Prevalence, Antibiotic Susceptibility Pattern and Associated Factors of Streptococcus pyogenes among Pediatric Patients with Acute Pharyngitis in Sidama, Southern Ethiopia

Background Streptococcus pyogenes is the most frequent cause of pharyngitis and skin infections in children and causes immune complications like rheumatic fever and rheumatoid heart disease (RHD), particularly in developing countries like Ethiopia. The aim of this study was to determine the prevalence, antibiotic resistance pattern, and associated factors of Streptococcus pyogenes among pediatric patients suspected of acute pharyngitis in Sidama Region, Southern Ethiopia. Methods A cross-sectional study was conducted on 213 acute pharyngitis suspected pediatric patients from April to September 2022 at Hawassa University Compressive Specialized Hospital and Yirgalem Hospital. Sociodemographic and clinical data were collected using a structured questionnaire. A throat swab was cultured to isolate S. pyogenes, and antimicrobial susceptibility testing was done using standard bacteriological techniques. Data were analyzed using SPSS version 25, and P value of <0.05 was considered as statistically significant. Result Out of 213 throat swabs cultured, 22 (10.3%) with 95% CI (6.6–14.6%) were S. pyogenes positive. All isolates of S. pyogenes were sensitive to penicillin and amoxicillin. In contrast, 8 (36.4%) isolates exhibited resistance to tetracycline, 7 (31.8%) to ceftriaxone, 6 (27.3%) to erythromycin, and 5 (22.7%) isolates showed multidrug resistance. The presence of palatal petechiae (P=0.037) and tonsillar swelling or exudate (P=0.007) were significantly associated with S. pyogenes carriage in children suspected of having acute pharyngitis. Conclusion In this study, the prevalence of S. pyogenes among children suspected with acute pharyngitis was low compared to other studies. The isolates showed a high level of resistance to commonly used antibiotics. Therefore, the treatment of pediatric acute S. pyogenes pharyngitis should depend on an antimicrobial susceptibility test. Furthermore, evaluation of S. pyogenes pediatric acute pharyngitis risk factors and tracking of antibiotic resistance are crucial in the controlling of pediatric acute S. pyogenes pharyngitis.


Introduction
Streptococcus pyogenes (S. pyogenes) is a Gram-positive, facultative anaerobic, β-hemolytic bacterium classifed as group A streptococcus based on carbohydrate it contains [1,2].It is the most frequent cause of skin and oropharyngeal infections, which can develop into more serious invasive and suppurative conditions like toxic shock syndrome and necrotizing fasciitis, which have been associated to increased rates of morbidity and mortality globally [3][4][5].Of these infections, pharyngitis is the most common among children, particularly those between the ages of 5 and 14 years.It causes 288.6 million episodes, with a high episode rate of 40 per 100 child in the low-and middle-income countries (LMICs), translating into 0.1 million disability-adjusted living years (DALYs) yearly [6].Te frequency of S. pyogenes pharyngitis was highest in sub-Saharan Africa, with a rate of 5.7 per 1000, as compared to 1.8 per 1000 in North Africa and 0.3 per 1000 in developed nations [7].For example, a 2017 study conducted in Uganda found that of 600 million cases of pharyngitis, 450 million cases were thought to be caused by S. pyogenes per year, with 20 to 40% of those cases being in children [8], despite, there are few publications of S. pyogenes in Africa [9].S. pyogenes is a contagious organism and can spread through direct or indirect contact with respiratory tract secretions, wounds, or sores on infected people's skin [10].Te infection varies depending on a number of factors, including age, gender, and standard of living in addition to seasonal variations and indoor air pollution [11].S. pyogenes remains susceptible to penicillin, and penicillin has proven to be an efective empirical treatment antibiotic for S. pyogenes infection.However, patients who have developed allergies to penicillin are treated with alternative antibiotics such as cephalosporin, lincosamides, and macrolides drugs [12,13].However, currently there have been varying reports of S. pyogenes resistance to cephalosporin, lincosamides, and macrolides (erythromycin and azithromycin) [4,12,14].Tis antibiotic resistance of S. pyogenes is mainly associated with drug overuses and poststreptococcus infection complication such as glomerulonephritis, rheumatic fever (RF), and rheumatic heart disease (RHD) which is the main concern worldwide [15,16].
In Ethiopia, data regarding S. pyogenes in children suspected with pharyngitis and antibiotic resistance are extremely limited, especially in the study area.Te earlier study conducted in the study area found that 12% of children had asymptomatic pharyngeal carriage of S. pyogenes [17].Tis was somewhat higher than the 9.1-11.3%prevalence of S. pyogenes among children in other regions who were suspected of having pharyngitis [17][18][19] and demand great attention.Additionally, both nationally and in the study area, empirical medications are the mainstay of treatment for S. pyogenes pharyngitis in Ethiopia.Despite S. pyogenes pharyngitis infection's high resistance to clindamycin, ceftriaxone, cefotaxime, cefepime, and erythromycin, antibiotics are prevalent in Ethiopia [18].Also, tetracycline and azithromycin resistance was highly observed in both asymptomatic and symptomatic S. pyogenes pharyngitis infections [17,19,20] in addition to multidrug resistant (MDR) cases [18].Terefore, the aim of this study was to determine the prevalence of S. pyogenes, antibiotic susceptibility pattern, and associated factors among children suspected with acute pharyngitis in Sidama, Southern Ethiopia. million peoples in Sidama region, Southern Nations, Nationalities, and Peoples (SNNPR), Southern Oromia, and some part of Somalia regions.Similarly, Yirgalem General Hospital which is located 322 km from Addis Ababa and 47 km from Hawassa City, the second largest hospital in Sidama region gives diferent health services in the catchment area and the community from surrounding.

Study Population.
All pediatric patients who were attending pediatric emergency wards in both hospitals during the study period were the study population.

Inclusion and Exclusion
Criteria.All children under 18 years of age with symptoms of acute pharyngitis whose parents accepted the consent were included in this study; however, children who have been on antibiotics in the last two weeks were excluded from the study [18].

Sample Size Determination and Sampling Techniques.
Sample size was determined using the following assumptions: 95% confdence interval (CI), power of 80%, and previous odds ratio of associated factors from Bahir Dar [18], by using EPI INFO stat calc version 7 software.To minimize errors arising from the likelihood of noncompliance, 10% of the sample size was added.Finally, by considering the 10% nonresponse rate, the sample size calculated was 213.A systematic random sampling technique was applied to select 213 respondents for interview.

Sampling Procedure and Sampling
Technique.Te participant's proportional allocation was calculated based on previous child fow data due to acute pharyngitis per month from each hospital's logbook.A systematic random sampling technique was applied to select 213 children who were suspected of having acute pharyngitis: 142 from HUCSH and 71 from Yirgalem General Hospital.To choose the frst participant, the K interval was calculated.After the K interval was calculated, the frst individual was selected by the lottery method and the others at a regular interval.

Operational Defnition. Multidrug resistance (MDR):
a species of microorganisms that is resistant to at least one agent in ≥3 antimicrobial categories [21].
Acute pharyngitis: a clinical feature characterized by sudden onset of sore throat, palatal petechiae, anterior cervical lymphadenopathy, and tonsillar swelling with or without exudates [22].

Collection of Sociodemographic and Clinical Data.
Sociodemographic characteristics data from the parents or children, as well as the children's clinical history, were collected by trained nurses using a structured questionnaire under physician supervision during children acute pharyngitis clinical examination.

2
International Journal of Microbiology 2.8.Sample Collection and Transportation Methods.Two throat samples were collected by trained nurses, one for Gram staining and the other for culture purpose, by using a sterile cotton swab and a sterile disposable tongue depressor from each child under physician supervision.Each collected swab was immediately immersed in the Amies transport medium (Oxoid, England).Samples were then transported to the Hawassa University School of Medical Laboratory, Microbiology laboratory within half an hour at room temperature from the Hawassa University Comprehensive Specialized Hospital, and using a cold bag from Yirgalem General Hospital.
2.9.Streptococcus pyogenes Identifcation.Te throat swab was inoculated on blood agar plates (blood agar base Oxoid UK) by rolling the swab over a small area of the plate and streaking the sample using a sterile loop.Te plates were then incubated for 24 hours at 37 °C in a candle jar, which can provide an atmosphere of 5% CO 2 .A plate culture that was negative for beta-hemolytic colonies after 24 hours was incubated for an additional 24 hours to allow the growth of slow growers.Beta-hemolytic streptococci were checked by their colony morphology, beta-hemolysis, and gram stain.Grampositive and catalase-negative streptococcus bacteria were confrmed by bacitracin disc sensitivity and pyrolidonyl arylamidase (PYR) tests.Bacitracin-sensitive and a purple color in PYR tests were identifed as S. pyogenes [23].

Statistical Analysis.
Data were entered into Epi Info version 3.1 and analyzed using SPSS version 25. S. pyogenes prevalence, variable frequency, and antimicrobial susceptibility pattern were determined by descriptive statistics.Bivariate logistic regression analysis was used to determine the associated risk factors.For those variables which have a P value <0.25 in the bivariate, the analysis was further entered into the multivariable logistic regression model and a P value of <0.05 was considered statistically signifcant at 95% CI.
2.12.Data Quality Control.Prior to actual data collection, the quality of the data was assured by pretesting 5% of questionnaires at Adare General Hospital, Hawassa City.
Training was given to the data collector's physician and nurses.For the laboratory test, the sterility of prepared culture media was checked by incubating the 5% of prepared media in a 5% CO 2 -enriched atmosphere at 37 °C for 24 hours.For each test, a quality control strain of S. pyogenes (ATCC19615) from the Ethiopian Public Health Institute (EPHI) was used as the positive control to check all media, biochemical tests, and antibiotics.To maintain the quality of the work and data management, the standard operating procedure for sample collection and laboratory analysis was strictly followed.1).Te overall prevalence of S. pyogenes was 22 (10.3%) and was more predominant in males, 17 (8.0%)than females.S. pyogenes acute pharyngitis was 12 (5.6%)more prevalent in children aged <5 years.Among the children with culture proven S. pyogenes pharyngitis, 15 (7.0%) of their parents were working in private occupations.Furthermore, S. pyogenes pharyngitis was more prevalent among children with a previous history of tonsil, 15 (7.0%), and tonsillar swelling or exudate, 14 (6.6) (Table 1).

Factors Associated with S. pyogenes Pharyngitis.
In bivariate analysis, sex, age, parent occupation, number of windows, tonsillar swelling or exudate, nasal fow, and vomiting were found to be statistically signifcant at a P value of <0.25 and were therefore taken into consideration as potential candidates for multivariate analysis.After adjusting other confounding variables, boys' children were 3.91 times more likely to develop S. pyogenes acute pharyngitis than girls' children (AOR � 3.91; 95% CI � 1.297-11.792;P � 0.015), children with palatal petechiae were 65% more likely to develop S. pyogenes acute pharyngitis than without (AOR � 0.35; 95% CI � 0.131-0.938;P � 0.037), and children with tonsillar swelling or exudate were 3.83 times more likely to develop S. pyogenes acute pharyngitis than those who have not developed tonsillar swelling or exudate (AOR � 3.83; 95% CI � 1.446-10.188,P � 0.007) (Tables 3 and 4).
In this study, all S. pyogenes isolates were sensitive to penicillin.Tis result was consistent with previous studies reported from diferent parts of Ethiopia [18][19][20] and studies from China [3], Iran [29], Israel [13], Zambia [22], and Tailand [26].In addition, the current study revealed that S. pyogenes was 100% sensitive to amoxicillin, which was similar to a previous study in Jimma, Ethiopia, that reported S. pyogenes 100% sensitivity to amoxicillin [19].In contrast, a study conducted in Sudan reported that 58.8% of the isolates were resistant to amoxicillin [1].Majority of the S. pyogenes isolates in the current study were also susceptible to the actions of azithromycin (86.4%), cefepime (90.9%), and clindamycin (95.5%).Tis result was higher than those study's results reported from Bahir Dar, Northern Ethiopia [18], and Gondar, Northwest Ethiopia [20].
Te relatively higher level of resistance in the study from Bahir Dar may be due to a diference in the level of antibiotic use in Bahir Dar [18] and Gondar [20] studies.
In the contrast, the isolates demonstrated resistance of 36.4%, 31.8%, and 27.3% to tetracycline, ceftriaxone, and erythromycin, respectively.Similarly, S. pyogenes was found to be resistant to those antibiotics in other studies.Te current tetracycline fnding was greater than studies conducted in Gondar, Northwest Ethiopia, 21.7% [20], and Bahir Dar, Northern Ethiopia, 14.3% [18], but lower than Jimma, Southwest Ethiopia, 52.5% [19].Additionally, the rate of erythromycin resistance is higher than in previous studies conducted in Ethiopia, Jimma [19], no resistance, Gondar 4.3% [20], and Bahir Dar 21.4% [18], and other countries like India with no resistance [27], Tailand 18.2% [26], but less than the fnding from Iran 37.2% [29].Similarly, the fnding of ceftriaxone resistance was greater than a study conducted in Gondor, Ethiopia, 13% [18], but lower than a study in Tailand with no resistance [27].Furthermore, 22.7% of the S. pyogenes isolates in this study were found to be multidrug resistant (MDR), which was marginally higher than the 21.3% found in a previous study conducted in Bahir Dar, Northern Ethiopia [18].Te observed variations in antibiotic sensitivity and phenotypic multidrug resistance in S. pyogenes could potentially be attributed to noncompliance with medication regimens or excessive drug consumption within the study area, as well as empirical pharyngitis treatment practices [33].Furthermore, the primary emergence of MDR secondary to drug resistance is maybe due to beta-lactamase enzyme production, which carries genes specifc to drug resistance, modifes antimicrobial targets, and activates the drug efux pump of S. pyogenes [2,12].
According to the associated factors, in the current study, boys were 3.91 times more likely than females to experience S. pyogenes acute pharyngitis (P value � 0.015).Tis result is in line with other studies reported from Taiwan [34] and Japan [30] which revealed that S. pyogenes pharyngitis is more associated with boys than girls.Similarly, the review International Journal of Microbiology studies conducted in China [35,36] and United States [37,38] also stated that S. pyogenes infection was more prevalent in boys than in girls.However, this result was at odds with previous Ethiopian studies carried out in Jimma [19], Gojam [20], and Bahir Dar [18] which, despite being statistically not signifcant, reported a higher prevalence of S. pyogenes pharyngitis in boys than in girls.Tis diference may be due to variations in study subjects, living standards, parental occupation, seasonal variations, and immune status between males and females [11,39].Concerning clinical factors, the presence of palatal petechiae and tonsillar swelling or exudate was signifcantly associated to children with S. pyogenes acute pharyngitis with P values of 0.037 and 0.007, respectively.Tis fnding was in agreement with the previous studies of Jimma, Southwest Ethiopia [19], and Gondar, Northwest Ethiopia [20], and the other study from the United States of America [40].However, our fnding was in contradiction with the study from California [41] that stated a patient with no palatal petechiae clinical symptom was positive for S. pyogenes.In fact, palate petechiae and swollen tonsils or exudate were the most prevalent symptoms of S. pyogenes pharyngitis with sudden onset of fever and sore throat [29,42,43].However, the geographical location and study subjects have an impact on the clinical characteristics of S. pyogenes pharyngitis [32].

Limitations of the Study.
We acknowledge that there are some limitations in the study.Te limitation of this study is its small size and small number of study sites which may underestimate the prevalence of S. pyogenes.Moreover, we did not perform the minimum inhibitory concentration (MIC) antimicrobial susceptibility test method rather than disc difusion.
Yirgalem General Hospital, in Sidama region, Southern Ethiopia, from April 15 to September 10, 2022.Hawassa University Comprehensive Specialized Hospital (HUCSH) is located at Hawassa City which is 275 km from Addis Ababa, the capital city of Ethiopia.It is the only comprehensive specialized hospital in Southern Ethiopia and provides diferent health services for more than 18 Consent to Participate.Ethical approval was obtained from the Institutional Review Board of Hawassa University, College of Medicine and Health Sciences (Ref.No: IRB/010/14).Permission was obtained from the clinical and academic director of HUCSH and Yirgalem General Hospital.Before enrolling, any of the eligible study participants' written informed consent was obtained from the children's parents or caregivers, and assent was also obtained from minors.Finally, all procedures were conducted based on the ethical Declaration of Helsinki guidelines, and all information was kept confdential.All positive results were reported to the Pediatrics Emergency Department of HUCSH and Yirgalem General Hospital physicians for appropriate antibiotic treatment.

Table 1 :
Prevalence of S. pyogenes with respect to sociodemographic and clinical characteristics of pediatric patients with acute pharyngitis at HUCSH and Yirgalem General Hospital, 2022.
Antibiotic susceptibility pattern of S. pyogenes among pediatric patients with acute pharyngitis at HUCSH and Yirgalem General Hospital, 2022.

Table 2 :
Multiple drug resistant of S. pyogenes isolated from pediatric with acute pharyngitis at HUCSH and Yirgalem General Hospital, 2022.

Table 3 :
Bivariate and multivariable logistic regression analysis of factors associated with S. pyogenes acute pharyngitis at HUCSH and Yirgalem General Hospital, 2022.
Key: AOR: adjusted odds ratio, COR: crude odds ratio, CI: confdence interval, Note.* Indicates the signifcant relation between variables, P over, continued surveillance of S. pyogenes prevalence, antibiotic resistance, and risk factors is very important in controlling S. pyogenes pediatric acute pharyngitis.Developing specifc guidelines for S. pyogenes and strengthening of laboratory infrastructure are signifcant to minimize the empirical treatment of S. pyogenes pediatric acute pharyngitis in Ethiopia.

Table 4 :
Bivariate and multivariable logistic regression analysis of the clinical factor with S. pyogenes acute pharyngitis at HUCSH and Yirgalem General Hospital, 2022.Indicates the signifcant relation between variables, P value <0.05.8 International Journal of Microbiology the original draft, project administration, and fnal version approval.DAF conducted analysis and interpretation of data, project administration, visualization, writing, reviewing, and editing, and fnal version approval.AAN collected materials, performed analysis and interpretation of data, writing, reviewing, and editing, and fnal version approval.BJH performed analysis and interpretation of data, visualization, writing, reviewing, and editing.