|Year : 2016 | Volume
| Issue : 2 | Page : 159-165
Genotyping and serotyping of macrolide and multidrug resistant Streptococcus pneumoniae isolated from carrier children
SF Swedan1, WA Hayajneh2, GN Bshara1
1 Department of Medical Laboratory Sciences, Faculty of Applied Medical Sciences, Jordan University of Science and Technology, Irbid 22110, Jordan
2 Department of Pediatrics, Faculty of Medicine, Jordan University of Science and Technology, Irbid 22110, Jordan
|Date of Submission||28-Aug-2014|
|Date of Acceptance||23-Jan-2015|
|Date of Web Publication||14-Apr-2016|
S F Swedan
Department of Medical Laboratory Sciences, Faculty of Applied Medical Sciences, Jordan University of Science and Technology, Irbid 22110
Source of Support: None, Conflict of Interest: None
Aims: Streptococcus pneumoniae, an opportunistic pathogen commonly carried asymptomatically in the nasopharynx of children, is associated with increasing rates of treatment failures due to a worldwide increase in drug resistance. We investigated the carriage of S. pneumoniae in children 5 years or younger, the identity of prevalent serotypes, the rates of resistance to macrolides and other antimicrobial agents and the genotypes responsible for macrolide resistance. Materials and Methods: Nasopharyngeal swabs were collected from 157 children under 5 years for cultural isolation of S. pneumoniae. Antibiogram of isolates was determined using the disk diffusion test, and the minimal inhibitory concentration to macrolides was determined using the E-test. Isolate serotypes and macrolide resistance genes, erm(B) and mef(E), were identified using multiplex polymerase chain reactions. Results: S. pneumoniae was recovered from 33.8% of children; 41.9% among males and 21.9% among females (P = 0.009). The highest carriage rate occurred among age groups 7-12 months and 49-60 months. Most frequent serotypes were 19F, 6A/B, 11A, 19A, 14 and 15B/C. Resistance to macrolides was 60.4%. Resistance to oxacillin, trimethoprim/sulfamethoxazole and clindamycin was present among 90.6%, 54.7% and 32.1% of isolates, respectively. All isolates were susceptible to chloramphenicol, levofloxacin and vancomycin. Isolates resistant to one or more macrolide drugs were more likely to be multidrug resistant. Resistance to clindamycin or oxacillin coexisted with macrolide resistance. Among the erythromycin-resistant isolates, erm(B), mef(E) and erm(B) and mef(E) genes were present at rates of 43.8%, 37.5% and 6.3%, respectively. Erm(B) and mef(E) were associated with very high level and moderate-to-high level resistance to macrolides, respectively. Conclusion: A significant proportion of children harboured macrolide and multidrug-resistant S. pneumoniae.
Keywords: Antimicrobial resistance, colonisation, Jordan, prevalence
|How to cite this article:|
Swedan S F, Hayajneh W A, Bshara G N. Genotyping and serotyping of macrolide and multidrug resistant Streptococcus pneumoniae isolated from carrier children. Indian J Med Microbiol 2016;34:159-65
|How to cite this URL:|
Swedan S F, Hayajneh W A, Bshara G N. Genotyping and serotyping of macrolide and multidrug resistant Streptococcus pneumoniae isolated from carrier children. Indian J Med Microbiol [serial online] 2016 [cited 2019 Mar 26];34:159-65. Available from: http://www.ijmm.org/text.asp?2016/34/2/159/176840
| ~ Introduction|| |
Streptococcus pneumoniae is an asymptomatic coloniser of the nasopharynx of healthy individuals with carriage rate higher among children (40%) than adults (10%).  Carriage of S. pneumoniae increases the risk of development of pneumococcal diseases, especially in high-risk groups such as the very young. It is responsible for a high percentage of cases of community-acquired pneumonia and is the most frequent cause of bacterial pneumonia in children under 5 years of age leading to death of more than 1 million children (under 5 years) per year. 
S. pneumoniae antiphagocytic polysaccharide capsule which determines the bacterial serotype is the most important virulence factor and is the component used for the synthesis of pneumococcal vaccines. Knowledge of the prevalent capsular serotypes is crucial for development of effective vaccines. Although more than 90 serotypes of S. pneumoniae have been identified to date, it is estimated that only a fraction of these is responsible for approximately 75% of cases of invasive pneumococcal diseases (IPD) worldwide. Serotype 19A alone is responsible for 50% of IPD cases in children.  In the Middle East, the most common serotypes associated with IPD, in order of decreasing prevalence are 14, 6B, 5, 6A and 19A. 
The worldwide variation in the prevalence of pneumococcal serotypes and drug resistance is related to variations in the use and misuse of antimicrobial agents and the implementation of pneumococcal vaccination programs. The use of pneumococcal vaccines has reduced carriage rates and the rates of drug resistance and IPDs caused by the serotypes covered by the vaccines. However, the prevalence of IPD and drug-resistance by non-vaccine serotypes has increased.  Hence, mapping of regional serotypes and the implementation of periodical surveillance studies is essential to identify possible serotype shifts.
In recent years, a worrying trend of multidrug resistance including that towards macrolides by S. pneumoniae has been observed.  Two major mechanisms are responsible for macrolide resistance; the modification of the drug binding site due to acquisition of erm(B) gene (erythromycin ribosome methylase) which is associated with the MLS B (macrolides, lincosamides and streptogramin B) phenotype, and the expression of an efflux pump system due to the presence of mef genes (mef(A) and mef(E)) that are associated with the M phenotype.  Mef(A) is predominant in Europe while mef(E) is predominant in the United States, Asia and South Africa.  Hence, this study investigated the presence of the erm(B) and mef(E) genes among the isolates.
Information regarding prevalence of S. pneumoniae in Jordan is lacking. The objectives of this study were to determine the carriage rate of S. pneumoniae in Jordanian children 5 years or younger, the identity of prevalent serotypes, the rates of resistance to macrolides and other antimicrobial agents and the genotypes responsible for macrolide resistance. This information will be essential for the implementation of treatment and prevention interventions.
| ~ Materials and Methods|| |
The study was approved by the institutional review board. One hundred fifty-seven children under 5 years of age attending the paediatrics clinic of a major hospital in Northern Jordan for routine checkup, between September 2012 and March 2013, were randomly recruited for the study. Informed written consent was obtained from the legal guardian. Medical history information was obtained from parents and medical records. Samples were collected from the left and right nasopharynx (one swab per side) of apparently healthy children, using sterile nasopharyngeal calcium alginate-tipped swabs (Puritan medical, Guilford, Maine, USA). The two samples from each child were cultured immediately on blood agar medium (supplemented with 2.5 mg/L gentamicin). Culture plates were incubated at 37°C with CO 2 provided by CO 2 bags (CampyGen sachets, Oxoid, Basingstoke, Hampshire, UK) for 18-24 h. Colonies having Gram-positive lanceolate-shaped diplococci demonstrating alpha-haemolysis, optochin susceptibility and bile solubility were identified as S. pneumoniae. DNA was obtained from each isolate using the Promega Wizard; genomic DNA purification kit (Fitchburg, Wisconsin, USA). All pure isolates of S. pneumoniae were stored in 500 μL of skim milk-tryptone-glucose-glycerine storage medium at −70°C.
Antimicrobial susceptibility testing
Nine antimicrobial disks: Azithromycin (15 μg), chloramphenicol (30 μg), clarithromycin (15 μg), clindamycin (2 μg), erythromycin (15 μg), levofloxacin (5 μg), oxacillin (1 μg), sulfamethoxazole/trimethoprim (1.25 μg/23.75 μg) and vancomycin (30 μg) were used to perform the Kirby-Bauer disk diffusion test using blood agar media. Growth inhibition results were interpreted according to the clinical and laboratory standards institute (CLSI) 2012 guidelines.
Three types of macrolide E-test strips; azithromycin, clarithromycin and erythromycin, all obtained from Liofilchem (Roseto Degli Abruzzi, Italy) were used for each pneumococcal isolate for minimum inhibitory concentration (MIC) determination using blood agar media. The plates were incubated aerobically overnight at 37°C. An isolate was considered resistant to macrolide drugs at MIC values of ≥2 mg/L for azithromycin and ≥1 mg/L for clarithromycin and erythromycin.
Streptococcus pneumoniae capsular serotyping
Seven sequential multiplex polymerase chain reactions (PCR) were performed in 25 μL final volumes, according to a previously published protocol and primers.  The protocol enabled the simultaneous and specific identification of 28 different S. pneumoniae capsular serotypes. A primer pair targeting the cpsA locus was included as an internal positive control in all reactions. Serotypes detectable were 1, 3, 4, 6A/B, 7F, 7C, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B/C, 16F, 17F, 18, 19A, 19F, 20, 22F, 23F, 31, 33, 34, 35B, 35F and 38. Amplification reactions were carried out in a Bio-Rad S1000™ thermal cycler (Hercules, California, USA). ATCC strains (Manassas, Virginia, USA) 49619, 700669 and 700673 having serotypes 19F, 23F and 19A, respectively, were used as positive controls.
Identification of the erm(B) and mef(E) resistance genes
Macrolide resistance genes, erm(B) and mef(E), were identified in S. pneumoniae using multiplex PCR according to a previously published protocol and primers.  PCR was performed in 25 μL final volumes. Control strains harbouring erm(B) (ATCC 700673), mef(E) (mef(E)-positive clinical isolate recovered from a nasopharyngeal swab from King Abdullah University Hospital, Jordan), or lacking macrolide resistance genes (ATCC 49619) were included with every reaction. Amplification reactions were carried out in a Bio-Rad S1000™ thermal cycler (Hercules, CA, USA) at 93°C for 3 min, followed by 35 cycles of 93°C for 60 s, 50°C for 60 s and 72°C for 60 s and final elongation at 72°C for 5 min. The specificity of PCR amplification was confirmed by sequencing several representative PCR product bands after separation on agarose gel electrophoresis.
Identification of polymerase chain reaction products
Amplified PCR products were separated on 2% agarose (with ethidium bromide) using 120 volts for 50 min. Product bands were visualised using a Bio-Rad ChemiDoc™ XRS gel documentation system and Quantity One; software (Hercules, CA, USA).
The IBM SPSS software version 21 (Armonk, NY, USA) was used to generate descriptive analysis of raw data, including generation of all frequency tables and cross tabulations. The Pearson Chi-square test and the one-sample binomial test were used, where appropriate, to compare frequency data. A P < 0.05 was considered statistically significant.
| ~ Results|| |
Streptococcus pneumoniae carriage
The 157 children recruited for the study had an age range of 1-60 months, a mean of 21.3 months, standard deviation (SD) of 18.2 months and a median of 18 months. The children consisted of 93 males (59.2%) and 64 females (40.8%). S. pneumoniae was recovered from 53 children (33.8%) [Table 1]. Of the 53, 41.9% (39/93) and 21.9% (14/64) of males and females, respectively, harboured S. pneumoniae (P = 0.009). Among the S. pneumoniae carriers, the age range was 3-57 months, with a mean of 27.3 months, SD of 17.3 months and a median of 24 months.
|Table 1: Distribution of children by age groups colonised with Streptococcus pneumoniae and attending kindergarten or daycare centres |
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The highest rate of S. pneumoniae carriage occurred in age groups 7-12 months and 49-60 months, at 50% each, while the lowest carriage rate occurred in age group 1-6 months at 11.1% (P = 0.242) [Table 1]. Carriage rate was higher in children attending kindergarten or day care centres (for more than 4 h a day, 5 days a week) than non-attending children (45% [9/20] vs. 32.1% [44/137], respectively). However, the difference was not statistically significant (P = 0.255) (data not shown).
Streptococcus pneumoniae serotypes
The serotypes of 43 (81.1%) isolates belonged to 15 different capsular serotypes. The serotypes of 10 (18.9%) isolates could not be determined. The serotype most frequently identified was 19F (9/43; 17.0%), followed by serotypes 6A/B (6/43; 11.3%); 11A and 19A (4/43; 7.5% each); 14 and 15B/C (3/43; 5.7% each); 9V, 15A and 23F (2/43; 3.8% each) and 8F, 16F, 18, 22F, 33F and 35B (1/43; 1.9% each). Two isolates (3.8%) expressed double serotypes, 6A/B and 35B and 11A and 14 [Table S1] . The mef(E)-positive control had serotype 8F.
|Table S1: Serotypes of Streptococcus pneumoniae isolates and the distribution of macrolide resistance genes|
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Out of the 53 S. pneumoniae isolates, all were susceptible to chloramphenicol, levofloxacin and vancomycin, 48 (90.6%) were resistant to oxacillin, 29 (54.7%) were resistant to azithromycin, clarithromycin and trimethoprim/sulfamethoxazole, 28 (52.8%) were resistant to erythromycin and 17 (32.1%) were resistant to clindamycin [Table 2].
|Table 2: Antimicrobial susceptibility results for the Streptococcus pneumoniae isolates |
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The S. pneumoniae ATCC 49619 strain was susceptible to levofloxacin and vancomycin and resistant to the remaining antimicrobials. The ATCC 700673 strain was intermediately susceptible to trimethoprim/sulfamethoxazole and susceptible to the remaining antimicrobials. The mef(E)- positive control was resistant to oxacillin and trimethoprim/sulfamethoxazole and susceptible to the remaining antimicrobials.
Thirty isolates in total were resistant to one or more macrolides [Table 3]. These isolates were significantly more likely to be additionally resistant to two or three non-macrolide drugs than the macrolide sensitive isolates, having resistance rates of 64% versus 36% and 100% versus 0%, respectively (P = 0.002) [Table 3].
|Table 3: Correlation between Streptococcus pneumoniae macrolide resistance and resistance to other antimicrobial agents |
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The isolates' MIC values for azithromycin ranged from 0.25 to >256 mg/L, for clarithromycin 0.047 to >256 mg/L and for erythromycin 0.094 to >256 mg/mL [Table S2]. Based on MIC interpretation criteria, 32 out of the 53 isolates (60.4%) were resistant to all three macrolide drugs. S. pneumoniae ATCC 700673 (erm(B) positive control) was resistant to all macrolide drugs with MIC values >256 mg/L. S. pneumoniae ATCC 49619 (macrolide resistance negative control) was intermediately susceptible to azithromycin (MIC = 1 mg/L) and susceptible to both clarithromycin and erythromycin (MIC = 0.047 and 0.19 mg/L, respectively). The mef(E)-positive control was resistant to azithromycin, clarithromycin and erythromycin (MIC = 8, 2 and 4 mg/L, respectively). Interestingly, for MIC values <256 mg/L, azithromycin consistently demonstrated higher MIC values than clarithromycin and erythromycin for almost all clinical isolates (data not shown) [Table S2].
|Table S2: Frequency of E - test MIC results for the Streptococcus pneumoniae isolates |
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Macrolide resistance genotypes
Of the 53 isolates, 14 (26.4%) had the erm(B) alone, 13 (24.5%) had mef(E) alone and 7 (13.2%) had both erm(B) and mef(E). Among the 32 erythromycin resistant isolates (MIC ≥1 mg/L), erm(B), mef(E) and erm(B) and mef(E) were present at rates of 43.8% (14/32), 37.5% (12/32) and 6.3% (2/32), respectively [Table 4].
Most isolates having erm(B) alone had an MIC value >256 mg/L for erythromycin, whereas, most isolates having mef(E) alone had MIC values between 3 and 24 mg/L for erythromycin [Table 4]. Most isolates (78.9%) having both genes had MIC values well below 1 mg/L (specifically ≤0.19 mg/L; data not shown) for erythromycin [Table 4]. The genotypes responsible for erythromycin resistance among four isolates could not be determined [Table 4]. No significant association between serotype and macrolide resistance genotype was observed (P = 0.363; Table S1).
| ~ Discussion|| |
Macrolide resistance among S. pneumoniae varies according to country or region due to worldwide variations in the prevalence of S. pneumoniae genotypes and phenotypes.  To the best of our knowledge, this is the first study in Jordan to investigate, not only the molecular prevalence of S. pneumoniae serotypes among asymptomatic carrier children, but also the association between macrolide resistance phenotypes and genotypes. In addition, the presence of multidrug resistance using antimicrobial agents recommended by the CLSI for the treatment of pneumococcal infections was investigated.
The overall S. pneumoniae carriage rate was 33.8%; 41.9% among males and 21.9% among females (P = 0.009). The difference in carriage according to gender cannot be explained due to attendance of kindergarten or daycare (P = 0.255) and might be related to factors not assayed by the study, such as the number of close contacts and level of social interaction. The highest carriage rates occurred in age groups 7-12 months and 49-60 months. In contrast, age group 1-6 months had a very low carriage rate, possibly due to having maternally-derived antibodies. This hypothesis is supported by the observation that children in the age group 7-12 months, in which the maternally-derived antibodies have weaned, demonstrated a very high carriage rate. Using an established multiplex PCR protocol for molecular serotyping of S. pneumoniae,  the following serotypes were most prevalent: 19F, 6A/B, 11A, 19A, 14 and 15B/C. The serotypes of 18.9% of the isolates could not be determined due to protocol limitations. Identified serotypes are consistent in type but not in frequency with reports from the Middle East and the Mediterranean region. In Lebanon, serotypes 19F, 2, 5, 9V/9A, 23, 6A/B/C, 14 and 19A were reported.  In KSA, serotypes 23F, 19F, 6B, 5 and 1 were reported.  In Iran, serotypes 19, 6, 14, 17, 20, 23, 21 and 11 were reported.  In Italy, serotypes 3, 19F, 23F, 19A, 6B and 14 were reported. 
Among isolates having the serotype 19F, 88.9% (8/9) were macrolide resistant (P = 0.039.) This suggests an association between the 19F serotype and presence of macrolide resistance. Macrolides resistance genes were detected in 75% (6/8) of the macrolide-resistant 19F isolates (P = 0.289) (data not shown). Interestingly, in Lebanon, 64.3% of the 19F isolates had a macrolide resistance gene.  The remaining 25% (2/8) of the macrolide-resistant 19F isolates lacked detectable macrolide resistance genes. Therefore, macrolide resistance within these isolates could be due to other mechanisms, such as mutations in the 23S rRNA or ribosomal proteins L4 and L22. , All study isolates were susceptible to chloramphenicol, levofloxacin and vancomycin. High rates of resistance were observed for oxacillin (90.6%), a predictor for β-lactam drug resistance. Moderate rates of resistance were observed for trimethoprim/sulfamethoxazole (54.7%) and clindamycin (32.1%). According to the disk diffusion test, macrolide resistance was observed at a rate of 52.8-54.7%. In contrast, the E-test identified macrolide resistance in 60.4% of isolates for each of the three macrolides. This small discrepancy in the rates macrolide resistance is likely attributed to the very high resolution of the E-test as 2-3 isolates were observed to have intermediate susceptibility to macrolides using the disk diffusion test while the E-test reported resistance-to-borderline resistance.
Antimicrobial susceptibility results were consistent with findings reported by others in the region. In Lebanon, 97.7% of S. pneumoniae isolated from clinical samples were susceptible to chloramphenicol, 70.5% were resistant to trimethoprim/sulfamethoxazole and 67.7% were resistant to erythromycin.  In KSA, 62% of the S. pneumoniae isolates were resistant to erythromycin.  In Iran, 57.2% and 54.9% of S. pneumoniae isolates were resistant to clarithromycin and azithromycin, respectively.  In contrast, in Turkey, all S. pneumoniae isolates were susceptible to vancomycin.  Countries from other regions had varying results. In the USA, 100% and >99% of S. pneumoniae isolates were susceptible to vancomycin and levofloxacin, respectively.  In Italy, 52% of S. pneumoniae isolates were macrolide resistant.  Much higher erythromycin resistance (90.5%) was reported among isolates from the nasopharynx of asymptomatic nonvaccinated children attending daycare centres in Korea. 
All 17 isolates resistant to clindamycin were also highly resistant to the three macrolides (MIC >256 mg/L each) (P < 0.001). Of these, 88.2% (15/17) had macrolide resistance genes (P = 0.002). Namely, 86.7% (13/15) had erm(B), 6.7% (1/15) had mef(E) and 6.7% (1/15) had both erm(B) and mef(E). In contrast, out of 35 clindamycin sensitive isolates, 34.3% (12/35) had mef(E) and of these, 91.7% (11/12) were macrolide resistant. Erm(B) and mef(E) and erm(B) were present in 17.1% (6/35) and 2.9% (1/35) of the isolates, respectively. No detectable genes were present in the remaining isolates, i.e. 45.7% (16/35) (data not shown). Taken together, findings suggest an association between clindamycin resistance and macrolide resistance and specifically due to erm(B) but not mef(E) (P < 0.001), which is in agreement with the MLS B phenotype. Consistent with these results, a German study found that 94.1% of the clarithromycin-resistant isolates harboring the erm(B) gene were also resistant to clindamycin. 
Interestingly, all 32 isolates resistant to the three macrolide drugs were resistant to oxacillin (data not shown). This strongly suggests that macrolide resistance likely coexists with resistance to oxacillin (P = 0.004). In support of the coexistence of macrolide resistance and resistance to clindamycin and oxacillin, the erythromycin-resistant isolates were more likely to be multidrug resistant than the macrolide sensitive isolates (P = 0.002) [Table 3].
Among the three macrolides used in the current study, erythromycin proved to be a good representative antimicrobial agent to identify macrolide resistance. The use of erythromycin as a representative for macrolide susceptibility testing was also recommended by the CLSI (2012).
All 24 isolates having erm(B) alone and 92.3% (12/13) of the isolates having mef(E) alone were resistant to erythromycin (P < 0.001 and P = 0.003, respectively). Surprisingly, but not statistically significant, isolates having both genes, only had a 28.6% (2/7) resistance rate to erythromycin (P = 0.453) [Table 4]. The majority (i.e. 71.4%; 15/21) of the erythromycin susceptible isolates lacked a macrolide resistance gene (P = 0.078) [Table 4]. The presence of macrolide resistance genes in the six isolates that were erythromycin susceptible [Table 4], might be explained by incomplete gene expression or the expression of inactive gene products, both of which may arise due to mutations. This was similarly reported previously. 
When present alone, erm(B) resulted in very high-level resistance to erythromycin (MIC >256 mg/L) (P = 0.002), while mef(E) alone, in most clinical isolates and in the positive control strain, resulted in moderate-to-high level resistance to erythromycin (MIC = 3-24 mg/L) (P < 0.001) [Table 4]. In support of these observations, erm(B) is associated with the MSL B phenotype which is associated with very high MIC values, while mef(E) is associated with the (M) phenotype, which is associated with moderate-to-high MIC values. One isolate possessing both erm(B) and mef(E) had an erythromycin MIC >256 mg/L suggesting an intact erm(B) and either an intact or defective mef(E) [Table 4]. A second isolate possessing both genes had an erythromycin MIC of 3-24 mg/L suggesting a defective erm(B) and an intact mef(E) [Table 4]. The remaining five isolates possessing both genes and demonstrating macrolide susceptibility could potentially be carrying defective copies of the two genes [Table 4].
Infants 7-12 months old and children under 5 years, in general, are good target groups for pneumococcal vaccination. This study identified the most prevalent S. pneumoniae serotypes that could be included in a pneumococcal vaccine if it is to be considered in Jordan in the future. Based on the finding of significant macrolide and multidrug resistance among the study isolates, we recommend the implementation of a pneumococcal vaccination program. However, this study only focussed on healthy children under 5 years attending a major hospital from Northern Jordan. Therefore, wider-scale future studies from throughout Jordan should include other age groups and larger numbers of isolates obtained from pneumococcal disease and asymptomatic carriers.
| ~ Conclusions|| |
This study identified S. pneumoniae carriage among one-third of children under 5 years of age with carriage varying according to age and gender. High rates of macrolide and multidrug resistance were documented. Macrolide resistance genes erm(B) and mef(E) were responsible for resistance to macrolides in most isolates. Prevalent serotypes were mostly consistent in type but not frequency with serotypes among surrounding countries. Significant rates of macrolide and multidrug resistance were documented among the isolates. Clindamycin resistance coexisted with macrolide resistance phenotype and erm(B) genotype and oxacillin resistance coexisted with macrolide resistance. No significant association between serotype and macrolide resistance genotype was observed potentially due to small sample size.
Financial support and sponsorship
This study was supported by the Research Deanship of Jordan University of Science and Technology (Grant Number 20120084).
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table S2]