Sudan Journal of Medical Sciences Volume 18, Issue no. 1, DOI 10.18502/sjms.v18i1.12861 Production and Hosting by Knowledge E Research Article Epidemiological Aspects and Antibiotics Susceptibility Patterns of Streptococcus pyogenes Isolated from Subjects with Tonsillitis, Sudan Elnaim Bushra Ahmed1,2, Elsir Ali Abu Groun3, Babiker Saad Almugadam4, Yousif Musa Alobaid Ahmed4, Aymen Mudawe Nurain Mudawe5, Nassir Abakar Babiker6, and Nadir Abuzeid1* 1Department of Microbiology, Faculty of Medical Laboratory Sciences, Omdurman Islamic University, Omdurman, Sudan 2Department of Medical Laboratory Investigations, Kosti Police Hospital, Kosti, Sudan 3Department of Microbiology, Faculty of Medical Laboratory Sciences, University of Khartoum, Khartoum, Sudan 4Department of Microbiology, Faculty of Medical Laboratory Sciences, University of El Imam El Mahdi, Sudan, Sudan 5Delta College of Science and Technology, Omdurman , Sudan 6Central Medical Laboratory, Wad Medani Teaching Hospital for Obstetrics & Gynecology, Wad Medani, Sudan ORCID: Elnaim Bushra Ahmed: https://orcid.org/0000-0003-1066-9245 Babiker Saad Almugadam: https://orcid.org/0000-0003-3014-8359 Yousif Musa Alobaid Ahmed: https://orcid.org/0000-0002-0863-3262 Nadir Abuzeid: https://orcid.org/0000-0003-2074-7892 Abstract Background: Globally, Streptococcus pharyngitis is a major public health challenge. The current study investigates the prevalence of Streptococcal pyogenes among children under 17 years old in ENT Kosti Teaching Hospital and examines the susceptibility of isolated S. pyogenes strains to commonly used antibiotics. Methods: A total of 384 throat swabs were obtained from children under the age of 17 who attended the Kosti Teaching Hospital between 2019 and 2021. Streptococcus pyogenes was isolated by conventional microbiology procedures. Each S. pyogenes strain was subjected to antibiotic susceptibility testing according to the CLSI guidelines. Results: Most participants of this study were females 219 (57%) and aged between 5 and 10 years 259 (67.4%). Out of the 384 participants, 134 (34.9%) and 255 (66.4%) suffered from lymphadenopathy and tonsil hyperplasia, respectively. Interestingly, lymphadenopathy and tonsil hyperplasia were more (P � 0.05) in the 5–10 age group than those aged 11–16 years. Moreover, 41.4% of the participants were infected by a GAS sore throat. GAS sore throat is significantly associated with lymphadenopathy (AOR: 2.375, 95% CI: 1.479–3.815, P � 0.000) and tonsil hyperplasia (AOR: 3.374, 95% CI: 1.939–5.874, P � 0.000). Notably, males (AOR: 0.853, 95% CI: 0.549–1.325, P 0.479) and individuals aged 5–10 years (AOR: 0.867, 95% CI: 0.464–1.618, P 0.654) were less likely to have a GAS sore throat. In our study, all isolated strains were sensitive to penicillin. Clindamycin, azithromycin, and erythromycin resistance were observed in 7 (4.4%), 44 (27.7%), and 47 (29.6%) isolates, respectively. Conclusion: The study displayed the current situation of GAS sore throat in the White Nile state. Penicillin was found to be the effective drug to cure S. tonsillitis but a high rate of resistance to macrolides was noticed which is an alarming sign. How to cite this article: Elnaim Bushra Ahmed, Elsir Ali Abu Groun, Babiker Saad Almugadam, Yousif Musa Alobaid Ahmed, Aymen Mudawe Nurain Mudawe, Nassir Abakar Babiker, and Nadir Abuzeid* (2023) “Epidemiological Aspects and Antibiotics Susceptibility Patterns of Streptococcus pyogenes Isolated from Subjects with Tonsillitis, Sudan,” Sudan Journal of Medical Sciences, vol. 18, Issue no. 1, pages 6–24. DOI 10.18502/sjms.v18i1.12861 Page 6 Corresponding Author: Nadir Abuzeid; email: nadirabuzeid@oiu.edu.sd Received 2 December 2021 Accepted 17 November 2022 Published 31 March 2023 Production and Hosting by Knowledge E Elnaim Bushra Ahmed et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Editor-in-Chief: Prof. Nazik Elmalaika Obaid Seid Ahmed Husain, MD, M.Sc, MHPE, PhD. http://www.knowledgee.com https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ Sudan Journal of Medical Sciences Elnaim Bushra Ahmed et al Keywords: azithromycin, clindamycin, erythromycin, GAS, Streptococcus pyogenes 1. Introduction Pharyngitis or tonsillitis (sore throat) is defined as an inflammatory process involving the mucous membranes of the oropharynx and tonsils [1]. Tonsillitis is a major problem that threatens the health and socioeconomic lives of many people [1, 2]. Viruses are a common cause of pharyngitis; however, >15–30% of this condition is associated with group A streptococcus (GAS) infection [3, 4]. Streptococcal pharyngitis is a highly important disease, and its ultimate frequency arises among children aged 5–15 years [5]. The common signs of illness are usually abrupt and include painful throat, rapid onset fever, discomfort swallowing, enlarged tonsils, and tender cervical lymph nodes [6]. Transmission of S. pyogene mainly occurs with direct contact, contaminated objects, or nasal secretions from infected persons or carriers [7]. The occurrence of the disease is varied according to seasonal and environmental conditions [8]. Streptococcus pyogenes (S. pyogene), also identified as Lancefield GAS, is a significant pathogenic bacteria associated with a wide range of human diseases. Much data collection and assessment of the global burden of GAS infection indicated that about 616 million suffer from pharyngitis, with a minimum of 111 million cases of skin diseases (pyoderma). More seriously, 18.1 million people complained of invasive diseases and not less than 517,000 deaths occurred due to serious invasive diseases and disease sequelae [9–13]. This data reflects the important rank of GAS among pathogenic bacteria and express the effect of GAS on worldwide mortality and morbidity [10, 12, 14]. GAS is an exceptional causative agent of diseases that require an etiologic identification and specific treatment [12]. The diagnosis of pharyngitis based on the history of disease with clinical remakes is useful, however, accurate diagnosis requires laboratory analysis [15, 16]. In children, the McIsaac scoring system is the most common clinical prediction procedure used for the identification of streptococcal pharyngitis. Culturing throat swab on sheep blood remains the perfect technique for the identification of GAS infections [5]. Rapid Antigen detection tests and serodiagnosis, including anti-deoxyribonuclease B (ADNase B) and antistreptolysin O (ASO), are additional diagnostic tools for GAS diseases [17]. Penicillin and its products are still the drugs of choice, however, in patients who are allergic to it, macrolide is the alternative drug prescribed to treat S. pyogenes infections [18]. Growing macrolide resistance has been stated. Furthermore, the fastest developing problem of antibiotic resistance of S. pyogenes is raised. Evidence about increasing minimum DOI 10.18502/sjms.v18i1.12861 Page 7 Sudan Journal of Medical Sciences Elnaim Bushra Ahmed et al inhibitory concentration or reduced susceptibility to penicillin has been documented [19]. Various resistance rates have been reported according to geographical location and researcher [20, 21]. Unfortunately, little data on GAS pharyngitis were available regarding low-income countries [22]. Sudan is a poor country with poor health resources, researches concerning S. pharyngitis among children remains an ignored problem with few data available on the topic [23–27]. Moreover, trial treatment of microbial infections including tonsillitis without culture to recognize the causative pathogens is current practice in our study area and all over Sudan. Furthermore, data on prevalence, antibiotic susceptibility testing, and features related to S. pharyngitis among children in our state were least studied. Thus, this study intended to investigate the prevalence of GAS, antibiotics susceptibility patterns, and related aspects of S. pyogenes among children with pharyngitis in White Nile State, Sudan. 2. Materials and Methods 2.1. Study design and period This cross-sectional study was conducted at the Kosti Teaching Hospital between 2019 and 2021. 2.2. Study area Situated in southern Sudan and located between latitudes 12 to 13.30º North and longitude 31 to 33.30º East, White Nile state shares its border with Khartoum state in the North, North Kordofan state in the West, South Kordofan and Upper Nile states in the South-west, the state of Southern Sudan in the South, and the states of Al-Gazira and Sennar in the East. The state has a total area of 16,000 km2. It has a population of 1.7 million according to the 2008 population census. Of this number, 85% live in rural areas and around 15% in urban areas. Agriculture is the main source of livelihood in the state, and 65% of the state population work either as farmers or as seasonal laborers. White Nile remains one of the least developed areas in the country. There are 3 teaching and 19 rural hospitals. The most important towns are Al-Dweim, Algabaleen, Al-Kawwa, Rabak the capital of the state, and Kosti which is the largest town housing the Kosti teaching hospital (the largest hospital in the state in which the current study was carried out). DOI 10.18502/sjms.v18i1.12861 Page 8 Sudan Journal of Medical Sciences Elnaim Bushra Ahmed et al 2.3. Ethical considerations The study approval was taken from Omdurman Islamic University (Khartoum). Authorities of Kosti Teaching Hospital granted permission to collect the specimens. Written consent was obtained from the investigated children’s parents or guardians. A verbal agreement was obtained from all children. 2.4. Study participants, data, and sample size The study participants were children with tonsillitis between the ages of 5 and 15 years who attended the Kosti Teaching Hospital ENT unit. Each participant underwent a general physical examination by an ENT consultant before being selected for the study. Patients with a history of respiratory surgery, autoimmune disease, radiotherapy, body abnormality, or chemotherapy were excluded. Children who were under antimicrobial use or those who had a history of antibiotic use at least seven days before the pre- sentation were excluded. Data collection was done by using a pre-test questionnaire that covered the patients’ demographical data (age, gender, and residence) and clinical information (sore throat, fever, tonsils hyperemia, and cervical lymphadenopathy). Figure 1 shows the study flow diagram. The sample size was estimated using the following formula: n = Z2 × P (P–1)/d2, where n is the sample size, Z is the standard normal variable (corresponding to the 95% level of significance = 1.96), P is the expected prevalence = 0.5% as determined by a preliminary study, and d is the precision corresponding to the effect size = 0.05. Based on the above data, n = (1.96)2 × P (P–1)/(0.05)2 = 384. 2.5. Samples collection and processing of specimens Under good light and using the tongue depressor, the inside of the mouth was examined for signs of inflammation and the presence of pus and exudates. Using a sterile cotton swab, the two infected tonsils were swabbed and care was taken to avoid contamination of swabs with saliva or touching any part of the mouse. All swabs collected were labeled and immediately transferred to the laboratory. DOI 10.18502/sjms.v18i1.12861 Page 9 Sudan Journal of Medical Sciences Elnaim Bushra Ahmed et al P a r t i c i p a n t ’ s s e l e c t i o n and data collection Study flowchart Statistical analysis and data presentation Sample collection and laboratory analysis P a rt i c i p a n t ’ s s e l e c ti o n b a s e d o n s tu d y exclusion and inclusion criteria and the main targeted criteria was age ≤ 16 years All data were analyzed using IBM SPSS Statistics for Windows (Version 21) and presented in figures and tables. The results were expressed as numbers and percentages Throat swab was collected from everyone.Subsequently, all the samples were cultivated and the isolated streptococcus pyogenes were tested for their susceptibility to antibiotics Figure 1: Study flow chart. 2.6. Inoculation of collected sample on culture media 5% sheep blood agar (Hi Media, India) was the growing medium used to isolate the organisms. Throat specimens were rolled firmly over one-sixth of the plate to deposit the specimen, the wire loop was used to streak the inoculum over the surface of the plate, and plates were incubated at 35ºC overnight. A candle jar was used to provide an atmosphere of CO2. After 24 hr of incubation, each plate was checked for colonies with 𝛽-hemolytic characteristics. Culture plates negative for β-hemolytic colonies were incubated for an additional 24 hr to allow for the recovery and detection of slow growers. Selected 𝛽-hemolytic colonies were subjected to another sub-culture using blood agar plates to obtain pure growth. After overnight incubation, the pure colonies were tested for their Gram reaction, catalase. All 𝛽-hemolytic and catalase-negative colonies were tested for Bacitracin susceptibility bacteria. The bacterial suspension was evenly spread onto a blood agar plate using a swab, and 0.05 U Bacitracin disc (Hi Media, India) was placed on the inoculated surface and incubated in a candle jar for 18–24 hr at 35ºC. Any zone of inhibition surrounding the disc was indicative of a presumptive GAS. 2.7. Bacterial isolates Three hundred eighty-four throat swabs were collected from subjects with tonsils infec- tion (tonsillitis) from 2019 to 2021 in Kosti, Sudan. The specimens were collected from the main hospital of the state ENT unit. Only 159 S. pyogenes out of inoculated bacteria were reported as S. pyogenes based on colony morphology, beta-hemolysis on sheep blood agar, and sensitivity to bacitracin disc (0.05 U, Himedia, India). DOI 10.18502/sjms.v18i1.12861 Page 10 Sudan Journal of Medical Sciences Elnaim Bushra Ahmed et al 2.8. Antibiotics susceptibility testing The sensitivity testing was done after sub-culturing the bacteria onto a 5% blood agar medium (Hi-Media, India) overnight incubation at 37ºC to yield a new growth of S. pyogenes. The test was performed according to the Clinical Laboratory Standards Institute (CLSI) [28]. About three to five well-defined colonies were picked using a sterile wire loop and emulsified into 3–5 ml sterile physiological saline to prepare a turbidity suspension equivalent to the 0.5 McFarland standards. Using a sterile cotton swab, the suspension of the test organism was inoculated into Mueller–Hinton sensitivity medium- based blood agar (Hi-Media, India). Next, sterile forceps were used to transfer the antimicrobial discs (erythromycin 15 mg, azithromycin15 mg, penicillin 10 mg, clindamycin 2 mg) onto the inoculated plate (all antibiotics were from Hi-Media, India). The inoculated plates were incubated overnight at 37ºC and in the presence of 5% CO2 provided by using a candle jar. Following the incubation, the zones of inhibition were measured by using a ruler and reported in mm. Compared with the manufacturer’s reference chart results, the generated results were provided (Hi-Media, India). The susceptibility of isolates to each antibiotic was recorded as sensitive, resistant, or intermediated. Streptococcus pyogenes ATCC1916 was used as a control. 2.9. Statistical analysis All data were analyzed using IBM SPSS Statistics for Windows (Version 21). The results were expressed as numbers and percentages. The statistical differences were evaluated using the Chi-squared and Fisher’s Exact tests. Multinomials and binary were involved in investigating the associations of dependent variables with the independent factors. A P-value � 0.05 was considered significant. 3. Results 3.1. Sociodemographic and clinical features of the study partici- pants Three hundred and eighty-four participants were enrolled in this research – 219 (57%) females and 165 (43%) males. The age of the studied participants ranged from 5 to 15 years with a mean age of 9.0 and a standard deviation (SD) of 3.0. The vast majority of study participants 259 (67.4%) were between the ages of 5 and 10 years. Moreover, about 296 (77.1%) participants were from urban and 88 (22.9%) were from rural areas. DOI 10.18502/sjms.v18i1.12861 Page 11 Sudan Journal of Medical Sciences Elnaim Bushra Ahmed et al Furthermore, 268 (69.8%) participants were in primary school, 59 (15.4%) in secondary school, and <57 (14.8) in kindergarten (Table 1). Out of the 384 participants, only 34.9% had lymphadenopathy and 66.4% suffered from tonsil hyperplasia. Fever and sore throat were found in all patients. Interestingly, there was a significant dissimilarity (P � 0.05) in the occurrence of lymphadenopathy and tonsil hyperplasia, which were more in the 5–10 years age group compared to individuals of 11–16 years (Table 2). Table 1: Characteristics of the study participants. Variable Frequency: N (%) Gender Male 165 (43) Female 219 (57) Age (yr) 5–10 259 (67.4) 11–16 125 (33.6) Residence Urban 296 (77.1) Rural 88 (22.9) Education level Kindergarten 57 (14.8) Primary school 268 (69.8) Secondary school 59 (15.4) N, Number. 3.2. Prevalence of GAS sore throat The overall prevalence of GAS associated with tonsillitis (sore throat) in this research was 159 (41.4%) and 225 (58.6%) due to non-GAS sore throat (Figure 2). There was no significant variation in the frequency of GAS sore throat among gender, age groups, education levels, and residence, P � 0.05. Regarding the link of GAS sore throat with the clinical features, we found that the frequency of GAS sore throat was significantly higher in individuals with lymphadenopathy or tonsil hyperplasia, P � 0.05. The findings of logistic regression analysis also revealed that GAS sore throat is significantly asso- ciated with lymphadenopathy (AOR: 2.375, 95% CI: 1.479–3.815, P � 0.000) and tonsil hyperplasia (OR: 3.374, 95% CI: 1.939–5.874, P � 0.000). Notably, males (OR: 0.853, 95% CI: 0.549–1.325, P 0.479) and individuals aged 5–10 years (OR: 0.867, 95% CI: 0.464–1.618, P 0.654) were less likely to be infected by a GAS sore throat, but it is not significant. Consequently, lymphadenopathy and tonsil hyperplasia (P < 0.05) were independent predictors of S. pyogenes pharyngitis in children (Table 3). DOI 10.18502/sjms.v18i1.12861 Page 12 Sudan Journal of Medical Sciences Elnaim Bushra Ahmed et al Table 2: Clinical features of the study subjects. Variable Frequency: N (%) Fever Sore throat Lymphadenopathy Tonsil hyperemia Yes No Yes No Yes No Yes No Overall 384 (100) 0 (0) 384 (100) 0 (0) 134 (34.9) 250 (65.1) 255 (66.4) 129 (33.6) Gender Male 165 (100) 0 (0) 165 (100) 0 (0) 62 (37.6) 103 (62.4) 106 (64.2) 59 (35.8) Female 219 (100) 0 (0) 219 (100) 0 (0) 72 (32.9) 147 (67.1) 149 (68) 70 (32) X2 – 0.915 0.607 P-value – 0.339 0.436 Age (yr) 5–10 259 (100) 0 (0) 259 (100) 0 (0) 110 (42.5) 149 (57.5) 206 (79.5) 53 (20.5) 11–16 125 (100) 0 (0) 125 (100) 0 (0) 24 (19.2) 101 (80.8) 49 (39.2) 76 (60.8) X2 – – 20.097 61.491 P-value – – �0.000 �0.000 Residence Urban 296 (100) 0 (0) 296 (100) 0 (0) 94 (31.8) 202 (68.2) 193 (65.2) 103 (34.8) Rural 88 (100) 0 (0) 88 (100) 0 (0) 40 (45.5) 48 (54.5) 62 (70.5) 26 (29.5) X2 – – 5.602 0.839 P-value – – 0.018 0.36 Education level Kindergarten57 (100) 0 (0) 57 (100) 0 (0) 29 (50.9) 28 (49.1) 48 (84.2) 9 (15.8) Primary school 268 (100) 0 (0) 268 (100) 0 (0) 95 (35.4) 173(64.6) 185 (69) 83 (31) Secondary school 59 (100) 0 (0) 59 (100) 0 (0) 10 (16.9) 49 (83.1) 22 (37.3) 37 (62.7) X2 – – 14.808 31.35 P-value – – 0.001 �0.000 Data assessed by Pearson Chi-Square.N, Number; X2, Chi-Square. 3.3. Antibiotics susceptibility findings 159 GAS isolates were tested for their susceptibility to erythromycin (15 μg), clindamycin (2 μg), penicillin (10 U), and azithromycin (15 μg). We found that all the isolated strains were sensitive to penicillin. However, the frequency of antibiotic resistance to clin- damycin, azithromycin, and erythromycin resistance was observed in 7 (4.4%), 44 (27.7%), and 47 (29.6%) of isolates, respectively (Figure 3). Additionally, the rate of clindamycin, erythromycin, and azithromycin resistance was more in individuals suffering from lym- phadenopathy (P � 0.05) or tonsil hyperplasia (P � 0.05). Furthermore, the rate of clindamycin and azithromycin resistance was also more in males than females but it was not significant (Table 4). DOI 10.18502/sjms.v18i1.12861 Page 13 Sudan Journal of Medical Sciences Elnaim Bushra Ahmed et al Prevalence: N (%) GAS Non-GAS 159 (41.4) 225 (58.6) Figure 2: Overall prevalence of streptococcal sore throat. GAS, Group A streptococci; Non-GAS, Non-group A streptococci; N, Number. 159 (100%) Clindamycin 152 (95.6) 7 (4.4) Erythromycin 112 (70.4) 47 (29.6) Azithromycin Sensitive Resistant 115 (72.3) 44 (27.7) Pencillin Frequency expressed as N (%) Figure 3: Susceptibility of the isolated GAS to antibiotics. GAS, Group A streptococci; N, Number. 4. Discussion Human infections caused by S. pyogenes are a common reason for morbidity and mor- tality globally [29]. The prevalence of acute pharyngotonsillitis caused by S. pyogenes is approximately 15 to 30%. This percentage varies from region to region [30]. In the DOI 10.18502/sjms.v18i1.12861 Page 14 Sudan Journal of Medical Sciences Elnaim Bushra Ahmed et al Table 3: Association of streptococcal sore throat with gender, age groups, residence, and clinical features of participants. Variable Prevalence Association Unadjusted Adjusted N (%) X2 P-value OR (95%CI) P-value AOR (95% CI) P-value Gender Female 95 (43.4) 1 1 Male 64 (38.8) 0.818 0.366 0.853 (0.549– 1.325) 0.479 0.853 (0.549– 1.325) 0.479 Age (yr) 11–16 49 (39.2) 1 1 5–10 110 (42.5) 0.372 0.542 0.867 (0.464– 1.618) 0.654 0.867 (0.464– 1.618) 0.654 Residence Rural 35 (39.8) 1 1 Urban 124 (41.9) 0.126 0.723 1.317 (0.783– 2.217) 0.300 1.317 (0.783– 0.217) 0.300 Education level Secondary school 29 (49.2) 1 1 Primary school 104 (38.8) 2.622 0.27 1.016 (0.547– 1.889) 0.959 0.398 (0.151– 1.048) 0.062 Kindergarten 26 (45.6) 0.398 (0.151– 1.048) 0.062 0.391 (0.183– 0.837) 0.016 Clinical features Lymphadeno- pathy No 83 (33.2) 1 1 Yes 76 (56.7) 19.886 0.000 2.375 (1.479– 3.815) 0.000 2.375 (1.479– 3.815) 0.000 Tonsil hyperplasia No 31(24) 1 1 Yes 128 (50.2) 24.173 0.000 3.374 (1.939– 5.874) 0.000 3.374 (1.939– 5.874) 0.000 Statistical analysis performed using Pearson Chi-square and binary and multinomial logistic regression. N, Number; X2 , Chi-square; OR, Odd ratio; AOR, Adjusted odd ratio; CI, Confidence interval. present study, we present the prevalence of S. pyogenes associated with tonsillitis among Sudanese children who attended the Kosti Teaching hospital. A total of 384 study participants were involved in this study, of which 165 (43%) were males and 219 (57%) were females. Our findings reveal that of the 384 patients, only 159 (41.4%) were infected by a GAS sore throat and 58.6% by a non-GAS sore throat. This was lower when compared with a previous study carried out in the study area [26]. It is also lesser than that found in Mohammed et al.’ study (2016) (86%), which was conducted in Khartoum city, Sudan [25]. In contrast, it is higher than the results of Al Fadhil et al. (35.5%) and Abdelwahab et al.’s (2014) (6%) studies, which were also conducted in Khartoum city, Sudan [23, 24]. The prevalence of S. pyogenes associated with pharyngitis among children in African studies compared to the current study was higher (66.7%) in a Nigerian study by Uzodimma et al. and lower in an Egyptian study by Sultan et al. (28%), a Kenyan study by Osowicki et al. (23% ), and an Ethiopian study by Tesfaw et al. (11.3%) [30–33]. DOI 10.18502/sjms.v18i1.12861 Page 15 Sudan Journal of Medical Sciences Elnaim Bushra Ahmed et al Table 4: Relationship of antibiotics susceptibility patterns of the GAS isolates (N = 159) with gender, age groups, residence, and clinical features of study subjects. Variable Number Clindamycin: N (%) Erythromycin: N (%) Azithromycin: N (%) S R S R S R Gender Male 64 61 (95.3) 3 (4.7) 47 (72.3) 18 (27.7) 46 (70.8) 19 (29.2) Female 95 91 (95.8) 4 (4.2) 65 (68.4) 30 (31.6) 69 (72.6) 26 (27.4) X2 0.000 0.278 0.066 P-value 1.000𝐶 0.598 0.797 Age (yr) 5–10 110 104 (94.5) 6 (5.5) 77 (70) 33 (30) 78 (70.9) 32 (29.1) 11–16 49 48 (98) 1 (2) 35 (70) 15 (30) 37 (74) 13 (26) X2 0.303 0.000 0.162 P-value 0.582𝐶 1.000 0.687 Residence Urban 124 118 (95.2) 6 (4.8) 85 (68.5) 39 (31.5) 88 (71) 36 (29) Rural 335 34 (97.1) 1 (2.9) 27 (77.1) 8 (22.9) 27 (77.1) 8 (22.9) X2 – – – P-value 1.000𝐹 0.404𝐹 0.528𝐹 Education level Kindergarten 26 24 (92.3) 2 (7.7) 17 (65.4) 9 (34.6) 17 (65.4) 9 (34.6) Primary school 104 99 (99.2) 5 (4.8) 75 (72.1) 29 (27.9) 77 (74) 27 (26) Secondary school 29 29 (100) 0 (0) 20 (69) 9 (31) 21 (72.4) 8 (27.6) X2 1.864 0.603 0.881 P-value 0.367𝐹 0.766𝐹 0.704𝐹 Clinical features Lymphadeno- pathy Yes 76 72 (94.7) 4 (5.3) 50 (65.8) 26 (34.2) 51 (67.1) 25 (32.9) No 83 80 (96.4) 3 (3.6) 62 (74.7) 21 (25.3) 64 (77.1) 19 (22.9) X2 – 1.512 1.983 P-value 0.710𝐹 0.219 0.159 Tonsil hyperplasia Yes 128 121 (94.5) 7 (5.5) 84 (65.6) 44 (34.4) 87 (68) 41 (32) No 31 31 (96.4) 0 (0) 28 (90.3) 3 (9.7) 28 (90.3) 3 (9.7) X2 – – – P-value 0.347𝐹 0.008𝐹 0.013𝐹 Data analysis performed using Pearson’s Chi-square and Fisher’s Exact test𝐹 . N, Number; X2 , Chi-Square; S, Sensitive; R, Resistant; GAS, Group A streptococci. Moreover, the occurrence of tonsils infection was predominant observed among urbanized patients 124 (41.9%), while the disease was found only in 35 (39.8) patients from rural areas. This could be due to the living conditions and personnel behavior or more likely due to the consumption of cold drinks and fast food, which are easily avail- able in the urbanized area compared to rural areas. The result of this research revealed that GAS tonsillitis predominately occurred in children aged 5–10 years (68%) and the incidence among those aged 11–16 years is relatively low (32%). Similar observations were reported for the age group of 6–12 years (61%) [34]. However, contrary to the DOI 10.18502/sjms.v18i1.12861 Page 16 Sudan Journal of Medical Sciences Elnaim Bushra Ahmed et al results of the current study, a previous study performed on this subject area showed a higher rate of streptococcal pharyngitis among those aged 16–20 (85.7%) than those who were 11–15 (70%) and 5–10 (56.2%) years old. The distribution of streptococcal pharyngitis was more in female patients (95 [43.4%]) than in male patients (64 [38.8%]). Similarly, Mohammed et al. and Ahmed et al.’s studies reported that the majority of those who complained of tonsillitis were females. This is probably because the number of female patients admitted was more than male patients. On the other hand, Singh et al. reported that the frequency of streptococcal pharyngitis was more in boys than girls [35]. Furthermore, it was noted that 64% of primary school students, 19% of secondary school students, and 17% from kindergarten were infected by GAS pharyngitis. Our finding was proportionally 19%, which is similar to the results of Sharma et al. who found a GAS frequency of 17.5% in children aged 11–15 years (secondary-school level) [59]. However, the current research showed a higher frequency of GAS in the age group 5–10 years. This may be due to cross-infection because of overloaded classrooms and poor air circulation in them, low immunity, and the nature of children’s activity. Probably, the variation between studies could be due to the difference in sample size, study subjects, climates, seasons, and diagnostic procedures. According to the AST results, all isolates were sensitive to penicillin, which is similar to the findings of other previous studies [36–39]. In contrast to these results, a previous study carried out in Egypt found that four strains of S. pyogene were resistant to penicillin [40], which is different from our findings and the available literature. The efficiency of this drug could be due to the incapability of GAS to generate β-lactam enzymes. However, penicillin can be ineffective in treating S. pyogenes infections, as its action is evaded through the introduction into the epithelial cells, which is difficult for penicillin to reach inside cells [41]. Moreover, the development of the biofilm phenomenon [42] and the presence of other β-lactamase- producing bacteria act as protective tools for S. pyogenes [43, 44]. Many previous studies showed comparable findings. Indeed, the rate of resistance to erythromycin was 21.3%, Egypt [40], Lebanon 23%, [45], and Greece 22.8 % [46], which is lower than our result. Sayyahfar et al.’s study reported that 33.9% of GAS isolates were resistant to erythromycin [47]. The findings of these studies are considered more relevant to our study in which the resistance rate to erythromycin was 29.6%. Compared to this, several studies showed a low rate of resistance to erythromycin as reported in Norway (2.7%) [ 48], the United States (5.2%) [49], France (6.5%) [50], Italy (7.4%) [29], Spain (2.8%) [51], Tunis (5.2%) [52], and Taiwan (10.7%) [53]. Additionally, the clindamycin resistance rate of GAS in our study was 4.4%, however, many studies have shown higher results DOI 10.18502/sjms.v18i1.12861 Page 17 Sudan Journal of Medical Sciences Elnaim Bushra Ahmed et al – for instance, Iran (13.5%) and Korea (32.5%) [54, 55]. On the other hand, a low rate of clindamycin was reported in Italy (1.4%) and the USA (0.5%) [56, 57]. Our study showed an azithromycin resistance of 27.7% which is similar to the results of Sharma et al. (28.6%) and Rijal et al. (24%) [58, 59]. In contrast to these results, recently Khademi et al. reported that the resistance of azithromycin was 12%. The variation of reports outcomes may be due to the diversity of genotypic and phenotypic characters of circulating strains, or guidelines of antibiotic utilization rules between the different sites of the earth. 5. Conclusion The high proportion of GAS was isolated from the throats of children suffering from tonsillitis in White Nile State. Regardless of the resistance of isolates to some antibiotics, penicillin still is the drug of choice for streptococcal tonsillitis with 100% sensitivity. GAS tonsillitis with lymphadenopathy or tonsil hyperplasia showed a significant frequency of macrolides resistance. The development of macrolides resistance among isolates of GAS reflects the need for alternative choices for the management of GAS tonsillitis in subjects with a penicillin allergy. Acknowledgments The authors are grateful to all those who participated in this study, namely, the staff of the Department of Medical Laboratory Investigations, ENT unit Hospital, Kosti, Sudan, National Public Health Laboratory, Khartoum, Sudan, and the Department of Medical Microbiology, Faculty of Medical laboratory of Sciences, Omdurman Islamic University, Omdurman, Sudan. Competing Interests None declared. Availability of Data and Material The dataset generated during this study are available from the corresponding author on reasonable request. DOI 10.18502/sjms.v18i1.12861 Page 18 Sudan Journal of Medical Sciences Elnaim Bushra Ahmed et al Funding None. References [1] Hurst, J. R., Kasper, K. J., Sule, A. N., & McCormick, J. K. (2018). Streptococcal pharyngitis and rheumatic heart disease: The superantigen hypothesis revisited. Infection, Genetics and Evolution, 61(1), 160–175. [2] Pfoh, E., Wessels, M. R., Goldmann, D., Lee, G. M. (2008). Burden and economic cost of group A streptococcal pharyngitis. Pediatrics, 121(2), 229–234. [3] May, P. J., Bowen, A. C., & Carapetis, J. R. (2016). The inequitable burden of group A streptococcal diseases in Indigenous Australians. Medical Journal of Australia, 205(5), 201–203. [4] Marx, J., Hockberger, R., & Walls, R. (2013). Rosen’s emergency medicine: Concepts and clinical practice. Elsevier Health Sciences E-Book: 2-Volume Set. [5] Danchin, M. H., Rogers, S., Kelpie, L., Selvaraj, G., Curtis, N., Carlin, J. B., Nolan, T. M., & Carapetis, J. R. (2007). Burden of acute sore throat and group A streptococcal pharyngitis in school-aged children and their families in Australia. Pediatrics, 120(5), 950–957. [6] Choby, B. A. (2009). Diagnosis and treatment of streptococcal pharyngitis. American Family Physician, 79(5), 383–390. [7] Makthal, N., Vander Wal, A. R., Saavedra, M. O., Olsen, R. J., Musser, J. M., & Kumaraswami, M. (2019). Environmental pH and peptide signaling control virulence of Streptococcus pyogenes via a quorum-sensing pathway. Nature Communications, 10(1), 1–14. [8] Carapetis, J. R., Currie, B. J., & Kaplan, E. L. (1999). Epidemiology and prevention of group A streptococcal infections: Acute respiratory tract infections, skin infections, and their sequelae at the close of the twentieth century. Clinical Infectious Diseases, 28(2), 205–210. [9] Bisno, A. L., Rubin, F. A., Cleary, P. P., & Dale, J. B. (2005). Prospects for a group A streptococcal vaccine: Rationale, feasibility, and obstacles—Report of a National Institute of Allergy and Infectious Diseases workshop. Clinical Infectious Diseases, 41(8), 1150–1156. [10] Carapetis, J. R., Steer, A. C., Mulholland, E. K., & Weber, M. (2005). The global burden of group A streptococcal diseases. The Lancet Infectious Diseases, 5(11), 685–694. DOI 10.18502/sjms.v18i1.12861 Page 19 Sudan Journal of Medical Sciences Elnaim Bushra Ahmed et al [11] Ralph, A. P., Fittock, M., Schultz, R., Thompson, D., Dowden, M., Clemens, T., Parnaby, M. G., Clark, M., McDonald, M. I., Edwards, K. N., & Carapetis, J. R. (2013). Improvement in rheumatic fever and rheumatic heart disease management and prevention using a health centre-based continuous quality improvement approach. BMC Health Services Research, 13(1), 1–13. [12] May, P. J., Bowen, A. C., & Carapetis, J. R. (2016). The inequitable burden of group A streptococcal diseases in Indigenous Australians. Medical Journal of Australia, 205(5), 201–203. [13] Oliver, J. R., Pierse, N., Stefanogiannis, N., Jackson, C., & Baker, M. G. (2017). Acute rheumatic fever and exposure to poor housing conditions in New Zealand: A descriptive study. Journal of Paediatrics and Child Health, 53(4), 358–364. [14] Parks, T., Smeesters, P. R., & Steer, A. C. (2012). Streptococcal skin infection and rheumatic heart disease. Current Opinion in Infectious Diseases, 25(2), 145–153. [15] Karacan, M., Karakelleoğlu, C., & Orbak, Z. (2007). Diagnosis of group A beta- hemolytic Streptococcus using the Breese clinical scoring system. Southern Medical Journal, 100(12), 1192–1197. [16] Dale, A. P., Marchello, C., & Ebell, M. H. (2019). Clinical gestalt to diagnose pneumonia, sinusitis, and pharyngitis: A meta-analysis. British Journal of General Practice, 69(684), e444–e453. [17] Karmarkar, M. G., Venugopal, V., Joshi, L., & Kamboj, R. (2004). Evaluation & revaluation of upper limits of normal values of anti-streptolysin O & anti- deoxyribonuclease B in Mumbai. Indian Journal of Medical Research, 119, 26–28. [18] Sharma, Y., Vishwanath, S., & Bairy, I. (2010). Biotype and antibiotic resistance pattern of group A streptococci. Indian Journal of Pathology and Microbiology, 53(1), 187. [19] Ray, D., Sinha, S., Saha, S., Karmakar, S., Dutta, R. N., Bhattacharya, S., Pal, N. K., & Bhattacharya, B. (2010). A preliminary sentinel surveillance report on antibiotics resistance trend of Streptococcus pyogenes in Kolkata region, India. Al Ameen Journal of Medical Sciences, 3(2), 146–151. [20] Bourbeau, P. P. (2003). Role of the microbiology laboratory in diagnosis and management of pharyngitis. Journal of Clinical Microbiology, 41(8), 3467–3472. [21] Zhou, W., Jiang, Y. M., Wang, H. J., Kuang, L. H., Hu, Z. Q., Shi, H., Shu, M., & Wa, C. M. (2014). Erythromycin-resistant genes in group A β-haemolytic Streptococci in Chengdu, Southwestern China. Indian Journal of Medical Microbiology, 32(3), 290– 293. [22] World Health Organization. (2015). The current evidence for the burden of group streptococcal diseases [Discussion papers on Child Health]. Department of DOI 10.18502/sjms.v18i1.12861 Page 20 Sudan Journal of Medical Sciences Elnaim Bushra Ahmed et al Child and Adolescent Health and Development, WHO. http://whqlibdoc.who.int/hq/ 2005/WHO_FCH_CAH_05.07.pdf [23] Al Fadhil, A. O. O. Bacteriology of sore throats in a Sudanese population. [24] Abdelwahab, O. I., Eljak, M. A., & Suliman, A. M. (2014). Isolation and identification of Streptococcus pyogenes in patients with sore throat [Doctoral dissertation, Sudan University of Science and Technology]. [25] Mohammed, N. A. (2016). Bacterial isolates associated with tonsillitis in Khartoum (Sudan). African Journal of Medical Sciences, 1(9), 1–5. [26] Ahmed, E. B., Adam, A. M., Bakhiet, A. A., & Almugadam, B. S. (2018). Association of Streptococcus pyogenes with symptomatic pharyngitis in Kosti City, Sudan. Research & Reviews: Journal of Microbiology and Biotechnology, 7(1), 19–21. [27] Clinical and Laboratory Standards Institute (CLSI). (2018). Performance standards for antimicrobial susceptibility testing. 28𝑡ℎ Ed. Wayne, Pennsylvania: Clinical and Laboratory Standards Institute. [28] Gherardi, G., Petrelli, D., Di Luca, M. C., De Araujo, F. P., Bernaschi, P., Repetto, A., Bellesi, J., & Vitali, L. A. (2015). Decline in macrolide resistance rates among Streptococcus pyogenes causing pharyngitis in children isolated in Italy. European Journal of Clinical Microbiology & Infectious Diseases, 34(9), 1797–1802. [29] Bisno, A. L., Brito, M. O., & Collins, C. M. (2003). Molecular basis of group A streptococcal virulence. The Lancet Infectious Diseases, 3(4), .200–191 [30] Uzodimma, C. C., Dedeke, F. I., Nwadike, V., Owolabi, O., Arifalo, G., & Oduwole, O. (2017). A study of group a streptococcal pharyngitis among 3–15-year-old children attending clinics for an acute sore throat. Nigerian Journal of Cardiology, 14(2), 97. [31] Sultan, A. M., & Seliem, W. A. (2018). Evaluating the use of dedicated swab for rapid antigen detection testing in group a streptococcal pharyngitis in children. African Journal of Clinical and Experimental Microbiology, 19(1), 24–29. [32] Osowicki, J., Azzopardi, K. I., Baker, C., Waddington, C. S., Pandey, M., Schuster, T., Grobler, A., Cheng, A. C., Pollard, A. J., McCarthy, J. S., & Good, M. F. ( 2019). Controlled human infection for vaccination against Streptococcus pyogenes (CHIVAS): Establishing a group A Streptococcus pharyngitis human infection study. Vaccine, 37(26), 3485–3494. [33] Tesfaw, G., Kibru, G., Mekonnen, D., & Abdissa, A. (2015). Prevalence of group A β- haemolytic Streptococcus among children with pharyngitis in Jimma town, Southwest Ethiopia. Egyptian Journal of Ear, Nose, Throat and Allied Sciences, 16(1), 35–40. DOI 10.18502/sjms.v18i1.12861 Page 21 Sudan Journal of Medical Sciences Elnaim Bushra Ahmed et al [34] Vijayashree, M. S., Viswanatha, B., & Sambamurthy, B. N. (2014). Clinical and bacteriological study of acute tonsillitis. IOSR Journal of Dental and Medical Sciences, 13(1), 37–43. [35] Singh, A. K., Kumar, A., Agarwal, L., Agarwal, A., & Sengupta, C. )2015(. Prevalence of group A streptococcal pharyngitis among schoolchildren of Barabanki district, Uttar Pradesh, India. Journal of the Academy of Clinical Microbiologists, 17(2), 110. [36] Le Hello, S., Doloy, A., Baumann, F., Roques, N., Coudene, P., Rouchon, B., & Bouvet, A. )2010(. Clinical and microbial characteristics of invasive Streptococcus pyogenes disease in New Caledonia, a region in Oceania with a high incidence of acute rheumatic fever. Journal of Clinical Microbiology, 48(2), .530–526 [37] Bahnan, W., Hashwa, F., Araj, G., & Tokajian, S. (2011), Emm typing, antibiotic resistance and PFGE analysis of Streptococcus pyogenes in Lebanon. Journal of Medical Microbiology, 60(1), 98–101. [38] Wu, P. C., Lo, W. T., Chen, S. J., & Wang, C. C. (2014). Molecular characterization of group A streptococcal isolates causing scarlet fever and pharyngitis among young children: A retrospective study from a northern Taiwan medical center. Journal of Microbiology, Immunology and Infection, 47(4), 304–310. [39] Khosravi, A. D., Ebrahimifard, N., Shamsizadeh, A., & Shoja, S. (2016). Isolation of Streptococcus pyogenes from children with pharyngitis and emm type analysis. Journal of the Chinese Medical Association, 79(5), .280–276 [40] Ibrahim, S. B., El-Sokkary, R. H., Elhewala, A. A., El-Anwar, M. W., Awad, W. M., Hamed, A. M., & Badawy, I. I. (2014). Emerging resistance to erythromycin and penicillin among Streptococcus pyogenes isolates in Zagazig, Egypt. International Journal of Current Microbiology and Applied Sciences, 3(10), .756–750 [41] Kaplan, E. L., Chhatwal, G. S., & Rohde, M. (2006). Reduced ability of penicillin to eradicate ingested group A streptococci from epithelial cells: Clinical and pathogenetic implications. Clinical Infectious Diseases, 43(11), 1398–1406. [42] Ogawa, T., Terao, Y., Okuni, H., Ninomiya, K., Sakata, H., Ikebe, K., Maeda, Y., & Kawabata, S. (2011). Biofilm formation or internalization into epithelial cells enable Streptococcus pyogenes to evade antibiotic eradication in patients with pharyngitis. Microbial Pathogenesis, 51(1–2), 58–68. [43] Brook, I., & Gober, A. E. (2008). Failure to eradicate streptococci and beta�lactamase producing bacteria. Acta Paediatrica, 97(2), 193–195. [44] Brook, I. (2013). Penicillin failure in the treatment of streptococcal pharyngo-tonsillitis. Current Infectious Disease Reports, 15(3), 232–235. DOI 10.18502/sjms.v18i1.12861 Page 22 Sudan Journal of Medical Sciences Elnaim Bushra Ahmed et al [45] Karaky, N. M., Araj, G. F., & Tokajian, S. T. (2014). Molecular characterization of Streptococcus pyogenes group A isolates from a tertiary hospital in Lebanon. Journal of Medical Microbiology, 63(9), 1197–1204. [46] Syrogiannopoulos, G. A., Grivea, I. N., Al-Lahham, A., Panagiotou, M., Tsantouli, A. G., Michoula Ralf René Reinert, A. N., & van der Linden, M. (2013). Seven-year surveillance of emm types of pediatric group A streptococcal pharyngitis isolates in Western Greece. PLoS One, 8(8), e71558. [47] Sayyahfar, S., Fahimzad, A., Naddaf, A., & Tavassoli, S. (2015).Antibiotic susceptibility evaluation of group A streptococcus isolated from children with pharyngitis: A study from Iran. Infection & Chemotherapy, 47(4), .230–225 [48] Littauer, P., Caugant, D. A., Sangvik, M., Høiby, E. A., Sundsfjord, A., & Simonsen, G. S. (2006). Macrolide-resistant Streptococcus pyogenes in Norway: Population structure and resistance determinants. Antimicrobial Agents and Chemotherapy, 50(5), 1896–1899. [49] Villaseñor-Sierra, A., Katahira, E., Jaramillo-Valdivia, A. N., de los Angeles Barajas- García, M., Bryant, A., Morfín-Otero, R., Márquez-Díaz, F., Tinoco, J. C., Sánchez- Corona, J., & Stevens, D. L. (2012). Phenotypes and genotypes of erythromycin- resistant Streptococcus pyogenes strains isolated from invasive and non-invasive infections from Mexico and the USA during 1999–2010. International Journal of Infectious Diseases, 16(3), e178–e181. [50] Plainvert, C., Doloy, A., Loubinoux, J., Lepoutre, A., Collobert, G., Touak, G., Trieu- Cuot, P., Bouvet, A., Poyart, C., CNR-Strep network. (2012). Invasive group A streptococcal infections in adults, France (2006–2010). Clinical Microbiology and Infection, 18(7), 702–710. [51] Montes, M., Tamayo, E., Mojica, C., García-Arenzana, J. M., Esnal, O., & Pérez- Trallero, E. (2014). What causes decreased erythromycin resistance in Streptococcus pyogenes? Dynamics of four clones in a southern European region from 2005 to 2012. Journal of Antimicrobial Chemotherapy, 69(6), 1474–1482. [52] Ksia, S., Smaoui, H., Hraoui, M., Bouafsoun, A., Boutiba-Ben Boubaker, I., & Kechrid, A. (2017). Molecular characteristics of erythromycin-resistant Streptococcus pyogenes strains isolated from children patients in Tunis, Tunisia. Microbial Drug Resistance, 23(5), 633–639. [53] Huang, C. Y., Lai, J. F., Huang, I. W., Chen, P. C., Wang, H. Y., Shiau, Y. R., Cheng, Y.-W., Hsieh, L.-Y., Chang, S.-C., & Lauderdale, T. L. Y. (2014). Epidemiology and molecular characterization of macrolide-resistant Streptococcus pyogenes in Taiwan. Journal of Clinical Microbiology, 52(2), .516–508 DOI 10.18502/sjms.v18i1.12861 Page 23 Sudan Journal of Medical Sciences Elnaim Bushra Ahmed et al [54] Sayyahfar, S., Fahimzad, A., Naddaf, A., & Tavassoli, S. (2015). Antibiotic susceptibility evaluation of group A streptococcus isolated from children with pharyngitis: A study from Iran. Infection & Chemotherapy, 47(4), .230–225 [55] Kim, H.Y., & Uh, Y. (2004). Macrolide resistance in β-hemolytic streptococci: Changes after the implementation of the separation of prescribing and dispensing of medications policy in Korea. Yonsei Medical Journal, 45(4), 591–597. [56] Michos, A. G., Bakoula, C. G., Braoudaki, M., Koutouzi, F. I., Roma, E. S., Pangalis, A., Nikolopoulou, G., Kirikou, E., & Syriopoulou, V. P. (2009). Macrolide resistance in Streptococcus pyogenes: Prevalence, resistance determinants, and emm types. Diagnostic Microbiology and Infectious Disease, 64(3), .299–295 [57] Richter, S. S., Heilmann, K. P., Beekmann, S. E., Miller, N. J., Miller, A. L., Rice, C. L., Doern, C. D., Reid, S. D., & Doern, G. V. (2005). Macrolide-resistant Streptococcus pyogenes in the United States, 2002–2003. Clinical Infectious Diseases, 41(5), 599– .608 [58] Sharma, S., Praveen, S., Devi, K. S., Sahoo, B., Singh, W. S., & Singh, T. D. (2014). Prevalance of Streptococcus pyogenes infection in children aged between 5 to 15 years with acute tonsillopharyngitis and its antibiogram. IOSR Journal of Dental and Medical Sciences (IOSR-JDMS), 1(13), .55–50 [59] Rijal, K. R., Dhakal, N., Shah, R. C., Timilsina, S., Mahato, P., & Thapa, S. (2009). Antibiotic susceptibility of group A Streptococcus isolated from throat swab culture of school children in Pokhara, Nepal. Nepal Medical College Journal, 11(4), 238–240. DOI 10.18502/sjms.v18i1.12861 Page 24 Introduction Materials and Methods Study design and period Study area Ethical considerations Study participants, data, and sample size Samples collection and processing of specimens Inoculation of collected sample on culture media Bacterial isolates Antibiotics susceptibility testing Statistical analysis Results Sociodemographic and clinical features of the study participants Prevalence of GAS sore throat Antibiotics susceptibility findings Discussion Conclusion Acknowledgments Competing Interests Availability of Data and Material Funding References