Genetic Diversity among Group A Streptococcus Isolated from Throats of Healthy and Symptomatic Children

Abstract

Background: Group A streptococcus (GAS) is the commonest bacterial cause of pharyngitis. Children in the age group of 5–15 years are most commonly affected. It can also colonize throats of healthy children in this age group. Both cases and carriers can transmit it in the community.

Methods: Throat swab samples were collected from 1849 asymptomatic and 371 symptomatic children.

Results: The rate of isolation of GAS was 1.41% among the asymptomatic group and 7.55% among the symptomatic group. Nine different emm types were encountered in the asymptomatic children and 14 among the symptomatic children.

Conclusion: Throat swab cultures must be used in all cases of pharyngitis. Early and appropriate antibiotic therapy will prevent complications. Asymptomatic throat carriage of GAS in children was low in our study. However, they can still act as reservoirs. Emm typing helps in understanding epidemiology and finding new types.

INTRODUCTION

Group A streptococcus (GAS) is considered one of the most aggressive microorganisms encountered in clinical practice. Humans are the only known reservoirs. It causes diseases that are progressive, involve deeper tissues and can be life-threatening. It is responsible for a wide variety of suppurative and non-suppurative infections in humans. These include pharyngitis, pyoderma, erysipelas, cellulitis, necrotizing fasciitis, toxic streptococcal syndrome, scarlet fever, sepsis, pneumonia and meningitis. Post infection sequelae due to GAS are acute rheumatic fever (ARF) and acute post streptococcal glomerulonephritis.

Acute pharyngitis is common among children and adults. A wide array of microbial agents are implicated in its etiology. Most cases are viral in origin. School children in the age group of 5–15 years are most commonly affected. In children, approximately 20% cases of pharyngitis are caused by GAS[1]. GAS also frequently colonizes throats of school children. Asymptomatic carrier rates of 1.3–25.9% have been reported among school children in various studies [2–5].

GAS spreads by respiratory droplets and can transmit to susceptible children and adults. GAS associated diseases and sequelae continue to have devastating effects on public health and national economy, as they mainly affect children and young adults[6].

M protein, a cell surface antigen, is the major virulence factor of GAS. One GAS produces only one type of M protein. The terminal sequence that projects from the cell surface, the N-terminus, varies among the clinical isolates of GAS and forms the basis of Lancefield serological classification[7]. Many GAS, however, remain untypeable by this method. Hence, cloning of M protein gene, the emm gene, has led to the identification of emm genotypes [7]. More than 180 emm types and 800 emm subtypes have been described and new ones are emerging [8]. Emm typing helps in understanding pathogenesis and defining epidemiology.

Patients with GAS pharyngitis can be effectively treated, hence emphasis is laid on its definitive identification. Treatment not only cures the infection but prevents further complications. Importance in its isolation and identification also lies in the fact that it commonly transmits among children at school and day care centres. Hence, treatment prevents spread and possible outbreaks.

MATERIAL AND METHODS

The study was approved by the institutional ethics committee. School-going children in the age group of 5–15 years attending various schools in the district of Shimla were included in the study. Informed consent was taken from parents. The study population was divided in two groups: healthy and symptomatic. Each selected child was subjected to a brief focused medical history and physical examination to look for fever, cough and other features of sore throat like tonsillar and/or pharyngeal erythema, tonsillar and/or pharyngeal exudates, tonsillar enlargement and cervical lymphadenopathy. Children without any features of sore throat were included in the healthy group, and children with any feature of sore throat were included in the symptomatic group. Throat swab specimens were collected from 1849 asymptomatic and 371 symptomatic children.

All samples were inoculated on sheep blood agar and incubated at 37°C in a candle jar. Blood agar plates showing beta hemolytic colonies with colony morphology suggestive of beta hemolytic streptococcus species were processed further. Screening for GAS was done by bacitracin discs (0.04U) and sulfamethoxazole–trimethoprim (SXT) susceptibility test. Latex agglutination kit, Streptex, manufactured by Remel laboratories, was used to identify GAS. All strains of Group A beta hemolytic streptococci isolated were tested for antibiotic susceptibility by Kirby Bauer’s disc diffusion method on Mueller Hinton blood agar with 5% sheep blood. Commercially available antibiotic discs (Hi-Media Laboratories Pvt. Ltd.) of penicillin (10U), erythromycin (15 µg), vancomycin (30 µg), clindamycin (2 µg), chloramphenicol (30 µg) and levofloxacin (5 µg) were used. GAS isolates were sent to Post Graduate Institute of Medical Education and Research, Chandigarh, for emm typing.

OBSERVATIONS AND RESULTS

Out of 2220 children in the study population, 83.29% (1849/2220) were asymptomatic and 16.71% (371/2220) were symptomatic. In all, 1074 children (48.38%) were male and 1146 (51.62%) were female.

Beta hemolytic streptococci were isolated from 2.43% (45/1849) of healthy children and 8.36% (31/371) of symptomatic children. Similarly, prevalence of GAS was 1.41% (26/1849) among carriers and 7.55% (28/371) among cases. In all, 2.70% (29/1074) male children and 2.18% (25/1146) female children had throat swab cultures positive for GAS.

Prevalence of beta hemolytic streptococci and GAS in the study population is given in Table 1. Sex-wise distribution is given in Table 2. Age-wise distribution of GAS isolates is given in Table 3.

Table 1.

Prevalence of beta hemolytic streptococci and GAS among healthy and symptomatic school children

Study group	Beta hemolytic streptococci isolated	Group A streptococci isolated
Healthy group	2.43%	1.41%
Symptomatic group	8.36%	7.55%
Total	3.42%	2.43%

Table 2.

Sex-wise distribution of GAS

	Asymptomatic group			Symptomatic group
Nil	Male	Female	Total	Male	Female	Total
No of children	881	968	1849	193	178	371
Positive for GAS	14	12	26	15	13	28
Percentage	1.59	1.24	1.41	7.77	7.30	7.55

Table 3.

Age-wise distribution of GAS in the study population

	Healthy group			Symptomatic group
	5–10 years	11–15 years	Total	5–10 years	11–15 years	Total
No of children	779	1070	1849	195	176	371
Positive for GAS	7	19	26	17	11	28
Percentage	0.9	1.78	1.41	8.72	6.25	7.55

A total of 76 beta hemolytic streptococci were isolated from the study population. In the asymptomatic group, out of 45 isolates of beta hemolytic streptococci, 26 were Group A streptococcus, nine were Group C, seven were Group G, two were Group D and one was Group F streptococci. In the symptomatic group, among the 31 beta hemolytic streptococci isolates, there were 28 Group A and 3 Group G streptococci. Distribution of various serogroups among the study population is given in Table 4.

Table 4.

Serogroups of various beta hemolytic streptococci isolated

Serogroups of beta hemolytic streptococci	Healthy group	Symptomatic group
Group A	26 (1.40%)	28 (7.55%)
Group C	9 (0.49%)	0 (0%)
Group D	2 (0.11%)	0 (0%)
Group F	1 (0.05%)	0 (0%)
Group G	7 (0.38%)	3 (0.81%)
Total	45	31

Fifty-one (94.44%) isolates of GAS were sensitive to bacitracin and all were resistant to SXT. All the GAS strains that were isolated were sensitive to penicillin, ampicillin and levofloxacin. One strain was resistant to clindamycin (98.15% sensitivity). Sensitivity of erythromycin was 94.44%, whereas that of azithromycin was 96.3%.

Emm typing results were available for a total of 39 GAS isolates. In the asymptomatic group, 9 different emm types were encountered out of 17 GAS strains (Table 5). Emm12 was most common (eight isolates), followed by emm42.2 (two isolates). There was one isolate each of emm1, emm1.9, emm12.9, emm27, emm55, emm76 and emmstC58. In the symptomatic group, 14 emm types were seen among 22 isolates (Table 6). There were four isolates of emm12.9, three each of emm2 and emm18.6 and two isolates of emm77. One isolate each belonged to emm1.9, emm2.2, emm12, emm42.1, emm42.2, emm44, emm49.4, emm66, emmstKNB3.2 and emmst4722.0.

Table 5.

Emm type distribution of GAS among healthy children

Emm type	Clinical status	No of isolates (17)	Percentage
emm12	Asymptomatic	8	47.1
emm 42.2	Asymptomatic	2	11.8
emm 1	Asymptomatic	1	5.88
emm 1.9	Asymptomatic	1	5.88
emm 12.9	Asymptomatic	1	5.88
emm 27	Asymptomatic	1	5.88
emm55	Asymptomatic	1	5.88
emm 76	Asymptomatic	1	5.88
emm stC58	Asymptomatic	1	5.88

Table 6.

Emm type distribution of GAS among symptomatic children

Emm type	Clinical Status	No of isolates (22)	Percentage
emm12.9	Symptomatic	4	18.2
emm 2	Symptomatic	3	13.6
emm 18.6	Symptomatic	3	13.6
emm 77	Symptomatic	2	9.09
emm 1.9	Symptomatic	1	4.55
emm2.2	Symptomatic	1	4.55
emm12	Symptomatic	1	4.55
emm42.1	Symptomatic	1	4.55
emm 42.2	Symptomatic	1	4.55
emm 44	Symptomatic	1	4.55
emm49.4	Symptomatic	1	4.55
emm 66	Symptomatic	1	4.55
emm stKNB3.2	Symptomatic	1	4.55
emm st4722.0	Symptomatic	1	4.55

DISCUSSION

Diseases caused by GAS are a worldwide problem. It is responsible for a number of clinical manifestations ranging from acute pharyngitis to life-threatening invasive and post-infectious diseases. The prevalence of GAS in throat is known to be highest in school-aged children, but it varies geographically in different countries and also by year[9].

Signs and symptoms cannot precisely predict the microbial etiology. Hence, it becomes imperative to invest timely in throat culture in all cases of pharyngitis. Carriers are an important source of GAS in the community. Insight into the epidemiology thus becomes necessary.

In our study, prevalence of beta hemolytic streptococci was more in the symptomatic group (8.36%) as compared with the asymptomatic group (2.43%). Similarly, prevalence of GAS was more in the symptomatic group (7.55%) than in the asymptomatic group (1.41%). Prevalence of GAS was slightly more in males in both groups, 1.59% (carriers) and 7.77% (cases), as compared with females, 1.24% (carriers) and 7.30% (cases).

Among studies from India, GAS carrier rates of 3.7–8.4% have been reported from school-going children [3, 10, 11]. Rates of isolation of GAS from children with pharyngitis from India have been 4.0–13.5%[11, 12]. In our study, carrier rate of GAS is on a lower side (1.41%); however, among the symptomatic group, the rate (7.55%) is comparable with other studies from India. A lower incidence of GAS carriers (2.5%) has been reported by Pichichero et al. [13].

Dumre et al. [14] isolated GAS from 10.9% (38/350) of healthy children from Nepal. Kim and Lee [9] conducted a study among healthy school children in Jinju, Southern Korea. The isolation rates of GAS were 16.9% in 2002 and 17.9% in 1995.

Age has been considered as an important factor in the microbiological etiology of pharyngitis. The peak incidence of GAS pharyngitis occurs in children aged 5 to 10 years. The age distribution in our study in children with pharyngitis showed that 8.72% of the GAS isolates (17/195) were from the age group of 5 to 10 years and 6.25% (11/176) were from the age group of 11–15 years. Comparison of analysis of prevalence of GAS in the age groups 5 to 10 years and 11–15 years did not reveal significant difference, with a P value of 0.7 by Fisher’s exact test. Prevalence of GAS isolates among symptomatic male and female children was statistically not significant on univariate analysis (p = 0.5).

Nandi et al. [12] in their study reported the incidence of GAS pharyngitis to be slightly higher among 11-year-olds. The occurrence of the peak incidence at 11 years in this study has been referred to the fact that at this age, children move from primary school to high school, thus being exposed to infection from other strains of GAS [12] Incidence of sore throat and GAS pharyngitis was not significantly different among males and females in this study.

Bacitracin sensitivity test is useful for presumptive identification of GAS. In our study, 94.44% (51/54) strains of GAS were sensitive to bacitracin. Bacitracin sensitivity test can be used as a simple method of distinguishing Group A strains and can be carried out in laboratories where facilities for carrying serological grouping are not available. Application of bacitracin disc on primary culture plate for throat swabs can also help in rapid identification of GAS.

The SXT test presumptively distinguishes Group A and B streptococci from other β-hemolytic streptococci. When used in conjunction with the bacitracin susceptibility test, it helps screen out those non-A, non-B streptococci that may be susceptible to bacitracin because both Group A and B strains are SXT-resistant, whereas Groups C, F and G are SXT-susceptible.

Penicillin is the drug of choice for GAS infections, as it is universally sensitive to it. In our study also, 100% GAS isolates were sensitive to penicillin. Antibiotic drug resistance was not a major problem in the present study, with all GAS isolates being sensitive to penicillin, ampicillin and levofloxacin. Only one strain was resistant to clindamycin (98.15% sensitivity), two to azithromycin (96.3% sensitivity) and three to erythromycin (94.44% sensitivity). Hence, penicillin must be used in all cases of GAS infections, and macrolides should be reserved for penicillin-allergic individuals to prevent emergence of resistance.

GAS produces a large number of virulence factors to overcome host immunity. M protein is a major surface antigen that is antiphagocytic and protective in some strains. It has also been associated with pathogenesis of ARF. M proteins are encoded by emm genes and sequencing of these genes can be used to classify GAS.

Nine different emm types were encountered out of 17 GAS strains in the asymptomatic group. Emm12 was most common (eight isolates), followed by emm42.2 (two isolates). Similar to our findings, emm12 (34.4%) was the most common GAS isolated from throats of healthy school children from Korea, followed by emm75 (10.4%), emm18 (9.4%), emm22 (8.3%) and emm1 (7.3%)[9]. Abdissa et al. [15] reported 8.75% carrier rate of GAS from Ethiopia and six different types, emm3.19, emm5.48, st62.0, emm29.2, st463.0 and st3757.0, listed in descending order of frequency, made up 35.4% of the isolates. These types are different from those reported in our study.

Fourteen emm types were seen among 22 isolates from the symptomatic group. There were four isolates of emm12.9, three each of emm2 and emm18.6 and two isolates of emm77. In children with symptomatic acute pharyngitis from Korea, most frequent emm type was emm12 (28.5%), followed by emm75 (18.7%), emm22 (13%), emm2 (12.2%) and emm8 (8.1%)[16]. Typing of 216 consecutive isolates of Streptococcus pyogenes from patients with pharyngitis in the region of Aachen, Germany, revealed emm1 to be most common (18.5%), followed by emm12 (15.7%), emm3 (14.4%) and emm28 (13.9%) [17]. Among 200 school children from South India with complaints of sore throat, the prevalence of GAS pharyngitis was found to be 6% (12 out of 200). Ten different emm types were found among the 11 isolates. They were emm1-2.2, emm55, emm57, emm71 (two isolates), emm74, emm82.1, emm95, emm112.2, st11014 and st2002.1 [18].

The emm types reported in our study show heterogeneous nature of GAS isolates. Few similar emm types have been reported from Korea and Germany. However, interestingly, emm types reported from South India are quite different. This points toward a diversity in nature of GAS in various parts of the world. Typing of isolates helps in understanding epidemiology. Developing countries are worse affected by ARF. It is important to understand epidemiology of GAS in these regions to help in prevention and cure. However, data from such regions are scarce and data from developed nations cannot be applied.

GAS carrier rates in our study are low (1.41%); however, a significant number of sore throat cases (7.55%) were caused due to GAS. Throat cultures are needed in patients with symptoms of sore throat to identify GAS infection for timely and adequate treatment. Clinical judgment to diagnose GAS pharyngitis could be erroneous. Microbiological confirmation to clinical features helps in preventing unnecessary antibiotic use. Additionally, throat culture is an easy tool. Higher prevalence of GAS infection among children and risk of complications make GAS infection case finding very important. Children with GAS pharyngitis have to be vigorously treated not only to prevent complication but also to limit its spread to susceptible individuals. Penicillin is the drug of choice for GAS infections and macrolides are commonly used for allergic individuals.

Genetic diversity among GAS bacterium contributes to variations in virulence. Knowledge about molecular pathogenesis of ARF and Rheumatic Heart Disease and its association with GAS strains can be supplemented by studying throat swab cultures in asymptomatic and symptomatic individuals, as throat swab cultures are usually negative in children with these manifestations.

Continuous surveillance is also needed because of changing epidemiology. Changes in prevalent strains with disappearance of certain serotypes and replacement with new ones need to be studied. Emm typing helps in understanding pathogenesis and allows finding new types.