Abstract
The presence of the anti-opacity factor (anti-OF) antibody (Ab) in the serum used for identifying the OF antigen (Ag) type represents previous or current infection with group A streptococci (GAS) of the OF Ag type. Throat cultures were taken from 172 elementary schoolchildren in Chinju, Korea, and venous blood samples were collected at the same time to screen for the frequency of the anti-OF Ab. After isolation of GAS, the OF Ag of each GAS was identified by inhibition of the opacity reaction with recognized anti-OF sera. The anti-OF Abs in the sera were screened with the six most common OF Ag types. OF22 and OF28 were high in prevalence (28.2 and 20.5%, respectively) among OF Ag types, while anti-OF types 4, 28, and 22 were frequently identified (39.5, 29.7, and 15.7%, respectively) in the sera. Thirty-two of 39 (82.1%) OF Ag-producing GAS carriers, 25 of 34 (73.5%) GAS carriers not producing OF Ag, and 72 of 99 (72.7%) throat culture-negative children harbored the anti-OF Ab. Forty-five (26.2%) of 172 children had two different anti-OF Abs, and 11 (6.4%) had more than three anti-OF Abs. Seventy-five percent of 172 elementary schoolchildren were shown to be previously or currently infected with GAS. The percentages of children positive for the anti-OF Ab were very high regardless of the result of throat culture or OF Ag production of GAS. We could also demonstrate (i) that the prevalent strains of GAS changed according to the time span by determining the difference between the frequencies of OF Ag and anti-OF Ab and (ii) that repeated infections were not uncommon in schoolchildren, as one-third had more than two different anti-OF Abs.
There has been a remarkable increase in the number of reports of severe invasive infections caused by group A streptococci (GAS) during the last decade (3). Serotypings are important not only for surveying the epidemiology of GAS infections but also for understanding the mechanism of invasive infections. Opacity factor (OF), a cell wall component of GAS, makes horse serum opaque (5, 20). The physicochemical properties of OF are almost similar to those of M protein (21). Generally OF-positive strains do not stimulate an immune response in a rabbit. OF is an apolipoproteinase which is inhibited by pepsin or trypsin but which is not affected by HCl or heating (6, 18). Recently Kreikemeyer et al. revealed that OF is sfbII, a fibronectin binding surface protein of GAS (11). OF antigen (Ag) has type specificity, and the OF type is concordant with the M type (20, 21). There are currently about 80 recognized and confirmed M types (8, 9). Of these, 27 are known to produce OF. More M types make it likely that we will identify more OF producers (4). OF typing is useful in the identification of new M types as well as in simplifying M typing by saving time, labor, and valuable sera. The OF type of the strain is determined by inhibition of the serum opacity reaction (SOR) after mixing OF protein and type-specific human anti-OF sera. We isolated GAS from healthy schoolchildren by throat culture and T, OF, and M serotypings to characterize the GAS carrier status. When someone is infected with GAS, he or she can make either anti-M or anti-OF antibody (Ab), depending on the serotype of the strain. The Ab seems to persist lifelong and to protect from reinfection with the same serotype (12). Detection of the anti-M Ab in the sera is very hard to perform. The anti-OF Ab type is rather easily determined in the same way as the OF Ag type is determined. The presence of the anti-OF Ab indicates previous or current GAS infection (12). The presence of multiple anti-OF Abs indicates that the person was infected several times with different serotypes. To evaluate the immune status of GAS infections, blood samples drawn from the same children were screened for anti-OF Abs. Most of the previous epidemiological studies have been performed with throat culture and serotypings (10, 17). The epidemiological usefulness of the anti-OF Ab for surveying the degree of past or present infection of GAS in the schoolchildren was compared with that of throat culture.
MATERIALS AND METHODS
Bacterial isolation and serotyping.
Throat cultures were taken from 172 healthy schoolchildren (ages 7 to 12) without any symptoms or signs of pharyngitis in Chinju, Korea. After incubation of the blood agar plate overnight at 37°C, beta-hemolytic streptococci were identified with a bacitracin disk (0.04 U) and a latex agglutination test (A Strep AD; Denka Seiken, Tokyo, Japan). After the T type of GAS was screened with anti-T sera (Sevac, Prague, Czech Republic) (1), cell wall protein extracted either by the Lancefield hot-acid method or with 1% sodium dodecyl sulfate was used for SOR screening (7). If the cell wall protein was SOR positive, it was mixed with type-specific human anti-OF sera. Anti-OF sera used included types 2, 4, 9, 11, 13, 22, 25, 28, 48, 49, 58 to 64, 66, 68, 73, 75 to 78, and 81 (1). Identification of OF Ag types was performed by a microwell technique using horse serum (Gibco Laboratories, Grand Island, N.Y.) according to the result of T Ag typing (8). Opacity was spectrophotometrically measured at 450 nm. If cell wall extract was SOR negative, M types were identified by Ouchterlony double immunodiffusion (19). The anti-M sera used were 1 to 6, 8, 12, 14, 15, 17 to 19, 22 to 26, 28, 36 to 41, 43, 47, 49, 51 to 53, 58, 60, 75, and 76.
Anti-OF Ab typing.
The sera of children were reacted with six representative OF Ags of GAS, which were commonly isolated from Chinju, including T4/OF4, T11/OF78, T12/OF22, T12/M nontypeable (NT), T13/OF13, and T28/OF28. The anti-OF Ab type was identified by a microwell technique (8). We classified the children into three groups: group I, children with OF-positive GAS; group II, children with OF-negative GAS; group III, children without GAS in the throats. The frequency and numbers of anti-OF Ab types in the sera were analyzed for each group of children. The percentages of children positive for anti-OF Abs for all school grades were compared.
RESULTS
Serotyping.
Cultures from 73 (42.4%) of 172 children tested grew GAS, of which 39 (53.4%) strains were SOR positive (Table 1). OF22 (28.2%) and OF28 (20.5%) were the most common OF types (chi square = 18.1; P = 0.0029). T types of 13 (33.3%) OF-NT strains were as follows: 6 were T12, 2 were T4, 1 was T28, and 4 were NT. Of the 34 SOR-negative strains, M12 (52.9%) was predominant and M6 (17.9%) was the next most common (chi square = 35.9; P = 0.0001). OF types 4, 13, and 78 were rare.
TABLE 1.
Frequencies of OF type and M type of GAS
| OF or M type | No. | % |
|---|---|---|
| SOR positive | ||
| OF4 | 2 | 5.1 |
| OF13 | 2 | 5.1 |
| OF22 | 11 | 28.2 |
| OF28 | 8 | 20.5 |
| OF78 | 3 | 7.7 |
| OF-NTa | 13 | 33.3 |
| Total | 39 | 100 |
| SOR negative | ||
| M1 | 3 | 8.8 |
| M3 | 1 | 2.9 |
| M5 | 1 | 2.9 |
| M6 | 6 | 17.7 |
| M12 | 18 | 52.9 |
| M-NTb | 5 | 14.7 |
| Total | 34 | 100 |
T types of OF-NT strains: T12, six; T4, two; T28, one; T-NT, four.
T types of M-NT strains: T11, two; T12, one; T28, one; T-NT, one.
Anti-OF Ab typing.
The frequencies of anti-OF Ab types are shown in Table 2. Anti-OF types 4 and 28 were predominant in all three groups. The frequencies of anti-OF types 4 and 28 were 39.5 and 29.7%, respectively. Anti-OF type 22 is more common (22.2%) in group III than in other groups. Anti-OF types 22, 78, and 13 were not common (likelihood chi square = 21.1; P = 0.0205).
TABLE 2.
Frequencies of anti-OF Ab types in the sera according to the throat culture and SOR of GAS
| Anti-OF Ab type | No. (%)b positive in:
|
|||
|---|---|---|---|---|
| Group I (n = 39) | Group II (n = 34) | Group III (n = 99) | Total (n = 172) | |
| 4 | 17 (43.6) | 9 (26.5) | 42 (42.4) | 68 (39.5) |
| 13 | 5 (12.8) | 1 (2.9) | 10 (10.1) | 16 (9.3) |
| 22 | 5 (12.8) | 22 (22.2) | 27 (15.7) | |
| 28 | 18 (46.2) | 11 (32.4) | 23 (23.2) | 51 (29.7) |
| 78 | 4 (10.3) | 3 (8.8) | 10 (10.1) | 17 (9.9) |
| NTa | 5 (12.8) | 1 (2.9) | 2 (2.0) | 8 (4.7) |
A T12 OF-NT strain was used for anti-OF Ab typing.
Group I, children GAS positive by SOR; group II, children GAS negative by SOR; group III, children without GAS in the throat culture.
Thirty-two (82.1%) of the children in group I had the anti-OF Ab (Table 3). Percentages of children positive for the anti-OF Ab in group II and group III were 73.5 and 72.7%, respectively (chi-square test; P > 0.05). Forty-five (26.2%) had two anti-OF types, while 11 (6.4%) harbored more than three anti-OF types.
TABLE 3.
Numbers of anti-OF Abs according to the throat culture results and SOR of GAS
| No. of anti-OF Abs | No. (%)a of positive samples in:
|
|||
|---|---|---|---|---|
| Group I (n = 39) | Group II (n = 34) | Group III (n = 99) | Total (n = 172) | |
| 0 | 7 (18.0) | 9 (26.5) | 27 (27.3) | 43 (25.0) |
| 1 | 13 (33.3) | 15 (44.1) | 45 (45.5) | 73 (42.4) |
| 2 | 14 (35.9) | 8 (23.5) | 23 (23.2) | 45 (26.2) |
| 3 | 4 (10.3) | 2 (5.9) | 4 (4.0) | 10 (5.8) |
| 4 | 1 (2.6) | 1 (0.6) | ||
| Total positive | 32 (82.1) | 25 (73.5) | 72 (72.7) | 129 (75.0) |
See Table 2 for an explanation of each group.
Sixty-two percent of the first-grade schoolchildren had the anti-OF Ab (Table 4). The percentages of children positive for the anti-OF Ab in the other school grades ranged from 70.4 to 82.8%, which showed no significant differences according to school grade (chi-square test; P > 0.05).
TABLE 4.
Frequency of anti-OF Ab according to school grade of children
| School grade | No. of children:
|
|
|---|---|---|
| Tested | With anti-OF Ab (%) | |
| 1 | 37 | 23 (62.3) |
| 2 | 29 | 24 (82.8) |
| 3 | 26 | 20 (76.9) |
| 4 | 23 | 19 (82.6) |
| 5 | 27 | 19 (70.4) |
| 6 | 30 | 24 (80.0) |
| Total | 172 | 129 (75.0) |
DISCUSSION
Although we do not know the full biological significance of the OF protein, OF Ag typing has been used for the epidemiological study of GAS infections, especially when the strains do not produce an immune reaction in a rabbit. The anti-OF Ab in the serum of a patient is a marker for a past or current infection with the specific OF type strain and has a preventive role for reinfection with the same type (13). There have as yet been no reports on the frequency of the anti-OF Ab, as analyzed either by the results of throat cultures or by the nature of cell wall protein of GAS in schoolchildren. Screening anti-OF Abs in the sera was very useful to elucidate previously widespread strains and the degree of infections in the community (14, 15). On the basis of the results for the distribution of anti-OF Abs in Chinju, Korea, OF types 4 and 28 might have been the most prevalent in the past. On the basis of the serotyping data, we assumed that an OF type 4 strain had been very common in the past but is now rare. Infections with OF type 28 were common in the past and continue to be. The most common OF types of GAS were OF22 (28.2%) and OF28 (20.5%), but the frequency of anti-OF22 (15.7%) did not use to be so high, which suggests that infections with OF22 are more frequent now than in the past. Many authors reported that the serotypings of GAS changed annually, which might be due to a change in bacterial invasiveness or host immunity (2). In the distribution of M types of SOR-negative strains, M12 (52.9%) was predominant. Group II had either anti-OF type 28 (32.4%) or 4 (26.5%) commonly. Group III frequently contained anti-OF types 4 (42.4%), 28 (23.2%), and 22 (22.2%), while anti-OF types 13 and 78 were less common (10.1%). Although group III showed no GAS in the throats, unexpectedly 72.7% have been infected with various types of GAS. There is no significant difference in the number of anti-OF types among the three groups. The percentage of children positive for the anti-OF Ab in the first grade was 62.3%, indicating that many children were already infected with GAS before admission to elementary school. But the percentages of samples positive for the anti-OF Ab in the other school grades did not significantly increase. Infections with a new serotype of GAS might not occur frequently in this age group. Anti-OF Ab screening of schoolchildren detected GAS infection in three-fourths of those tested. On the basis of throat cultures, the carrier rate of GAS in schoolchildren ranged from 10 to 20%. Although their population and OF Abs studied are different from ours, Prakash and Dutta demonstrated that 39% of 235 subjects had at least one type of OF Ab (16). They found that ∼48% had two or more Abs and that certain types of Abs were prevalent in rheumatic fever or acute glomerulonephritis patients. Differences among studies may be due to the populations investigated and are not due to the methods employed. Considering that we did not detect the anti-M Ab in the sera, it is probable that the percentage of those actually infected with GAS is greater than 75%. One-third (32.6%) of schoolchildren were infected with more than two different OF types, which indicates that multiple infections were not uncommon in schoolchildren.
In conclusion, GAS infections were quite common in Korean schoolchildren. The spectra of previously prevalent strains were different from those of the currently widespread ones. Multiple infections were not uncommon. Sixty-two percent of the first-grade schoolchildren were already infected with GAS. Screening the anti-OF Ab was quite valuable for understanding the epidemiology of GAS infections. Because anti-OF Ab screening is simpler and easier to perform than the serotyping of GAS, screening anti-OF Abs can be effectively used for the epidemiological study of GAS in the community.
ACKNOWLEDGMENTS
We appreciate the technical advice from and provision of anti-OF and anti-M sera by Dwight R. Johnson and Edward L. Kaplan at the Department of Pediatrics and World Health Organization Collaborating Center for References and Research on Streptococci, University of Minnesota Medical School.
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