DOI: https://doi.org/10.21203/rs.2.12479/v1
Streptococcus pyogenes (S. pyogenes) is a beta-hemolytic streptococcus which is classified as Lancefield Group A Streptococcus (GAS). S. pyogenes has remained a significant human pathogen for centuries. It causes a wide variety of infections in humans ranging from mild skin and upper respiratory tract (URT) infections to severe life-threatening conditions such as Rheumatic fever, glomerulonephritis, septicemia, pneumonia and streptococcal toxic shock syndrome (1).
Infection begins with colonization of the URT or injured skin surfaces. All age group may carry GAS on throat and epidermis of skin, however, children aged 5-15 years old are a major reservoir of pharyngeal carriage of GAS (2).
GAS is highly communicable and can cause disease in individuals of all ages. School-age children (5-15 years) are considered as the major reservoir of GAS, with a prevalence of 2.5-25% or more depending on the study setting (3). In Ethiopia, the asymptomatic carriage rate of GAS among healthy schoolchildren was 9.7-16.9% (4, 5).
An educational status, employment status of the family, separation of mother and father (6), sex (7-10), socioeconomic and environmental factors, lack of awareness of disease transmission are the risk factor for colonization of the S. pyogenes among school children.(11, 12).
GAS has not been developed resistance to any of the penicillin’s over the last decades. Nowadays, it is starting to appear antimicrobial drug resistant strain from asymptomatic children (13) including penicillin (6). However, in Ethiopia, antimicrobial resistance pattern to the GAS isolated from throat/phyeranax were not adequately explicated. Therefore, the present study was aimed to determine the carriage rate of S. pyogenes, associated risk factors and antibiotic susceptibility pattern among school children in Hawassa, South Ethiopia, 2018.
Hawassa is the capital city of the Southern Nation Nationalities and people’s Government. It is located 275km South of Addis Ababa, and has an altitude of 1665m above sea level with mean annual temperature and rainfall of 20.9OC and 997.6mm, respectively.
Study Design
A school based cross-sectional study design was conducted from May-October 2018 in Hawassa, South Ethiopia.
Source population
All school children who attached governmental primary schools found in Hawassa town during the study period.
All children aged 5-15years who attend the class in selected schools during the study period and children whose parents had accepted the consent to participate in the study.
Exclusion Criteria
All children who were on antibiotics for the last two weeks those with any signs and symptoms of respiratory diseases such as fever, soreness and throat, cough and watery nasal discharge were excluded.
Study population
A total of 295 school children were enrolled in this study. The study participants were selected by using a multi-stage stratified sampling technique. From the total of 19 governmental primary schools 30%(5 of the schools) were included. Simple random sampling technique was used to select the schools. Proportionate amount of samples were assigned to each selected schools. Then study participants were identified by the lists of students using simple random sampling technique.
Specimen collection
All the swabs were immediately transferred to Amies transport medium (Oxiod, UK). Each sample was labeled very well. Within 2 hours the collected swab were transported to the Microbiology laboratory (9, 14, 15).
Isolation of GAS
The throat swabs were directly inoculated to 5% sheep blood agar plates (Blood agar base, Oxiod UK) by rolling the swab over a small area of the plate and streaking the sample using a sterile loop and incubated at 37ºC with 5% CO2 atmosphere and examined beta-haemolytic colonies after 24hrs and 48 hrs. Beta-haemolytic streptococci were identified by their colony morphology and beta-haemolysis.
All plates with beta-haemolytic colonies were sub-cultured with 0.04U Bacitracin disks (Oxoid, Uk) in blood agar plates similar manner as an earlier. All grams positive, catalase negative and any zone of inhibition around the disk were candidate for Pyrrolidonyl arylamidase (PYR) tests. A purple color in PYR tests were identified as S. pyogenes according to publications (16-18).
Antimicrobial susceptibility testing
Antibiotic susceptibility test (AST) was performed on disc diffusion method for all S. pyogenes using Penicillin(10U), Erythromycin(15µg), Amoxicillin(10µg), Chloramphenicol(30µg), Ceftriaxone(30µg), Vancomicin(30µg) and Tetracycline(10µg).
Data management and quality control
Quality of the data was ensured by using pre-structure questionnaire. For laboratory analysis the sterility of the prepared media was checked by incubating 5% by of prepared with in 5% CO2 enriched atmosphere at 37oc for 24hrs before using it. A quality control strain of S. pyogenes (ATCC19615) was used a positive control for each test.
Data processing and analysis
Data entry and analysis was performed by using SPSS version 20. The frequency of variables the prevalence of S. pyogens, and antibiotic susceptibility pattern was determined. The association between risk factors and S.pyogenes colonization was determined by using logistic regression. A p-value <0.05 at 95% confidence interval (CI) was considered statistically significant.
Socio-demographic characteristics
Out of 295 school children who participated in the present study, 147(51.2%) were males, 250(87.8%) were within the age of 5-12 years. About 41.8% of the study participants found to have no formal education. A fifty percent of students’ mothers were a house-wife. About 162(56.4%) of a total children’s parents/guardian had a monthly income between 500-900ETB, 33(11.5%) had 1000-1500 ETB and 92(32.1%) had higher than 1500ETB per month.
Among 287 school children 35(12.2%) 95% CI [19-27.8] were confirmed to have S. pyogenes in throat swabs. A colonization rate of S. pyogene among children who were 5-8 years old, 9-12 years old, 13-15 years old, those who live with employed mother, those who live with poor income source were 12(17.1%), 18(10.0%), 5(13.5%), 8(17.0%), and 26(16%) respectively (Table 3).
The prevalence of S. pyogenes was higher among children with employed mother 8(17.0%) than other occupations. Highest carriage rate was detected in low socioeconomic class 500-900ETB per month 26(16.0%) followed by 1000-1500ETB 4(12.1%). Among a total of 35(12.2%) S. pyogenes isolates, the highest carriage rate was observed in student’s family size more than 5 person per house 23(12.6%).
Out of 35 S. pyogenes isolated in this study, 35(100%), 26(74.3%), and 15(42.9%) were susceptible to penicillin, Vancomycin and Tetracycline respectively. About 34(97.1%) of S. pyogens isolates were sensitive to Erythromycine, Chloramphenicol, Ceftraxone and Amoxacline (Table 1 and 2).
Table 1 Antimicrobial susceptibility pattern of S. pyogens isolated from school children at Hawassa city from May-October 2018 (n=35)
|
Antimicrobial agents |
Resistant n (%) |
Susceptible n (%) |
|
Penicillin |
0(0.0%) |
35(100%) |
|
Vancomycin |
9(25.7%) |
26(74.3%) |
|
Erythromycin |
1(2.9%) |
34(97.1%) |
|
Chloramphenicol |
1(2.9%) |
34(97.1%) |
|
Ceftriaxone |
1(2.9%) |
34(97.1%) |
|
Amoxacline |
1(2.9%) |
34(97.1%) |
|
Tetracycline |
20(57.1%) |
15(42.9%) |
Table 2 The predominant multiple antibiotic resistant phenotypes for S. pyogenes isolated from school children at Hawassa city from May-October 2018 (n=24)
|
Phenotypes |
Isolates tested N (%) |
|
Tetracycline |
13(54.2) |
|
Vancomycine |
4(16.7) |
|
Tetracycline, Amoxicillin |
1(4.2) |
|
Tetracycline, Ceftriaxone |
1(4.2) |
|
Tetracycline, Vancomycine |
4(16.7) |
|
Tetracycline, Vancomycine, Erythromycin |
1(4.2) |
Risk factor analysis for pharyngeal carriage
The possible risk factors such as age, sex, children living status, parents/guardians occupation, parents/guardians education, income of parents, family size, person per bed room sharing, and past history of recurrences of URTI were evaluated for pharyngeal carriage of S. pyogenes. It was observed in bivariate analysis that, female children (COR =2.212; 95%CI= 1.055-.638; p=.013), low income of parents (COR =3.326; 95%CI= 1.231-8.990; p=0.001), children being with mother(COR=0.34; 95%CI=0.2-1.6; p=0.301), and occupational status of mothers (COR =1.8(1.2-4.40) 95%CI=1.2-4.40; p=0.02 were observed.
The female children (AOR=2.730; 95%CI=1.24-6.037; p=0.013), and low income of parents (AOR=11.917; 95%CI=2.729-2.032; p=0.001) were associated with S. pyogenes carriage. Conversely, Occupational status of mothers (AOR=100; 95%CI=.023-.437; p=0.002) was associated with reduced likelihood of risk for asymptomatic pharyngeal carriage of S. pyogenes (Table 3).
Table 3 Distribution and association of S. pyogenes among school children in Hawassa from May-October 2018
|
Variables |
Total S. pyogenes |
|
Total N=287(100%) |
P value |
||
|
Present n=35(12.2%) |
Absent n=252(87.8%) |
COR(95% CI) |
||||
|
Sex |
Female |
23(16.4) |
117(83.6) |
1 |
140(48.8) |
0.013 |
|
Male |
12(8.2) |
135(91.8) |
2.21(1.8-3.14) |
147(51.2) |
||
|
Age |
5-8 |
12(17.1) |
58(82.9) |
0.76(0.5-1.8 |
70(24.4) |
0.123 |
|
9-12 |
18(10.0) |
162(90.0) |
1.41(0.8-3.20) |
180(62.7) |
0.626 |
|
|
13-15 |
5(13.5) |
32(86.5) |
1 |
37(12.9) |
||
|
Children Living Status |
With others* |
7(14.9) |
40(85.1) |
0.64(0.3-1.90) |
47(16.4) |
0.427 |
|
Mother only |
7(25.0) |
21(75.0) |
0.34(0.2-1.6) |
28(9.8) |
0.301 |
|
|
Father Only |
0(0.0) |
4(100.0) |
0 |
4(1.4) |
0.264 |
|
|
Both parent |
21(10.1) |
187(89.9) |
1 |
208(72.5) |
||
|
Educational status of parents |
Illiterate |
19(15.8) |
101(84.2) |
0.89(0.5-2.20) |
120(41.8) |
0.365 |
|
1-4 |
1(5.0) |
19(95.0) |
3.2(1.8-4.80) |
20(6.7) |
0.309 |
|
|
5-12 |
10(8.9) |
102(91.1) |
1.7(0.9-2.6) |
112(39.0) |
0.546 |
|
|
>12 |
5(14.3) |
30(85.7) |
1 |
35(12.2) |
||
|
Occupational status of Mothers |
Others** |
10(10.2) |
88(89.8) |
1.8(1.2-4.40) |
98(34.1) |
0.002 |
|
House Wife |
17(12.0) |
125(88.0) |
1.5(0.7-3.20) |
142(49.5) |
0.002 |
|
|
Employed |
8(17.0) |
39(83.0) |
1 |
47(16.4) |
||
|
Occupational status of Fathers |
Others*** |
21(14.8) |
121(85.2) |
0.35(0.2-1.50) |
142(49.5) |
0.446 |
|
Farmer |
7(10.8) |
58(89.2) |
0.26(0.18-1.94) |
65(22.6) |
0.602 |
|
|
Employed |
7(8.8) |
73(91.2) |
1 |
80(27.9) |
||
|
Family income |
500-900 |
26(16.0) |
136(84.0) |
0.3(0.2-1.68) |
162(56.4) |
0.001 |
|
1000-1500 |
4(12.1) |
29(87.9) |
0.42(0.3-2.20) |
33(11.5) |
0.042 |
|
|
>1500 |
5(5.4) |
87(94.6) |
1 |
92(32) |
||
|
Past history of recurrent of URTI/RHD |
Yes |
6(14.6) |
35(85.4) |
0.78(0.6-1.70) |
41(14.3) |
0.607 |
|
No |
29(11.8) |
217(88.2) |
1 |
246(85.7) |
||
|
Number of family per house |
<5 |
12(12.5) |
84(87.5) |
1 |
96(33.4) |
0.208 |
|
>5 |
23(12.0) |
168(88.0) |
10.9 (4.0-22.0) |
191(66.6) |
|
|
|
Residence |
Urban |
16(22.9) |
54(77.1) |
1 |
70 (24.4) |
|
|
Rural |
19(8.8) |
198(91.2) |
3.1 (1.7-5.8) |
217(75.6) |
0.002 |
|
|
Cooking in bed room |
Yes |
10(7.0) |
133(93.0) |
2.79(1.2-6.4) |
143(49.8) |
0.321 |
|
No |
25(17.4) |
119(82.6) |
1 |
144(50.2) |
|
|
|
Previous any disease |
Yes |
25(12.8) |
170(87.2) |
0.83(0.6-2.1) |
195(67.9) |
|
|
No |
10(10.9) |
82(89.1) |
1 |
92(32.1) |
- |
|
|
History of Hospitalization |
Yes |
22(9.1) |
220(90.9) |
4.1(1.9-9.2) |
44(15.33) |
0.000 |
|
No |
13(28.9) |
32(71.1) |
1 |
45(15.7) |
|
|
*=guardians/care givers, **= both mother and father, *** =daily labor, merchant, students
The overall asymptomatic pharyngeal carriage rate of S. pyogenes among school children was 12.2% which is higher than the reports in Ethiopia 9.7%(4), in Tunisia 9.0%(19), in Nigeria 10% (20), Pemba 8.6%(21), India 8.4% (12), and Mangalore 5%(7) and lower than the report of Ethiopia 16.9%(5), Turkey 13.9%(22), Pennsylvania 15.9%(11), Australia19.5%(23) and Turkey 25.9%(15). The possible explanation for the variation might be due to vaccination status and age differences. Moreover, sample size, seasonal variation and method if used, geography and socio-demographic variation are another possible explanation of the difference (4, 11, 21, 24).
In the present study we assessed different factors that could possibly increase colonization rate of S. pyogens. Having female children 23(16.4%) were 2.21 times more risk than male children for S. pyogens colonization (p=0.013). Similar result was reported from India(6), Turkey(4) and Nepal(5) and Ethiopia(4, 11, 21, 24). This is might be due to social attitude towards female children or high contact with the others during supporting their mother in daily tasks.
The detection rate of S. pyogenes were high in children who had illiterate parents 19(15.8%) (p>0.05). It was in-lined with study reveled in India 44.9%(14), and in Iraqi 66.7%(25). This might be reflects the literate parents had better awareness of the disease transmission than the illiterate parents.
There was clear association of pharyngeal carriage rate and children from low income families in our finding which 26 were (16.0%). It was in accordance with the earlier finding in Ethiopia(5) and other studies carried out in many parts of the world (7, 25).
The carrier group of S. pyogenes was higher 10.9 times more among children living within families of more than 5 members than less family members in our study (p>0.05). Similarly, S. pyogenes carriage was significantly higher in large family size as reported in different authors (p< 0.05)(2, 23, 26). This might be an increasing the number of family members increases the rate of prevalence of many infectious diseases including GAS.
A Children with a history of hospital admission had 4.1 times chance to be colonized with GAS (p=0.00) which is comparable to other’s studies(27). A children with history of recurrent pharyngitis show higher incidence 8(80%) of carrier than those with no history of pharyngitis16(17.8%)(25).
A children from rural resident were 3 times (p=0.002) more likely to have S. pyogens carriage. This finding is similar to that the report found in Uganda at Wakisso district (28). In the current study, a high proportion (12.8%) of previously diseased children was colonized with S. pyogens even though it was not statistically significant (p>0.05).
Out of 35 of S. pyogens isolated in this study, 35(100%), 26(74.3%) and 15(42.9%) were susceptible to Penicillin, Vancomycin and Tetracycline respectively. However, 34(97.1%) of S. pyogens were susceptible to Erythromycin, Ceftriaxone, Chloramphenicol and Amoxacline. The finding is similar as compared with the report found in Ethiopia (4) and in different parts of the world (7-9, 12-14, 29-34). About 7(29.2%) of isolated GAS strains were showed multiple drug resistant (two or more drugs). This is might be due to the ease of availability of antibiotics and misuse of the drugs.
The prevalence of S. pyogenes among school children in this study was high. The gender difference and low income of parents, residence of the children and occupational status of the mother are the prime risk factors associated with the carriage rate. All S. pyogenes isolates were susceptible to penicillin but most isolates were susceptible to chloramphenicol, Erythromycin and Ceftriaxone. Low level of resistant was observed against vancomycin and high level was observed against tetracycline. Further study in the area by using large sample size and all predisposing factors should be investigated.
Serotyping of Group A streptococci and ASO titer was not performed due to lack of antisera.
Acknowledgements
We would like to acknowledge data collector, all staff working at kindergarten school who facilitated data collection, Hawassa University College of Medicine and Health Sciences, Jimma University Health Science institute for their support during the study. We would also acknowledge parents who allowed their children to participate in this study.
Authors’ contributions:
AA: Conceived and designed the study, performed the laboratory work, analyzed the data, involved in manuscript preparation. GB and ZS: Involved in protocol development and manuscript write up. DDG: Conceived and designed the study, supervised the study, involved in analysis and manuscript preparation. All authors read and approved the final manuscript.
Funding:
This study was supported by Jimma University, institute of Health Sciences. The support included payment for data collectors and purchase of materials and supplies required for the study. The support did not include designing of the study, analysis, and interpretation of data, and manuscript preparation.
Availability of data:
The data used/analyzed during the current study available from the corresponding author on reasonable request.
Ethics approval and consent to participate:
This study was ethically cleared from the Institutional Review Board (IRB) of the College of Medicine and Health Sciences, Hawassa University. Official permission was obtained from the study site and written informed consent was obtained from all parents/guardians of the children.
Consent for publication:
Not applicable.
Competing interests
The authors declare that they have no competing interests.