Abstract
Background
Group A streptococcus is human-restricted gram-positive pathogen, responsible for various clinical presentations from mild epidermis infections to life threatened invasive diseases. Under COVID-19 pandemic,. the characteristics of the epidemic strains of GAS could be different.
Purpose
To investigate epidemiological and molecular features of isolates from GAS infections among children in Beijing, China between January 2020 and December 2021. Antimicrobial susceptibility profiling was performed based on Cinical Laboratory Sandards Institute. Distribution of macrolide-resistance genes, emm types, and superantigens was examined by polymerase chain reaction.
Results
114 GAS isolates were collected which were frequent resistance against erythromycin (94.74%), followed by clindamycin (92.98%), tetracycline (87.72%). Emm12 (46.49%), emm1 (25.44%) were dominant emm types. Distribution of ermB, ermA, and mefA gene was 93.85%, 2.63%, and 14.04%, respectively. Frequent superantigenes identified were smeZ (97.39%), speG (95.65%), and speC (92.17%). Emm1 strains possessed smeZ, ssa, and speC, while emm12 possessed smeZ, ssa, speG, and speC. Erythromycin resistance was predominantly mediated by ermB. Scarlet fever strains harbored smeZ (98.81%), speC (94.05%). Impetigo strains harbored smeZ (88.98%), ssa (88.89%), and speC (88.89%). Psoriasis strains harbored smeZ (100%).
Conclusions
Under COVID-19 pandemic, our collections of GAS infection cutaneous diseases decreased dramatically. Epidemiological analysis of GAS infections among children during COVID-19 pandemic was not significantly different from our previous study. There was a correlation among emm, superantigen gene and disease manifestations. Long-term surveillance and investigation of emm types and superantigens of GAS prevalence are imperative.
Keywords: Group A streptococcus, Emm type, Superantigen, Antimicrobial resistance, Scarlet fever, Children, COVID-19 pandemic, China
Introduction
Streptococcus pyogenes (GAS) is vital human pathogen responsible for a wide spectrum of infectious diseases, not only infection on skin and respiratory, but also invasive diseases, such as streptococcal toxic shock syndrome, necrotizing fasciitis as well as triggered autoimmune diseases [1, 2]. Human immunity to GAS may be related with disease manifestations after GAS infection [3]. Severe GAS infection diseases account for 18.1 million cases around the world, with 1.78 million new cases and 500,000 deaths every year [4, 5]. Li analyzed epidemiological characteristics and changes in incidence of GAS infection diseases in China after SARS outbreak. The yearly incidence was 2.44 cases per 100,000. Case-fatality ratios was 0.03 case per 1000 people. Significant seasonal features were May to June and November to December. Scarlet fever in children was high incidence and case-fatality [6]. Comparing with USA, GAS infection in China was usually presented in non-invasive GAS infection [7, 8]
Antibiotic resistance increases gradually, causing global concern [9]. Resistance of isolates to antibiotic varies in different countries and regions [10, 11]. In China, GAS was high frequent resistance to macrolides and clindamycin [12]. M protein is an important virulence factor of GAS coded by emm gene. Depending on variation of N-terminal, more than 250 emm types have been identified. Surveillance on GAS emm types in a long period can give a valuable clue for prediction of future emm clones [13]. The prevalent emm types vary over time in different countries and regions [14]. In China, in the year of 2011, emm12 was the most prevalent type in scarlet fever, with high resistance to erythromycin, tracycline, and clindamycin. However, epidemiological characteristics of M protein changed with time [15].
Sixteen known sAgs have been identified in GAS, including speA, speC, speG-M, smeZ, ssa, speQ, and speR [16], responsible for GAS virulence and successful infection pathogenesis [17].
Researches on GAS epidemiological features have been attracted great attention around the world. Relationship among GAS infection diseases, emm types, and sAgs distribution has not been identified [18–20]. Because COVID-19 pandemic has changed our lifestyle, molecular characteristics of GAS isolated from Chinese children may be different.
In this study, we analyzed emm types, sAgs, and antimicrobial susceptibility resistance of GAS isolates as well as GAS infection categories to find differences among GAS infected cutaneous diseases before and under COVID-19 pandemic.
Materials and methods
Strain collection
Our patients were from outpatient department of Dermatology in Children’s Hospital, Capital Institute of Pediatrics in Beijing China. This study was approved by the Ethics Committee of the Capital Institute of Pediatrics. Between January 2020 and December 2021, 114 GAS isolates were recovered from throat swabs and skin infections. Throat and skin swabs were obtained from patients by two physicians for routine microbiologic analysis.
Bacterial identification
The samples were incubated in a CO2 incubator at 37℃ for 24–36 h on Colombian blood plate (BD, USA). Morphologically suspected GAS colonies were confirmed by Gram’s staining and latex agglutination with the Streptococcus grouping kit (Oxoid, Basingstoke, UK).
Antimicrobial susceptibility testing
The antibiotic susceptibility testing was performed for 10 antibiotics by K-B method. Protocols followed our previous study. Susceptibility of bacteria was determined by diameter of bacteriostatic ring and CLSI standard. Streptococcus pneumoniae ATCC 49,619 was used as control strain.
DNA extraction
DNA extraction of GAS genome was performed according to the recommended method by the Center for Disease Control and Prevention.
Emm genotypes
All isolates were performed emm genotypes according to protocols and recommendations of CDC. Sequence data were compared with emm typing database (https://www2.cdc.gov/vaccines/biotech/strepblast.asp).
Erythromycin-resistance gene detection
Erythromycin resistance genes ermB, ermA, and mefA were performed for all isolates. Primer sequences for ermB, ermA and mefA were designed by Suvorov [10]. Protocol and reaction mixture followed our previous studies [21, 22].
Superantigen detection
Eleven virulence genes, consisting of speA, speC, speG, speH, speI, speJ, speK, speL, speM, ssa, and smeZ were amplied by PCR with primers presented by Green [23]. Protocol and reaction mixture followed our previous study.
Results
Clinical data
One hundred fourteen isolates were received including throat samples (n = 84) and skin samples (n = 30). Of 114 isolates, 84 strains were collected from scarlet fever, 17 strains from impetigo, 10 strains from psoriasis, 1 strain from allergic purpura, and 2 strains from suppurative tonsillitis. 43 strains (37.72%) were recovered from girls, and 71 strains (62.28%) were from boys.. Patients aged from 22 days to 11 years old (median 6.25 years).
Antimicrobial susceptibility testing results
All GAS isolates were sensitive to penicillin, ceftriaxone, cefotaxime, vancomycin, and cefepime. The highest rate of resistance was against erythromycin (94.74%), followed by clindamycin (92.98%), tetracycline (87.72%). Distribution of antimicrobial susceptibility was presented in Table 1.
Table 1.
Antimicrobial susceptibility test of 114 isolates of GAS from 2020 to 2021, compared with our previous studies [21, 22]
| Antibiotic | Susceptibility (n %) | Intermediate (n %) | Resistant (n %) | ||||||
|---|---|---|---|---|---|---|---|---|---|
| 2020–2021 | 2019 | 2016–2017 | 2020–2021 | 2019 | 2016–2017 | 2020–2021 | 2019 | 2016–2017 | |
| Penicillin | 114/100 | 271/100 | 297/100 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 |
| Ceftriaxone | 114/100 | 271/100 | 297/100 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 |
| Levofloxacin | 110/96.49 | 271/100 | 297/100 | 4/3.51 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 |
| Cefotaxime | 114/100 | 271/100 | 297/100 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 |
| Vancomycin | 114/100 | 271/100 | 297/100 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 |
| Cefepime | 114/100 | 271/100 | 297/100 | 0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 |
| Chloramphenicol | 103/90.35 | 261/96.31 | 283/95.29 | 9/7.89 | 6/2.21 | 12/4.04 | 2/1.75 | 4/1.48 | 2/0.67 |
| Tetracycline | 6/5.26 | 23/8.49 | 16/5.39 | 8/7.01 | 11/4.06 | 13/4.38 | 100/87.72 | 237/87.45 | 268/90.23 |
| Erythromycin | 5/4.39 | 7/2.58 | 3/1.01 | 1/0.88 | 7/2.58 | 2/0.67 | 108/94.74 | 257/94.83 | 292/98.3 |
| Clindamycin | 5/4.39 | 30/11.1 | 8/2.69 | 3/2.63 | 3/1.11 | 2/0.67 | 106/92.98 | 238/87.82 | 287/96.6 |
Erythromycin-resistant gene distributions
One hundred eight erythromycin resistance isolates were found. 107 erythromycin resistance isolates (93.86%) harbored ermB gene, 2 isolates (1.75%) harbored ermA gene, and 16 isolates (14.04%) harbored mefA gene. The distribution of erythromycin resistance genes in GAS isolates from different diseases is presented in Table 2.
Table 2.
Distribution of erythromycin-resistant genes GAS isolates in different diseases, compared with our previous studies [21, 22]
| Diseases | ermB (n/%) | ermA (n/%) | mefA (n/%) | ||||||
|---|---|---|---|---|---|---|---|---|---|
| 2020–2021 | 2019 | 2016–2017 | 2020–2021 | 2019 | 2016–2017 | 2020–2021 | 2019 | 2016–2017 | |
| Scarlet fever (n = 84) | 80/95.24 | 199/90.87 | 292/97.64 | 3/3.57 | 7/3.20 | 0/0 | 13/15.48 | 19/8.68 | 5/1.68 |
| Impetigo (n = 17) | 17/100 | 25/86.31 | 0/0 | 0/0 | 1/3.45 | 0/0 | 2/11.76 | 1/3.45 | 0/0 |
| Psoriasis (n = 10) | 8/80 | 16/84.21 | 0/0 | 0/0 | 2/10.53 | 0/0 | 1/10 | 2/10.53 | 0/0 |
| Allergic purpura (n = 1) | 1/100 | 1/100 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 |
| Suppurative tonsillitis (n = 2) | 2/100 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 |
Emm molecular typing
The 114 isolates exhibited a high genetic diversity. 13 emm types accounted for 114 isolates. The majorities of cases were emm12 (53), emm1 (29), emm12.19 (5), and emm12.67 (5). Distribution of emm types and erythromycin-resistant genes in 114 GAS is presented in Table 3.
Table 3.
Distribution of emm types and erythromycin-resistant genes in 114 GAS isolates, compared with our previous studies [21, 22]
| emm types | emm subtypes | Count | ermB (n/%) | ermA (n/%) | mefA (n/%) | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 2020–2021 | 2019 | 2016–2017 | 2020–2021 | 2019 | 2016–2017 | 2020–2021 | 2019 | 2016–2017 | 2020–2021 | 2019 | 2016–2017 | ||
| emm1.0 | 31 | 89 | 78 | 27/23.68 | 84/31 | 78/26.3 | 0/0 | 2/0.74 | 0/0 | 4/3.51 | 10/3.7 | 2/0.67 | |
| emm12.0 | emm12.0 | 62 | 93 | 139 | 51/44.74 | 85/31.37 | 136/45.79 | 1/0.88 | 3/1.11 | 0/0 | 10/8.77 | 5/1.85 | 1/0.34 |
| emm12.19 | 5 | 22 | 27 | 5/4.39 | 21/7.75 | 27/9.09 | 0/0 | 1/0.37 | 0/0 | 0/0 | 1/0.37 | 0/0 | |
| emm12.21 | 4 | 2 | 2 | 3/2.63 | 2/0.74 | 2/0.67 | 0/0 | 0/0 | 0/0 | 1/0.88 | 1/0.37 | 0/0 | |
| emm12.29 | 3 | 0 | 0 | 3/2.63 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | |
| emm12.37 | 3 | 4 | 8 | 3/2.63 | 4/1.48 | 6/2.02 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | |
| emm12.65 | 2 | 0 | 0 | 1/0.88 | 0/0 | 0/0 | 1/0.88 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | |
| emm12.67 | 5 | 0 | 0 | 4/3.51 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 1/0.88 | 0/0 | 0/0 | |
| emm12.69 | 2 | 13 | 2 | 2/1.75 | 12/4.43 | 2/0.67 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | |
| emm4.0 | 2 | 4 | 1 | 1/0.88 | 2/0.74 | 1/0.34 | 1/0.88 | 1/0.37 | 0/0 | 0/0 | 1/0.37 | 0/0 | |
| emm11.0 | 1 | 0 | 0 | 1/0.88 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | |
| emm75.0 | 3 | 8 | 8 | 3/2.63 | 7/2.58 | 8/2.69 | 0/0 | 1/0.37 | 0/0 | 0/0 | 0/0 | 0/0 | |
| emm89.0 | 3 | 2 | 4 | 3/2.63 | 1/0.37 | 4/1.35 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | |
| Total | 114 | 271a | 297a | 107/93.85 | 243/89.67 | 97/64 | 3/2.63 | 10/3.69 | 0/0 | 16/14.04 | 22/8.12 | 5/1.68 | |
aTotal number of cases in that year, some emm subtypes were not found in the year 2020-2021
Thirteen different emm types were identified. The most common emm types were emm12.0 (53/114, 46.5%), emm1.0 (29/114, 25.4%). The most prevalent emm subtypes in GAS strains from scarlet fever were emm12.0 (37/114, 32.5%), emm1.0 (25/114, 21.9%), emm12.19 (5/114, 4.4%), and emm12.67 (4/114, 3.5%). The most predominant emm subtype in impetigo was emm12.0 (13/114, 11.4%). The most common emm subtypes from psoriasis were emm12.0 (2/114, 1.8%), emm12.29 (2/114, 1.8%), and emm89.0 (2/114, 1.8%). Distribution of GAS emm genotypes of strains in different diseases is presented in Table 4.
Table 4.
Distribution (%) of GAS emm genotypes of strains in different diseases among children in Beijing from 2020 to 2021, compared with our previous studies [21, 22]
| emm types | Scarlet fever | Impetigo | Psoriasis | Allergic purpura | Suppurative tonsillitis | Total | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 2020–2021 | 2019 | 2016–2017 | 2020–2021 | 2019 | 2016–2017 | 2020–2021 | 2019 | 2016–2017 | 2020–2021 | 2019 | 2016–2017 | 2020–2021 | 2019 | 2016–2017 | 2020–2021 | 2019 | 2016–2017 | |
| emm1.0 | 25 | 72 | 78 | 1 | 9 | 0 | 1 | 6 | 0 | 0 | 0 | 0 | 2 | 0 | 0 | 29 | 89 | 78 |
| emm12.0 | 37 | 78 | 139 | 13 | 11 | 0 | 2 | 4 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 53 | 93 | 138 |
| emm11.0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 |
| emm12.19 | 5 | 17 | 27 | 0 | 3 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 5 | 21 | 27 |
| emm12.21 | 3 | 1 | 2 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 3 | 2 | 2 |
| emm12.29 | 1 | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 3 | 0 | 0 |
| emm12.37 | 2 | 3 | 8 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 3 | 4 | 8 |
| emm12.65 | 2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 0 |
| emm12.67 | 4 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 5 | 0 | 0 |
| emm12.69 | 1 | 12 | 2 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2 | 13 | 2 |
| emm4.0 | 1 | 3 | 1 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 4 | 1 |
| emm75.0 | 2 | 5 | 8 | 0 | 1 | 0 | 1 | 2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 3 | 8 | 8 |
| emm89.0 | 1 | 1 | 4 | 1 | 0 | 0 | 2 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 4 | 2 | 4 | |
| Total | 84 | 219 | 297 | 17 | 29 | 0 | 10 | 19 | 0 | 1 | 1 | 0 | 2 | 0 | 0 | 114 | 271 | 297 |
Emm types and superantigen distribution
Among 114 GAS isolates, the most predominant superantigen genes detected were smeZ (112/114, 98.25%), speG (110/114, 96.49%), and speC (106/114, 92.98%). Among 53 emm12.0 isolates, the most prevalent superantigen genes detected were speG (51/114, 44.74%), ssa (51/114, 44.74%), and smeZ (51/114, 44.74%). Among 29 emm1.0 GAS isolates, the most predominant superantigen genes detected were speG (29/114, 25.44%), ssa (29/114, 25.44%), smeZ (29/114, 25.44%), speC (29/114, 25.44%). and speA (24/114, 21.05%). The distributions of emm types and superantigens in GAS isolates is shown in Table 5.
Table 5.
Distributions of emm types and superantigens in GAS isolates
|
emm Types |
Distribution of superantigens (n) | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Count | specA | speC | speH | speI | speJ | speK | speL | speM | speG | ssa | smeZ | |
| emm1.0 | 29 | 24 | 29 | 5 | 3 | 27 | 0 | 0 | 1 | 29 | 29 | 29 |
| emm12.0 | 53 | 4 | 49 | 45 | 43 | 3 | 0 | 0 | 2 | 51 | 51 | 51 |
| emm11.0 | 1 | 0 | 1 | 1 | 1 | 0 | 0 | 0 | 0 | 1 | 0 | 1 |
| emm12.19 | 5 | 1 | 5 | 0 | 0 | 2 | 0 | 0 | 0 | 5 | 5 | 5 |
| emm12.21 | 3 | 1 | 3 | 2 | 2 | 1 | 0 | 0 | 1 | 3 | 3 | 3 |
| emm12.29 | 3 | 0 | 3 | 3 | 3 | 0 | 0 | 0 | 1 | 3 | 3 | 3 |
| emm12.37 | 3 | 0 | 3 | 3 | 3 | 0 | 0 | 0 | 0 | 3 | 3 | 3 |
| emm12.65 | 2 | 0 | 2 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 2 | 2 |
| emm12.67 | 5 | 1 | 3 | 4 | 4 | 0 | 0 | 0 | 0 | 5 | 3 | 5 |
| emm12.69 | 2 | 1 | 2 | 0 | 0 | 1 | 0 | 0 | 0 | 2 | 2 | 2 |
| emm4.0 | 1 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1 |
| emm75.0 | 3 | 0 | 3 | 3 | 3 | 0 | 0 | 0 | 3 | 3 | 0 | 3 |
| emm89.0 | 4 | 0 | 2 | 0 | 0 | 0 | 1 | 0 | 0 | 4 | 1 | 4 |
| Total | 114 | 32 | 106 | 66 | 62 | 34 | 1 | 0 | 8 | 110 | 103 | 112 |
| Percentage (%) | 28.03 | 92.98 | 57.98 | 54.38 | 29.82 | 0.88 | 0 | 7.02 | 96.49 | 90.35 | 98.25 | |
Discussion
Streptococcus pyogenes is bacterial pathogen worldwide responsible for a broad spectrum of infection diseases as well as autoimmune sequelae [1, 4]. Epidemiological and molecular features of GAS isolates are quite different in different countries. COVID-19 pandemic has already changed our lifestyle. People pay more attention to protective social distance, wearing masks, personal hygiene, and frequent hand washing [24]. Respiratory infection diseases have been reduced dramatically as well as GAS-related respiratory infection diseases. Because of these, isolates collected in our study were much fewer compared with our previous study. Our present study offered insights into antibiotic resistance, virulence genes of GAS under COVID-19 pandemic.
In our present research, male-to-female ratio was 1.65:1. Kim analyzed children suffered scarlet fever in Jeju in Korea between 2002 and 2016. He presented male-to-female ratio was 1.3:1[25]. In Shanghai, during 2011 to 2015, scarlet fever usually affected children aged three to nine [12]. Patients from our present study, aged from 22 days to 11 years old, with median 6.58 years old.
Resistance rate of macrolides in our present study was still high compared with our previous studies from 2016 to 2017, and 2019. Yu found that from 2016 to 2018, 342 GAS strains were highly susceptible to penicillin, levofloxacin, and chloramphenicol, whereas most of strains were resistant to azithromycin, erythromycin, clarithromycin, clindamycin, and tetracycline [9]. Since 1990, the resistance rate of GAS against clindamycin and macrolides has been high [7]. Chinese strains mainly harbored ermB gene. In our study, 93.86% stains harbored ermB gene. Distribution of ermB gene in our GAS strains among scarlet fever, impetigo, psoriasis, allergic purpura, and suppurative tonsillitis was 95.24%, 100%, 80%, 100%, and 100% respectively (Table 3). In our previous study from 2016 to 2017, 97.64% GAS strains harbored ermB gene. In the year of 2019, we found 89.67% isolates harbored ermB gene.
M protein is immune-dominant GAS protein, locating on surface of bacterial cell wall [26], which adhering to host cell and block phagocytosis, aiding GAS colonization [27]. Macrolide resistance in GAS links to some emm types. In our study, emm12.0 and emm1.0 were predominant types in macrolide resistance GAS. Emm12.0 carried ermB was the most frequent macrolide resistance isolates, which was consistent with Liang’s study between 2005 and 2008 as well as our study in 2009 [22].
M protein and sAgs play an important role in GAS infection pathogenesis. There is a close relationship between emm types and sAgs [28]. In this study, we presented distribution of emm types including 13 emm types and 11 sAgs. Types emm12.0 and emm1.0 exhibited higher polymorphism rate which were similar with our previous study as well as Yu’ study from 2016 to 2018 [9]. They were responsible for about 73.81% of scarlet fever cases in our present study. Tsai collected 320 GAS strains from 339 children in Southern Taiwan. Emm12 (63.8%) was dominant type, following emm1 (16.9%), emm4 (11/0.9%) during 2000 to 2019 [29].
The dominant emm12.0, emm1.0, emm12.19, and emm12.67 types in this study were similar to those in Southeast Asia, UK and Southern Taiwan [30], but were different from results presented in Portugal and Canada. Ana exhibited markers of invasive GAS were emm1 and emm64, speA, and speJ independently, However, GAS carried emm4, emm75, ssa, speL/M genes were independent markers in pharyngitis [31]. In Canada, since 2010, emm1 has been the most frequent type. Epidemic scarlet fever has been reported in China, United Kingdom. In China, UK. GAS isolates were emm1, emm12, emm3, and emm4 respectively carrying speA, speC, ssa [32]. Our research was a little different from previously epidemic reports. Emm12 strains had been major epidemic isolates.
GAS M protein has been surveillance in Beijing from 2011 to 2018, meanwhile, M 12 stains began to decrease from 2011, and the lowest point was in 2014. Meanwhile, M 1 stains began to raise, and reached to the highest point in 2014, and then exceed M 12 from 2013 to 2014 [33]. However, our present research was different form Yu’ research. During 2019–2021, 2016–2017, we found GAS from scarlet fever and impetigo carried emm12, predominantly. In psoriasis, GAS carried emm1 in 2019 (Table 4), however, between 2020 and 2021, the isolates carried emm12, emm12.29 and emm89 predominantly [21, 22]. Patricia found emm70, emm33, emm25, emm93.3,and emm11 were the most frequent emm types among impetigo, pharyngitis, and asymptomatic throat [3].
Liang and Luca found emm1.0 isolates harbored speA, speC with similar frequencies, meanwhile, emm12.0 carried low frequencies speA, and high frequencies speC. The frequencies of speA, speC among emm1.0, emm12.0 isolates in present study were consistent with Liang’s results[20], while that in our previous study were in agreement with Luca’s results[34].
In our present study, 11 sAgs were detected in GAS isolates. SmeZ, ssa, speC were the most common sAgs. Emm1 carried speG, ssa, smeZ, speC, and speA. However, content of speH, speI, and speM was less. Emm12 harbored speG, ssa, smeZ and speG, with little speA, speJ and speM. Both emm1.0 and emm12.0 had no speK, speL. Lu found among invasive or not GAS isolates harbored speB, and slo, meanwile, smeZ, speC, and speF were determined in more than 90% isolates from 2009 to 2016 in 7 cities in China. These isolates carried emm12.0 (42.9%) and emm1.0 (30.7%) [35].
Liang found scarlet fever isolates carried speA (52.4%), and speC (79.3%) from 2005 to 2008 in mainland China [20]. SAg distribution was varied in different geographic areas. In France, Plainvert exhibited GAS strains carried speA (59%), speC (37%), ssa (13%), and smeZ (92%) in meningitis from 2003 to 2013. During 2006 to 2009, Friaes presented more than 90% GAS isolates carried speG and smeZ. In Ireland, Mary exhibited invasive emm types were emm1, emm3, meanwhile, in non-invasive GAS isolates were emm4, emm28, and emm3. SpeA, speG and speJ were related with invasive GAS isolates, whereas speC, speI, and ssa with non-invasive GAS infections [36]. According to our present data, we found scarlet fever isolates harbored speC (94.05%), and smeZ (98.81%), psoriasis isolates carried speC (80%), and smeZ (100%). Impetigo isolates carried speC (88.89%), ssa (88.89%), and smeZ (88.89%). In our previous study from 2016 to 2017, we found that the most prevalent scarlet fever isolates carried smeZ (96.97%), speC (92.59%) and speG (91.58%), presented in Table 6. However, in our study of 2019, the most prevalent GAS carried smeZ (94.46%), speC (91.14%) and ssa (74.91%). Scarlet fever isolates prevalently harbored smeZ (93.6%), speC (90.4%). Psoriasis isolates harbored smeZ (100%), speC (100%), and impetigo isolates harbored smeZ (100%), ssa (89.7%), and speC (89.7%) [22]. Catarina collected 303 GAS strains from scarlet fever, tonsilla-pharyngitis patients between 2002 and 2008. Isolates from scarlet fever carried smeZ, ssa, speG and speC. Strains from pharyngitis carried smeZ, speG, speC, and ssa [37].
Table 6.
Distribution of superantigens and emm types in isolates among different GAS infected cutaneous diseases
|
emm types |
Scarlet fever (n = 84) |
Impetigo (n = 17) |
Psoriasis (n = 10) |
Count 114 |
||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| speA | speC | speH | speI | SpeJ | ssa | smeZ | speA | speC | speH | speI | speJ | ssa | smeZ | speA | speC | speH | speI | speJ | ssa | smeZ | ||
| emm1.0 | 21 | 25 | 4 | 2 | 23 | 25 | 25 | 0 | 1 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 0 | 0 | 1 | 1 | 1 | 134 |
| emm12.0 | 2 | 34 | 32 | 31 | 1 | 36 | 36 | 0 | 12 | 11 | 10 | 0 | 12 | 12 | 2 | 2 | 1 | 1 | 2 | 2 | 2 | 241 |
| emm11.0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1 | 1 | 0 | 0 | 1 | 4 |
| emm12.19 | 1 | 5 | 0 | 0 | 2 | 5 | 5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 18 |
| emm12.21 | 1 | 3 | 2 | 2 | 1 | 3 | 3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 15 |
| emm12.29 | 0 | 1 | 1 | 1 | 0 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2 | 2 | 2 | 0 | 2 | 2 | 15 |
| emm12.37 | 0 | 2 | 2 | 2 | 0 | 2 | 2 | 0 | 1 | 1 | 1 | 0 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 15 |
| emm12.65 | 0 | 2 | 0 | 1 | 0 | 2 | 2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 7 |
| emm12.67 | 1 | 2 | 3 | 3 | 0 | 2 | 4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1 | 1 | 0 | 1 | 1 | 20 |
| emm12.69 | 0 | 1 | 0 | 0 | 0 | 1 | 1 | 1 | 1 | 0 | 0 | 1 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 8 |
| emm4.0 | 0 | 1 | 0 | 0 | 0 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 3 |
| emm75.0 | 0 | 2 | 2 | 2 | 0 | 0 | 2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1 | 1 | 0 | 0 | 1 | 12 |
| emm89.0 | 0 | 1 | 0 | 0 | 0 | 0 | 1 | 0 | 1 | 0 | 0 | 0 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 2 | 7 |
| totle | 26 | 79 | 46 | 44 | 27 | 78 | 83 | 1 | 16 | 12 | 11 | 2 | 16 | 16 | 3 | 8 | 6 | 6 | 2 | 6 | 10 | 114 |
| Percentage% | 30.95 | 94.05 | 54.76 | 52.38 | 32.14 | 92.86 | 98.81 | 5.55 | 88.89 | 66.67 | 61.11 | 11.11 | 88.89 | 88.98 | 30 | 80 | 60 | 60 | 20 | 60 | 100 | |
Our study has several limitations. Firstly, our research was conducted at a single center, which could have made biases to occurrence of GAS infected cutaneous diseases. Under COVID-19 pandemic, outpatients deceased dramatically. Atypical symptoms might have been misdiagnosed. Secondly, our study had small GAS isolates which might not fully represent GAS types under COVID-19 pandemic.
In summary, our study exhibited epidemiology and molecular characteristics of GAS infection cutaneous diseases in a children’ hospital in Beijing under COVID-19 pandemic. We compared our research with researches before COVID-19 pandemic. Collections of GAS infected cutaneous diseases decreased dramatically. M proteins in psoriasis were different in the year of 2019 and 2020 to 2021. There were no significant changes in epidemiology and molecular characteristics of GAS in children with scarlet fever, impetigo before and during COVID-19 pandemic. Long-term surveillance and investigation of emm types and superantigens of GAS prevalence are necessary.
Disclaimer
The study sponsors had no role in study design; collection, analysis, and interpretation of data; writing the report; or the decision to submit the report for publication.
Acknowledgements
Our deepest gratitude goes to the study participants, data collectors, and supervisors.
Authors’ contributions
Hongxin Li, Lin Zhou, Yong Zhao designed the study; Hongxin Li, Lin Zhou collected data; Hongxin Li, Lin Zhou, and Lijuan Ma, Xiaoyan Liu, Jin Hu, Haihua Zhang, Yan Liu coordinated and supervised the data collection; Hongxin Li, Lin Zhou, Yong Zhao analyzed the data; Lin Zhou, Yong Zhao participated in the interpretation of data; Hongxin Li, Yong Zhao drafted the initial manuscript. All authors approved the final manuscript as submitted and agree to be accountable for all aspects of work.
Funding
This work was supported by Research Foundation of Capital Institute of Pediatrics (No. PY-2020–05) and supported by Research Foundation of Capital Institute of Pediatrics (No. GZ-2021–10). The study sponsors had no role in study design; collection, analysis, and interpretation of data; writing the report; or the decision to submit the report for publication.
Availability of data and materials
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.
Declarations
Competing interests
The authors declare no competing interests.
Ethics approval and consent to participate
This study was approved by the ethics committee of the Capital Institute of Pediatrics. Informed written consent were obtained from the participants’ guardians before collecting samples, and anonymity of the participants was guaranteed. This study was conducted in accordance with the ethical principles that have their origin in the Declaration of Helsinki and that are consistent with Good Clinical Practice and applicable regulatory requirements.
Consent for publication
Not applicable.
Conflicts of interest
The authors declare no competing interests.
Footnotes
Publisher’s Note
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References
- 1.Walker MJ, Barnett TC, McArthur JD, Cole JN, Gillen CM, Henningham A, Sriprakash KS, Sanderson-Smith ML, Nizet V. Disease manifestations and pathogenic mechanisms of Group A Streptococcus. Clin Microbiol Rev. 2014;27(2):264–301. doi: 10.1128/CMR.00101-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Wijesundara NM, Lee SF, Cheng Z, Davidson R, Rupasinghe HPV. Carvacrol exhibits rapid bactericidal activity against Streptococcus pyogenes through cell membrane damage. Sci Rep. 2021;11(1):1487. doi: 10.1038/s41598-020-79713-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Campbell PT, Tong SYC, Geard N, Davies MR, Worthing KA, Lacey JA, Smeesters PR, Batzloff MR, Kado J, Jenney AWJ, McVernon J, Steer AC. Longitudinal Analysis of Group A Streptococcus emm Types and emm Clusters in a High-Prevalence Setting: Relationship between Past and Future Infections. J Infect Dis. 2020;221(9):1429–1437. doi: 10.1093/infdis/jiz615. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Carapetis JR, Steer AC, Mulholland EK, Weber M. The global burden of group A streptococcal diseases. Lancet Infect Dis. 2005;5(11):685–694. doi: 10.1016/S1473-3099(05)70267-X. [DOI] [PubMed] [Google Scholar]
- 5.Anderson J, Imran S, Frost HR, Azzopardi KI, Jalali S, Novakovic B, Osowicki J, Steer AC, Licciardi PV, Pellicci DG. Immune signature of acute pharyngitis in a Streptococcus pyogenes human challenge trial. Nat Commun. 2022;13(1):769. doi: 10.1038/s41467-022-28335-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Yang S, Wu J, Ding C, Cui Y, Zhou Y, Li Y, Deng M, Wang C, Xu K, Ren J, Ruan B, Li L. Epidemiological features of and changes in incidence of infectious diseases in China in the first decade after the SARS outbreak: an observational trend study. Lancet Infect Dis. 2017;17(7):716–725. doi: 10.1016/S1473-3099(17)30227-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Yu D, Liang Y, Zheng Y, Yang Y. Clindamycin-resistant Streptococcus pyogenes in Chinese children. Lancet Infect Dis. 2021;21(12):1631–1632. doi: 10.1016/S1473-3099(21)00699-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Villalon P, Saez-Nieto JA, Rubio-Lopez V, Medina-Pascual MJ, Garrido N, Carrasco G, Pino-Rosa S, Valdezate S. Invasive Streptococcus pyogenes disease in Spain: a microbiological and epidemiological study covering the period 2007–2019. Eur J Clin Microbiol Infect Dis. 2021;40(11):2295–2303. doi: 10.1007/s10096-021-04279-2. [DOI] [PubMed] [Google Scholar]
- 9.Yu D, Liang Y, Lu Q, Meng Q, Wang W, Huang L, Bao Y, Zhao R, Chen Y, Zheng Y, Yang Y. Molecular Characteristics of Streptococcus pyogenes Isolated From Chinese Children With Different Diseases. Front Microbiol. 2021;12:722225. doi: 10.3389/fmicb.2021.722225. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Perez-Trallero E, Montes M, Orden B, Tamayo E, Garcia-Arenzana JM, Marimon JM. Phenotypic and genotypic characterization of Streptococcus pyogenes isolates displaying the MLSB phenotype of macrolide resistance in Spain, 1999 to 2005. Antimicrob Agents Chemother. 2007;51(4):1228–1233. doi: 10.1128/AAC.01054-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Ripa S, Zampaloni C, Vitali LA, Giovanetti E, Montanari MP, Prenna M, Varaldo PE. SmaI macrorestriction analysis of Italian isolates of erythromycin-resistant Streptococcus pyogenes and correlations with macrolide-resistance phenotypes. Microb Drug Resist. 2001;7(1):65–71. doi: 10.1089/107662901750152828. [DOI] [PubMed] [Google Scholar]
- 12.Chen M, Cai J, Davies MR, Li Y, Zhang C, Yao W, Kong D, Pan H, Zhang X, Zeng M. Increase of emm1 isolates among group A Streptococcus strains causing scarlet fever in Shanghai, China. Int J Infect Dis. 2020;98:305–314. doi: 10.1016/j.ijid.2020.06.053. [DOI] [PubMed] [Google Scholar]
- 13.Wang HB, Song YY, You YH, Wang HW, Han QH, Zhao JH, Zhang XX. Molecular epidemiological analysis of group A Streptococci isolated from children in Chaoyang District of Beijing, 2011: emm types, virulence factor genes and erythromycin resistant genes. Biomed Environ Sci. 2013;26(9):782–784. doi: 10.3967/0895-3988.2013.09.012. [DOI] [PubMed] [Google Scholar]
- 14.Steer AC, Law I, Matatolu L, Beall BW, Carapetis JR. Global emm type distribution of group A streptococci: systematic review and implications for vaccine development. Lancet Infect Dis. 2009;9(10):611–616. doi: 10.1016/S1473-3099(09)70178-1. [DOI] [PubMed] [Google Scholar]
- 15.You Y, Peng X, Yang P, Wang Q, Zhang J. 8-year M type surveillance of Streptococcus pyogenes in China. Lancet Infect Dis. 2020;20(1):24–25. doi: 10.1016/S1473-3099(19)30694-2. [DOI] [PubMed] [Google Scholar]
- 16.Reglinski M, Sriskandan S, Turner CE. Identification of two new core chromosome-encoded superantigens in Streptococcus pyogenes; speQ and speR. J Infect. 2019;78(5):358–363. doi: 10.1016/j.jinf.2019.02.005. [DOI] [PubMed] [Google Scholar]
- 17.Zeppa JJ, Kasper KJ, Mohorovic I, Mazzuca DM, Haeryfar SMM, McCormick JK. Nasopharyngeal infection by Streptococcus pyogenes requires superantigen-responsive Vbeta-specific T cells. Proc Natl Acad Sci U S A. 2017;114(38):10226–10231. doi: 10.1073/pnas.1700858114. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Gonzalez-Abad MJ, Alonso Sanz M. Invasive Streptococcus pyogenes infections (2011–2018): EMM-type and clinical presentation. Anales de pediatria. 2020;92(6):351–358. doi: 10.1016/j.anpedi.2019.10.014. [DOI] [PubMed] [Google Scholar]
- 19.Liang Y, Shen X, Huang G, Wang C, Shen Y, Yang Y. Characteristics of Streptococcus pyogenes strains isolated from Chinese children with scarlet fever. Acta Paediatr. 2008;97(12):1681–1685. doi: 10.1111/j.1651-2227.2008.00983.x. [DOI] [PubMed] [Google Scholar]
- 20.Liang Y, Liu X, Chang H, Ji L, Huang G, Fu Z, Zheng Y, Wang L, Li C, Shen Y, Yu S, Yao K, Ma L, Shen X, Yang Y. Epidemiological and molecular characteristics of clinical isolates of Streptococcus pyogenes collected between 2005 and 2008 from Chinese children. J Med Microbiol. 2012;61(Pt 7):975–983. doi: 10.1099/jmm.0.042309-0. [DOI] [PubMed] [Google Scholar]
- 21.Li H, Zhou L, Zhao Y, Ma L, Liu X, Hu J. Molecular epidemiology and antimicrobial resistance of group a streptococcus recovered from patients in Beijing. China BMC Infect Dis. 2020;20(1):507. doi: 10.1186/s12879-020-05241-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Li H, Zhou L, Zhao Y, Ma L, Xu J, Liu Y, Qin Q, Hu J, Liu X. Epidemiological analysis of Group A Streptococcus infections in a hospital in Beijing, China. Eur J Clin Microbiol Infect Dis. 2020;39(12):2361–2371. doi: 10.1007/s10096-020-03987-5. [DOI] [PubMed] [Google Scholar]
- 23.Green NM, Beres SB, Graviss EA, Allison JE, McGeer AJ, Vuopio-Varkila J, LeFebvre RB, Musser JM. Genetic diversity among type emm28 group A Streptococcus strains causing invasive infections and pharyngitis. J Clin Microbiol. 2005;43(8):4083–4091. doi: 10.1128/JCM.43.8.4083-4091.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Wang C, Horby PW, Hayden FG, Gao GF. A novel coronavirus outbreak of global health concern. Lancet. 2020;395(10223):470–473. doi: 10.1016/S0140-6736(20)30185-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Kim J, Kim JE, Bae JM. Incidence of Scarlet Fever in Children in Jeju Province, Korea, 2002-2016: An Age-period-cohort Analysis. J Prev Med Public Health. 2019;52(3):188–194. doi: 10.3961/jpmph.18.299. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Happonen L, Hauri S, Svensson Birkedal G, Karlsson C, de Neergaard T, Khakzad H, Nordenfelt P, Wikstrom M, Wisniewska M, Bjorck L, Malmstrom L, Malmstrom J. A quantitative Streptococcus pyogenes-human protein-protein interaction map reveals localization of opsonizing antibodies. Nat Commun. 2019;10(1):2727. doi: 10.1038/s41467-019-10583-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Castro SA, Dorfmueller HC. A brief review on Group A Streptococcus pathogenesis and vaccine development. Royal Society open science. 2021;8(3):201991. doi: 10.1098/rsos.201991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Imohl M, Fitzner C, Perniciaro S, van der Linden M. Epidemiology and distribution of 10 superantigens among invasive Streptococcus pyogenes disease in Germany from 2009 to 2014. PLoS ONE. 2017;12(7):e0180757. doi: 10.1371/journal.pone.0180757. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Tsai WC, Shen CF, Lin YL, Shen FC, Tsai PJ, Wang SY, Lin YS, Wu JJ, Chi CY, Liu CC. Emergence of macrolide-resistant Streptococcus pyogenes emm12 in southern Taiwan from 2000 to 2019. J Microbiol Immunol Infect. 2021;54(6):1086–93. [DOI] [PubMed]
- 30.Turner CE, Pyzio M, Song B, Lamagni T, Meltzer M, Chow JY, Efstratiou A, Curtis S. Sriskandan S (2016) Scarlet Fever Upsurge in England and Molecular-Genetic Analysis in North-West London. Emerg Infect Dis. 2014;22(6):1075–1078. doi: 10.3201/eid2206.151726. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Friaes A, Pinto FR, Silva-Costa C, Ramirez M, Melo-Cristino J. Group A streptococci clones associated with invasive infections and pharyngitis in Portugal present differences in emm types, superantigen gene content and antimicrobial resistance. BMC Microbiol. 2012;12:280. doi: 10.1186/1471-2180-12-280. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Walker MJ, Brouwer S, Forde BM, Worthing KA, McIntyre L, Sundac L, Maloney S, Roberts LW, Barnett TC, Richter J, Cork AJ, Irwin AD, You Y, Zhang J, Dougan G, Yuen KY, Nizet V, Beatson SA, Grimwood K, Davies MR. Detection of Epidemic Scarlet Fever Group A Streptococcus in Australia. Clin Infect Dis. 2019;69(7):1232–1234. doi: 10.1093/cid/ciz099. [DOI] [PubMed] [Google Scholar]
- 33.Okabe T, Norose Y, Hida M, Takeda S, Takase M, Suzuki Y, Ohkuni H. Change during an 8-Year Period in Streptococcus Pyogenes emm Types in Pharyngeal Isolates from Children with Noninvasive Infections. J Nippon Med Sch. 2020;87(4):211–214. doi: 10.1272/jnms.JNMS.2020_87-502. [DOI] [PubMed] [Google Scholar]
- 34.Luca-Harari B, Straut M, Cretoiu S, Surdeanu M, Ungureanu V, van der Linden M, Jasir A. Molecular characterization of invasive and non-invasive Streptococcus pyogenes isolates from Romania. J Med Microbiol. 2008;57(Pt 11):1354–1363. doi: 10.1099/jmm.0.2008/001875-0. [DOI] [PubMed] [Google Scholar]
- 35.Lu B, Fang Y, Fan Y, Chen X, Wang J, Zeng J, Li Y, Zhang Z, Huang L, Li H, Li D, Zhu F, Cui Y, Wang D. High Prevalence of Macrolide-resistance and Molecular Characterization of Streptococcus pyogenes Isolates Circulating in China from 2009 to 2016. Front Microbiol. 2017;8:1052. doi: 10.3389/fmicb.2017.01052. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Meehan M, Murchan S, Gavin PJ, Drew RJ, Cunney R. Epidemiology of an upsurge of invasive group A streptococcal infections in Ireland, 2012–2015. J Infect. 2018;77(3):183–190. doi: 10.1016/j.jinf.2018.05.010. [DOI] [PubMed] [Google Scholar]
- 37.Silva-Costa C, Carrico JA, Ramirez M, Melo-Cristino J. Scarlet fever is caused by a limited number of Streptococcus pyogenes lineages and is associated with the exotoxin genes ssa, speA and speC. Pediatr Infect Dis J. 2014;33(3):306–310. doi: 10.1097/INF.0000000000000088. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.