Activity of lymphocyte transformation (LTF) to streptococcal T antigens was studied in 7 asthmatic children and normal subjects included 21 children and 12 neonates (cord blood).T antigens used were Ti, T4, T6, T8, T12 and T22.
The results are summarized as follows.
1.In unstimulated lymphocyte cultures, no significant difference was found out between asthmatic (mean, 74.9±28.0 cpm) and normal children (mean, 83.5±43.3 cpm) in 3H-thymidine uptake.
2.The LTF activity to these T antigens in asthmatic children was significantly higher than that of normal subjects.The stimulation index (SI), especially, to T12 antigen in asthmatic children (mean SI=132.2) revealed remarkable elevation as compared with that in normal children (mean SI=4.5).

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The counterimmunoelectrophoresis method (CIE) was studied applicability to typing of group B streptococci.Types obtained by CIE were compared with those by the capillary precipitin method (CPP) and the slide agglutination method (SAG).
Preparation of antigens for typing of group B streptococci (GBS) has been performed by hot HC1 extraction at 50°C or 100°C, saline extraction and autoclave extraction method.The antigens prepared by 50C HC1 extraction and autoclave extraction showed identical reactivity and specificity, whereas the other antigens showed poor reactivity and many cross-reaction. Autoclave extraction is recommend for typing of GBS by CIE, because preparation of the antigens is rapid and relatively easy.
The antigens prepared by autoclave extraction from 16 strains of GBS isolated from patients were typed by CIE and the results were compared with types obtained by CPP and SAG.Polysaccharide antigen types obtained by CIE were in agreement with the results obtained by CPP and SAG.In 15 out of 16 strains “c” protein antigen type agreed with the results obtained by CPP and in the remaining one “c”protein antigen was not detected by CPP and SAG, but it was typed by CIE.This finding suggests a higher sensitivity of CIE in comparison with CPP and SAG.By the SAG method, 9 strains, in which “c” protein antigen was detected by CPP and CIE, showed various heat-labile protein antigen types, Q, S, or W.In the remaining 5 strains, in which “c” protein antigen was not detected by CPP and CIE, possessed no heat-labile protein antigens.
Detection of type antigen from three specimens of serum and concentrated urine obtained from neonates suffering from meningitis caused by GBS performed by CIE.Type III antigen was detected in the two concentrated urine samples and it was the same serological type as the strain isolated from the patient.In the serum obtained from GBS type Ic patient, no type antigen was detected.
The CIE method as applied to group B streptococci typing is specific, highly sensitive, rapid and relatively easy.

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In order to isolate β-hemolytic streptococci effectively from clinical specimens mixed with a large number of other bacteria, a new selective enrichment medium (SEB medium) was invented.
The growth promoting effect to β-streptococci of SEB medium and the selective effect to other bacteria were examined.
All standard strain examined and fresh clinical strain of group A, B. C, and G streptococci enriched in the SEB medium by a factor 10⁴-10⁶ after 24 hrs of incubation.In contract, growth of all the strain of staphylococci tested was inhibited in this medium.
The growth of Neisseria, E.coil, Pseudomonas, Candida albicans and some strains of a α-streptococci was inhibited out that of some other strain of streptococci such as S.faecalis grew in this medium.
We found SEB medium was most effective to inhibit the growth of staphylococci or other bacteria and to enhance the growth of fl-hemolytic streptococci as compared with other commercially available media.
β-Hemolytic streptococci inoculated in SEB medium could stroed at least one week at 4°C whereas the number of staphylococci decreased during the storage.SEB medium was stable and could be stored for at least 10 month at 5-7°C.

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In order to investigate statistical relation between numbers of patients with streptococcal infectious disease and meteorological data, we carried out multiple regression analysis on mumbers (dependent variable) of the patients per a hospital per a decade of a month and meteorological data (independent variables) of mean temperature (°C), max.temperature (°C), min. temperature (°C), relative humidity (%), mean vapor pressure (mb), wind speed (m/sec), total radiation on a horizontal surface (MJ/m²), no. of hours with sunshine (h), and precipitation (mm) per a decade of a month during the period from September in 1976 through August in 1977 and from September in 1978 through September in 1984, respectively, using a personal computer of type Canon CX-1 and its program for multiple regression analysis.
Obtained multiple regression equation with high variance ratio (43.8971), multiple correlation coefficient (0.9219) and proportion (0.8499) was Y=2.7415 log T_MAX+8.7521 log RH-3.4656 log MVP -4.4631 log TRH -12.6089, when all meteorological data were transformed to logarithmic values. Namely, selected independent variables in the equation were max. temperature, relative humdity, mean vapor pressure and total radiation on a horizontal surface, and one-sided probabilities of t-distribution of their partial regressin coefficients were from P=0.0000 to P=0.0055.As far as the above results are concerned, it seems to be able to estimate numbers of the patients from meteorological data by the multiple regression equation.

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Group A streptococci were isolated from 564 (20.9%) out of 2, 694 throat swab samples collected from patients with various infectious diseases during the period from April in 1976 through November in 1984 in Akita Prefecture. When rates of isolation positive by clinical diagnosis were compared, the highest rate (63.5%) was observed in 1st group including scarlet fever and streptococcal infectious disease.The rates (6.7-36.2%) from tonsillitis, angina, pharyngitis and other respiratoty infectious diseases in 2nd group were also significantly higher than those from 3rd group of viral erruptive diseases and other infectious diseases as control.
375 out of 564 isolates were typed into 14 serotypes and 73.9% out of serotyped islates were type 12 (41.9%), type 4 (21.6%) and type 6 (10.4%).However, 189 (33.5%) out of group A streptococci isolated were untypable.

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Determination of ASO and other serum antibodies to extracellular antigens has been widely employed for the diagnosis of infection of hemolytic streptococcus.A new laboratory microtiter technique for determining antibodies to a bacterial somatic component (anti-streptococcal-polysaccharide (ASP)) has been developed.This technique permits an easy detection of antibodies to C polysaccharide that is a component of the cell wall of Group A hemolytic streptococcus.The present study was designed to evaluate diagnostic value of ASP determination.
Serum samples were obtained from 538 children aged between 6 and 15 years (patients in Osaka municipal children's hospital and elementary school children in Osaka City) from April, 1979 to January, 1983 and serum ASP, ASO and ASK titers were determined.
The mean ASP level was 1: 64 for the age group between 6 and 9 years, 1: 43, between 10 and 12 years, and 1: 50, between 13 and 15 years, and the cutoff value between a normal and an abnormal level would be between 1: 32 and 1: 64.The coefficents of correlation was 0.214 between ASP and ASO, 0.103 between ASP and ASK, and 0.705 between ASO and ASK, which indicated that ASO and ASK had the most correlative, value.
Clinical isolates were obtained from a throat of 109 elementary school children in Osaka City six times over the period of one year and the relationship between Group A hemolytic streptococcus and titers of these antibodies was studied.The proportion of cases with elevated titers increased in all antibodies as the incidence of positive bacterial cultures increased.Of 50 patients from whom Group A hemolytic streptococcus isolated at least once, 23 (46.0%) showed elevations in ASO and ASK titers but only 18 (36.0%) had elevation in ASP titer.Among 28 children with normal ASO titer of below 1: 240 despite a positive bacterial culture, the numbers of cases with elevated ASP and ASK levels were 10 (38.5%) and 7 (25.0%), respectively.The determination of ASP was slightly more beneficial for the diagnosis of infections with Group A hemolytic streptococcus.

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During 1.5 months from late April through early June in 1983, we have conducted a survey of an isolation of hemolytic streptococci in throats of elementary school pupils (1st-6th grade) in Akita, Saitama, Niigata, Osaka, Shimane and Saga and of junior high school pupils (freshman) in Kochi. Detection rate of group-A, B, C and G hemolytic streptococci was compared as under the same conditions as possible for preparation of culture medium, collection of samples, culture methods and identification.We have innoculated streptococci on our N-O medium at the place where samples were collected from throats and identified them in our laboratory.The detection rate was higher than that previously reported.The detection rate was the highest in group-A, high in group-B and low in group-G in all schools and their classes, but extremely low or zero in some classes in group-C.The detection rate of group-A varied in every class of elementary and junior high school and the difference of the rate between the highest and the lowest group was 2 to 3 fold, and was high in male whereas that of group-B was high in female.The detection rate of the group-B streptococci was low in the lower grade of pupils and increased as their grade increased, the rate being the highest (19.7%) in the freshman of junior high school at puberty.The detection rate was higher in rural area (Akita, Shimane and Saga) than in urban area (Niigata, Saitama and Osaka).

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We have isolated group-A hemolytic streptococci (Streptococcus pyogenes) from throats of elementary and junior high school pupils in 7 prefectures as described in the Part I and classified them into T types by agglutination reaction using anti-streptococci rabbit sera.The classified 14 T-types were as follows; T-1, T-3, T-4, T-6, T-8, T-11, T-12, T-13, T-18, T-22, T-23, T-28, T-B3264 and T-5/27/44.Strains belonged to T-13, T-12 and T-4 types were frequently detected in this order in 7 regions.However, the main epidemic type detected at the highest rate varied in every region as follows;T-13, in Akita and Niigata, T-28 in Saitama, T-12 in Osaka, T-8 in Shimane, T-11 and T-12 in Saga, and T-28, T-1 and T-13 in Kochi.The result suggested that the main epidemic type of hemolytic streptococci was not the same one, but varied in every region in Japan.

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We have isolated group-B hemolytic streptococci (Streptococcus agalactiae) from throats of elementary and junior high school pupils in 7 prefectures as described in the Part I and classified them into types by agglutination reaction and in part by precipitation reaction using anti-streptococci rabbit sera.The type detected at highest rate was Ia type followed by Ic type in 6 regions except Shimane.In Shimane, the detection rate was higher in Ic type than in Ia type.Isolation rate of type II was low, and Ib type was isolated in place of III type.Although several strains of II type was isolated, X and R types were not isolated at all.

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During 1.5 months starting from late April through early June in 1983, we have isolated hemolytic streptococci group-A (413 strains), group-B (197 strains), group-C (12 strains) and group-G (63 strains) from throats of elementary and junior high school pupils in 7 prefectures as described in the Part I and tested their susceptibility to antibiotics.
1. Penicillins: In general, benzyl penicillin (PCG) was the most effective among PCG, ampicillin (ABPC) and amoxicillin (AMPC), and sulbenicillin (SBPC) was the least effective.The group-B was less susceptible than the group-A to penicillins.
2. Cephems: Cephaloridine (CER) was the most effective, followed by cephalothin (CET), and latamoxef (LMOX) was the the least effective among cephems.The group-B streptococci was the most resistant to cephems as well as PCG, and a strain resistant to LMOX (MIC25μg/ml) was isolated.
3.Macrolides: Erythromycin (EM) was effective among EM, oleandomycin (OL) and josamycin (JM).The group-A streptococci resistant or highly resistant (MIC 100-≥200μg/ml) to EM, OL and JM were found in 6.7, 8.7 and 8.5%, or in 5.8, 4.6and5.8% of the isolated strains, respectively. Strains highly resistant to OL or resistant to EM and JM were found in group-B.
4.Tetracyclin (TC): Resistance to TC varied among group-A, B and G streptococci, showing a biphasic resistant pattern.Strains resistant and highly resistant to TC were found in the above 3 groups, but not in group-C.
5.Chloramphenicol (CP): Strains resistant and highly resistant to CP were found in group-A and -B.
6.Comparison of microbial susceptibility to antibiotics among T types. Strains resistant and highly resistant to EM, OL and JM were found frequently in many of T-12type and in one of T-11 type. Strain resistant or highly resistant to TC was found in T-1, T-3, T-4, T-12, T-13, and T-28 types, or in T-4 and T-12 types, respectively. Strain resistant or highly resistant to CP was found in T-11 and T-12 types, respectively.
7. Comparison of strains resistant to TC, CP and macrolide antibiotics from standpoint of types.
Strain resistant to TC, CP and macrolide was found in many of T-12 type and in one of T-11 type of group A, and one of III type of group B. Strain resistant only to macrolide was found in Ic and Ia types of group B, and in none of group A. Strain resistant only to TC was found in many, especially of T-13, T-12 and T-4 types. Strain resistant only to CP or to both TC and CP was few.
8.Regional comparison of detection rate of strains resistant to antibiotics.T-12 type strain resistant to antibiotics was frequently found in Akita and Osaka but less frequently in Shimane.Strains resistant only to macrolide were found frequently in the north region and less frequently in the south regions.

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Hemolytic streptococci isolated from clinical specimen in Toyama Prefecture from 1980 to 1984 were investigated for the relationship between the age distribution of patients, the difference in the source of specimen, and the distribution of serological groups or types. The patterns of antibiotic sensitivity were also studied in relation to the serological groups or types of the isolates.
1.Most of the isolates from the upper respiratory tract or the suppurative region from children belonged to group A, while most of those from urine or vaginal discharge from adult or aged women, as well as those from newborn babies, belonged to group B.
2.Yearly changes in the distribution of types among group A isolates were observed regardless of the source of specimen, type 12 prevailing throughout the investigation period and the secondly dominant type alternating from type 6 (1980) to type 1 (1981), type 1 to type 13 (1982) and type 13 to type 4 (1983-84). On the other hand, the type distribution of group B isolates was invariable regardless of the year or the source of specimen, both Ia and III being consistently dominant.
3.All the isolates were sensitive to Penicillin G, Cephaloridin and Cephalexin. The minimum inhibitory concentration (MIC) of these antibiotics, however, was significantly higher to group B strains than to group A or G isolates. Among these antibiotics, Cephalexin always showed the highest MIC against the same strains. Most of the Erythromycin-registant strains were identified as group A type 12, and remainders as the other types of group A or as group B and G. With regard to group B isolates, no characteristic patterns of antibiotic sensitivity were observed among different types.

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Group B streptococci is becoming a problem as a cause of neonatal sepsis and meningitis in recent year.
The authors have recently studied the distribution of age and sex of patient, suorces of isolates, type of clinical specimen and serological type among 1, 944 strains of group B streptococci (GBS) isolated from clinical specimens at hospitals in Japan during the period of 1977-1983.
The results were as fillows;
1) Distribution of age of patients from whom GBS was isolated; Among 1, 233 cases, who had been well documented, the rates of each age group were 0 year, 3.9%; 1-5 years6.9%; 6-10years, 6.9%; 11-20 years, 9.9%; 21-30 years, 16.5; 31-40 years, 18.2%; 41-50 years, 14.6%; 51-60 years, 8.0%; 61-70 years, 5.7%;and over 70 years, 9.3%.
2) Isolation rates of GBS arranged according to sex; Among 1, 694 cases, who had been well documented, the 547 cases (32.3%) were from male and the 1, 147 (67.7%) cases were from female.
3) Type of clinical specimen from which GBS was isolated; Among.1, 850 cases, who had been well documented, the isolation rates by type of specimen were urine, 36.5%;throat swab, 31.4%; Vaginal swab, 17%;sputum, 4.2%; pus, 3.6%; semen, 1.8;cerebrospiral fluid, 1.2%; blood, 1.1%;and others, 3.3%.
4) Distribution of serological type of GBS isolrated; The rates of serotype of 1944 strains were type Ia, 28%;type Ib, 11%;type Ic, 15%;type II, 4%;type III, 24%;type IIIR, 9%;type R, 3%;and untypable, 7%.
Of 14 cases isolated from clinical specimens (blood or cerebrospinal fluid) with neonatal sepsis and/or meningitis, 13 cases (93%) were type III and tyee IIIR (type III 11 strains, type IIIR 2 strains).

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