWLEDGMENT
This work was supported in part by Biokit Inc., New Britain,
Conn.
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