Follow-Up Study of Pharyngeal Carriers of Beta-Hemolytic

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, June 1981, p /81/ $02.00/0 Vol. 13, No. 6 Follow-Up Study of Pharyngeal Carriers of Beta-Hemolytic Streptococci Among School Children in Sapporo City During a Period of 2 Years and 5 Months SHIZUE MAEKAWA,`* KATSUHIRO FUKUDA,2 TERUMASA YAMAUCHI,' TOMO YAMAGUCHI,' KEIKO TAKAHASHI,' AND KAZUAKI SUGAWA2 Department of Microbiology, Sapporo Medical College,' and Department of Public Health, School of Medicine, Hokkaido University,2 Sapporo, Japan Received 23 September 1980/Accepted 25 February 1981 A total of 124 normal school children in three classes (aged 8 to 9) were examined for beta-hemolytic streptococci for 29 months from August 1977 to December 1979 by means of serial monthly throat cultures. No significant difference between the carrier rate of boys and that of girls was observed. Although no monthly variation of carrier rates appeared in class II, marked temporal increases of carrier rates appeared from June 1978 (in Class I) and from August 1978 (in class III) to August These periodical variations of carrier rates observed in this investigation do not coincide with the low-in-summer and high-in-winter seasonal variation which has generally been observed by many other investigators. The average values of carrier rates throughout the period of this investigation were 30.9, 29.7, and 24.5% in classes 1, II, and III, respectively. The carrier rate of group A streptococci by month and by class showed some positive correlation with the carrier rate of total beta-hemolytic streptococci. T- type 4 and T-untypable strains were not identified up to a certain period; then each strain appeared at a given time during the course of our investigation in one class, spread to all three classes, and continued to be isolated until the end of this investigation. The origin of these types of strains could not be identified in this study. Of the children, 36 (29.0%) never became streptococcal carriers during the period of examination and 88 (71.0%) became streptococcal carriers at least one time during the 29 examinations; among these positive carriers, 29 children (23.0%) showed positive results in more than 50% of the examinations. The correlation between the asymptomatic colonization with beta-hemolytic streptococci and the occurrence of scarlet fever and other streptococcal diseases is still obscure, although investigations on these problems have been conducted by many investigators. Previous studies in the City of Nemuro, which is known as one of the districts in Japan having a high prevalence of scarlet fever, reported that the carrier rate of beta-hemolytic streptococci at schools in Nemuro did not differ from that in other districts (2, 4). Takizawa, in collaboration with the members of our laboratory, investigated the carrier rate of beta-hemolytic streptococci in school children in Makomanai Elementary School, Sapporo City, on an occasion of a scarlet fever epidemic, and observed that the carrier rate of beta-hemolytic streptococci in the class with the scarlet fever epidemic was significantly higher than the carrier rates of the other two classes in the 2nd grade, which were free from the epidemise (13). Successive examinations of carrier rates in those three classes were not done. This study is a follow-up examination of elementary school children in Sapporo City with beta-hemolytic streptococci by successive monthly throat cultures during a period of 2 years and 5 months (29 months). MATERIALS AND METHODS Subjects of the study. The subjects of this study consisted of healthy pupils in three classes in an elementary school in an urban district of Sapporo. The total number of pupils in the three classes was 106 (aged 8 to 9) when this study was begun. Each class contained almost the same number of pupils, and the ratio of boys to girls was approximately 1:1. During the 29 months of the study, 20 pupils moved to other schools and were thereafter eliminated from the follow-up study, and 18 pupils moved into these classes and were added to the study at the time of their entrance. Therefore, the total number examined was 124, and the number of pupils who could be observed throughout the whole study was 86. An interclass 1017

2 1018 MAEKAWA ET AL. exchange was made of about two-thirds of the pupils in April 1979, and three new classes were formed at that time. Isolation of streptococci from the throats of children. Throat swabs were taken from the surface of the tonsils with sterilized cotton swabs, rubbed softly on a part of the marginal area of a horse blood agar plate, and spread over the surface of the plate by streaking from that part with a wire inoculating loop. Beta-hemolytic colonies with typical characteristics of streptococci were fished from the blood agar plate after 24 h of incubation at 37 C in an aerobic incubator İsolation of streptococci and preservation of strains. Two or more colonies were taken from the blood agar plate and transferred to two tubes containing 2 ml of Todd-Hewitt broth. After incubation at 37 C for 24 h, the growth was transferred to a twocompartment blood agar plate in which one compartment contained 1% glucose and the other contained none. After 24 h of incubation at 37 C, the plates were inspected for characteristic colonies, that is, the appearance of a hemolytic zone and the influence of glucose on hemolysis (see below for interpretation of results on the two-compartment plate). This procedure was done for a confirmation of pure culture. If colonies with different characteristics were found on a plate, the same procedure was repeated until a pure culture of a streptococcal strain could be isolated. Horse blood containing Todd-Hewitt broth (1:4) was used for the preservation of isolated streptococcal strains. After a 24-h incubation at 37 C, the stock culture was stored at -75 C. Grouping and typing of streptococcal strains. Grouping sera of eight groups (A, B, C, D, E, F, G, and L) and T-typing sera of 22 types (Tl, T2, T3, T4, T5, T6, T8, T9, Tii, T12, T13, T15, T18, T22, T23, T25, T27, T28, T44, T49, B3264, and Impl9) were prepared in our laboratory by the method reported by Takizawa et al. (12). The standard streptococcal strains used for the preparation of these standard sera were kindly supplied by the same investigator, Institute of Public Health in Kanagawa Prefecture (World Health Organization Streptococcus Reference Center in Japan). Agglutinogens used for grouping and typing were prepared by the following procedure. The colony isolated from the above-mentioned two-compartment plate was transferred to 5 ml of 0.1% trypsin containing Todd-Hewitt broth and incubated at 30 C overnight. The streptococcal cells were collected by centrifugation and resuspended in 0.5 ml of Todd-Hewitt broth. One drop of a 1% trypsin solution (Difco Laboratories; 1:250) was added to the cell suspension and incubated at 37 C for 60 min. If a homogeneous cell suspension could not be obtained, further trypsinization was performied until the suspension was homogeneous. Homogeneous trypsinized cell suspensions were used as agglutinogens. Slide agglutination techniques were employed for grouping and typing. A 5-,ul amount of a grouping or T-typing serum was dropped onto the slide, and one loop (3-mm diameter) of cell suspension was mixed with the drop of serum. If the reaction was intensely positive, a marked agglutination reaction usually occurred within 1 min. Thereafter, observation J. CLIN. MICROBIOL. was continued for 2 min to avoid missing a weakly positive reaction. The following classifications were made from the results obtained by the above-mentioned procedures. Group A streptococci were identified when the isolate produced beta-hemolysis on horse blood agar, when beta-hemolysis was inhibited by the addition of glucose, and when positive agglutination occurred in antigroup A serum. Group B streptococci were identified when the colony gave a brownish color on horse blood agar containing glucose and was surrounded by a greenish-brown hemolytic zone and when positive agglutination occurred in anti-group B serum. Group C streptococci were identified when the isolate produced beta-hemolysis on horse blood agar, when beta-hemolysis was not inhibited by the addition of glucose, and when positive agglutination occurred in anti-group C serum. Group G streptococci were identified when the colony gave the same appearance as that of Group C on glucose-containing horse blood agar and when positive agglutination occurred in anti-group G serum. Several strains were isolated during the course of the study which showed a positive agglutination reaction to an anti-group F or anti-group D serum. However, since the grouping serum was not completely reliable, these isolated strains were eliminated from further analysis. No strains which showed positive agglutination reactions to either an anti-group E serum or an anti-group L serum were isolated throughout this study. Statistical analysis of the data. Since the data belonged to counts or proportions in nature, a yes-no type analysis of variance (11) with Scheffé's test (10) was utiized for analysis of the data. RESULTS Rate of streptococcal carriers among children examined by monthly throat cultures. The number of pupils examined for betahemolytic streptococci by serial monthly throat cultures ranged from 96 to 106 and averaged 101. A three-way analysis of variance (class, sex, and month) with relevant contrasts revealed no significant differences in the carrier rate by class between the period before the time of class member exchange and the period after the exchange. It also revealed that there was no sex difference with regard to the carrier rate but that there were statistically significant differences between the classes [Fo = 5.57 > F (2, 2,747, 0.05) = 2.99] and between the months of examinations [Fo = 2.96 > F (28, 2,747, 0.05) = 1.48] if each class was treated as if no class member had been exchanged. Class I showed a carrier rate of approximately 17% on the average from August 1977 to May 1978, and its rate increased beginning in June 1978 and maintained a relatively high level, averaging 41.6%, until August 1979 (Fig. 1). This difference was statistically significant [Fo = 2,020 > 28 x F(1, 2,834, 0.05) = 41.44]. Although class III showed

3 VOL. 13,1981 STREPTOCOCCI IN THE THROATS OF CHILDREN O F *,4 0 a 301 à o A S O N D J F M A M J J A b S O N D J F M A M J J A S O N D FIG. 1. Carrier rate of beta-hemolytic streptococci by class and by month during theperiod ofaugust 1977, to December (a) Class number; (b) the time of class member change. a trend similar to that of class 1, class II did not show such a trend during the study period. The average carrier rates during the 29 months were 30.9% (ranging from 11.4 to 52.0%), 29.7% (12.9 to 45.5%), and 24.5% (8.8 to 48.6%) for classes I, II, and III, respectively, and the overall average rate was 28.3%. These differences between the classes were statistically significant [Fo = 6.05 > F(2, 2,834, 0.05) = 3.00]. The highest carrier rate of the three classes combined was 45% in November 1978, and the lowest carrier rate was 19% in September Groups and types of beta-hemolytic streptococci isolated from carriers. The carrier rate of group A streptococci by month and by class varied from 2.9 to 40.6, and the mean carrier rate of group A streptococci by class was 21.4, 23.9, and 20.6 for classes 1, II, and III, respectively; the overall average was 21.9%. Therefore, the proportion of group A streptococci to total beta-hemolytic streptococci isolated was 77.4%, on the average. The carrier rate of group A streptococci by month and by class showed a statistically significant positive correlation with the carrier rate of total beta-hemolytic streptococci; r = 0.954, 0.841, and for classes I, II, and III, respectively. The group A streptococcal strains isolated from carriers in this study included six T-types (Ti, T4, T12, T13, T28, B3264) and some untypable strains. The proportion of T12, T13, T28, and B3264 strains to all of the group A streptococci differed between the classes [F(2, 554,0.05) = 3.01 < Fo 7.52, 4.85, 4.47, for T12, T13, and = B3264, respectively; Fo = > 2 x F(2, 554, 0.05) = 6.02 for T28] (Table 1) but not between the months. The proportion of TI, T4, and T- untypable strains varied depending on class and month, but the proportion of T-untypable strains appeared to have a pattern similar to that of T4 strains. T4 strains suddenly appeared in class Il in February 1978 and spreading to the remaining two classes later, and thereafter they were isolated continuously up to the end of the study. The difference between these two durations (August 1977 to January 1978 and February 1978 to December 1979) with regard to the proportion of T4 strains to the total beta-hemolytic streptococcal strains isolated (0/142 versus 248/686) was statistically significant [Fo = > 28 x F(28, 741, 0.05) 41.72]. = The proportion of group B, C, and G strains to the total number of beta-hemolytic streptococcal strains showed statistically significant differences between the classes [Fo = > F(2, 741, 0.05) 3.01]. = Frequency of the streptococci carriers and the groups and T-types of strains isolated from these carriers. Beta-hemolytic streptococci were not isolated from 36 children (29.0%) at any time during the 29 monthly examinations. Beta-hemolytic streptococci were isolated at least one time during the 29 examinations from 88 children (71.0%) and from 29 l I I a

4 1020 MAEKAWA ET AL. J. CLIN. MICROBIOL. TABLE 1. Carrier composition by streptococcal group and type and by class during the period ofaugust 1977 to December 1979 ' T-untypable strain. b Number within parentheses is the percentage of the total number of children examined. C Number within parentheses is the percentage of the total number of carriers of group A strains. The sum of the percentage figures is over 100 because it includes carriers who carried two strains simultaneously. TABLE 2. Frequency with which beta-hemolytic streptococci were carried during the period of August 1977 to December 1979 Frequency (%) No. (%) of carriers 0 36 (29) (10) (10) (7) (13) (8) (11) (4) (2) (4) (2) children (23.4%) in more than 50% of the examinations (Table 2). Of the 88 children, 44 carried only one kind of streptococcus but 8 children carried three kinds (Table 3). Of the 29 children who showed positive throat cultures in more than 50% of the examinations, 13 (44.8%) carried one strain, 14 (48.3%) carried two strains and 2 (6.9%) carried more than three strains. The corresponding figures with the remaining 59 carriers who showed positive results in 1 to 49% of the examinations were 31 (52.5%), 21 (35.6%), and 7 (11.9%), respectively. There was no statistically significant difference in the above proportions between the two groups of carriers. DISCUSSION Streptococcal carrier rate. The reported streptococcal carrier rate among normal school children varies from 10 to 70%, according to different investigators (3-7). The average of carrier rates calculated from the results of our serial monthly examinations was 28.3%, and this value was similar to that obtained from the examination of 181 school children by Sakurai (9) in Kawasaki City, Japan. It is commonly observed that although no monthly variation of carrier rate can be observed for the whole group, some specific monthly variations can be demonstrated if the results are analyzed by subgroup, that is, school class. These results were also obtained in this study. In our investigations, although no significant monthly variation of carrier rate was found in the case of class II, the level of carrier rate could be divided into two phases at certain points in the cases of class I and class III, and the differences in carrier rates were statistically significant. One phase was from August 1977 to May TABLE 3. Number of carriers, by streptococcal group and type No. of strep- Group tococcal No. (%) of groups or carriers A types isolated from carrier Ti T4 T12 T13 T28 B3264 UT" B C G 1 44 (50) (40) (9) (1) Total' (30) (38) (11) (5) (7) (8) (11) (30) (14) (9) 'Figures within parentheses indicate the percentage of the total carriers. The sum of these percentage figures is not 100.

5 VOL. 13, ; and the other was June 1978 to August Although some investigators have reported that the carrier rate becomes higher in the winter and lower in the summer, this seasonal variation could not be observed in this investigations. The administration of antibiotics during the period of this study was investigated by questionnaires to the parents of these children. Some antibiotics were given to six children among our subjects examined, and three out of the six children were streptococcal carriers. Although three children became noncarriers after the administration of antibiotics, the influence of antibiotic treatment can be neglected for the analysis of the carrier rates, because of the infrequency of such treatment. Groups and types of the isolated betahemolytic streptococcal strains. Zanen et al. (14) reported that the ratio of group A streptococcal strains to the total beta-hemolytic streptococcal strains isolated was 46% in preschool children (aged 1 to 7) by bimonthly serial throat cultures during a period of 29 months. On the other hand, Cornfeld et al. (1) reported the ratios of 63, 75, and 81%, respectively, in three separate schools in a 1-year follow-up study with serial monthly throat cultures. The figure 77.4%, calculated from the results of our investigation, coincides with the figures given by the latter author. The monthly fluctuation of carrier rate of all beta-hemolytic streptococcal strains was influenced mostly by the carrier rate of group A streptococci. The results of this investigation showed that the ratio of the sum of Tl and T4 strains to the total group A strains isolated was about twothirds. Other investigators (5, 8) have reported that these types have generally been isolated from patients with scarlet fever or pharyngitis and are also widely distributed in the throats of asymptomatic carriers in Japan. T4 strains were not isolated until a definite period in our study, but then they were isolated at every examination up to the end of our investigation. The appearance and the mode of spread of T4 strains were similar to those of T-untypable strains. The T-untypable strains, which failed to react with any of the 22 routinely-used antisera, were isolated 1 month after the appearance of the T4 strains in the subjects of our investigation. However, the source of the T4 strains was not detected. Scarlet fever was not observed in children during the period of our study. The reason for this rapid spread of T4 STREPTOCOCCI IN THE THROATS OF CHILDREN 1021 strains through the whole group of children in our study may be because these children had never been exposed to the T4 strains before and therefore had no immunity to them. ACKNOWLEDGMENTS We are much indebted to the teaching staff in Kitakujo Elementary School for their assistance in our examination. We are grateful to them for their kindness. We also thank T. T. A. Hayashi in the Department of Microbiology, Sapporo Medical College, Sapporo, Japan, for his comments and criticima of the manuscript. This study was partly supported by a grant from Oyama Health Foundation, Japan, to K.F. LITEEIATURE CITED 1. Cornfeld, D., G. Werner, R. Weaver, M. T. Bellows, and J. P. Hubbard Streptococcal infection in a school population: preliminary report. Ann. Int. Med. 49: Fukuda, K., H. Miyake, S. Maekawa, H. Kimura, S. Chiba, T. Yamashita, and K. Ishii Factors related to the incidence of symptomless infections with haemolytic streptococcus. Public Health London 92: Kaneko, Y., T. Oba, and T. Sakurai A follow-up study of throat carriers of streptococci among schoolchildren, p In M. J. Haverkorn (ed.), Streptococcal disease and the community. Elsevier Publiahing Co., Inc., New York. 4. Maekawa, S., and Y. Takizawa A continuous study of hemolytic streptococci in the throats of normal school children in the City of Nemuro, Hokkaido. Sapporo Med. J. 46: Miyamoto, Y., K. Takizawa, A. Mataushima, Y. Asai, and T. Takahashi Transition of the distribution of types of group A streptococci (from April 1969 to March 1974) and annual change of the main predominant types for the past 10 years. J. Jpn. Assoc. Infect. Dis. 49: Nakajima, K., T. Adachi, and M. Hada Yearly epidemiological survey of group A hemolytic streptococci isolated from the throats of healthy children. J. Jpn. Assoc. Infect. Dis. 53: Noguchi, M., Y. Wakamatsu, R. Chisuwa, H. Kusama, M. Ohashi, H. Fukumi, L. Tadokoro, M. Kudo, M. Yoshioka, T. Kasuga, and T. Hirayama Immunological significance of anti-streptolysin O in streptococcal infections. II. A two-year study of streptococcal infections in primary school children. Jpn. Med. Sci. Biol. 15: Parker, M. T International survey of the distribution of serotypes of streptococcus pyogenes (group A streptococci). Bull. W.H.O. 37: Sakurai, T An epidemiological study of streptococcal infection A follow-up study of healthy carriers among school children. Jpn. Public Health 18: Scheffé, H The analysis of variance. John Wiley & Sons, New York. 11. Taguchi, G A new statistical analysis method for clinical data, the accumulating analysis, in contrast with the chisquare test, p Shinjuku Shobo, Tokyo. 12. Takizawa, K., S. Akiyama, and Y. Miyamoto Reexamination and characterization of the T-agglutination complex or pattern or streptococcus pyogenes: preparation of anti-t sera. Jpn. J. Microbiol. 14:

6 1022 MAEKAWA ET AL. 13. Takizawa, Y., A. Takase, K. Konishi, and I. Tomizawa Studies on pharyngeal hemolytic streptococci in a classroom outbreak of scarlet fever in an elementary school of Sapporo. J. Jpn. Assoc. Infect. Dis. 51: J. CLIN. MICROBIOL. 14. Zanen, H. C., S. Ganor, and M. J. Van Toorn A continuous study of hemolytic streptococci in the throats of normal children, adults and aged men. Am. J. Hyg. 69: