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ORIGINAL ARTICLE
Year : 2016  |  Volume : 29  |  Issue : 4  |  Page : 812-817

Methicillin-resistant Staphylococcus aureus: A challenge for infection control


Department of Botany, Faculty of Science, Port Said University, Port Said, Egypt

Date of Submission20-Mar-2014
Date of Acceptance20-Jun-2014
Date of Web Publication21-Mar-2017

Correspondence Address:
Ahmed A Ahmed
Elgazayr, Belqas, Daqahlia, 35511
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.202519

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  Abstract 

Objective
The aim of this study was to evaluate a method for rapid and direct detection of methicillin-resistant Staphylococcus aureus (MRSA), allowing clinicians to make prompt decisions for the management of patients with staphylococcal bacteremia.
Background
The prevalence of methicillin resistance in nosocomial staphylococci has increased in the past decade. There has also been a recent increase in community-acquired MRSA infections in patients without apparent recognized risk factors.
Patients and Methods
In this work, blood culture was performed for all cases for isolation of the causative organisms. Staphylococcal isolates were then identified by standard methods including Gram stain, catalase, and bacitracin susceptibility tests. This was followed by testing the staphylococcal isolates with oxacillin using the disc diffusion method. All positive blood cultures were subjected to a multiplex PCR assay targeting 16S rRNA, nuc, and mecA genes.
Results
Our results showed that blood cultures were positive for 85% of the patients. Staphylococci accounted for 58.8% of the positive cases. In total, 52% of the isolated staphylococci were S. aureus and 48% were coagulase-negative staphylococci (CoNS). 76.9% of S. aureus isolates were MRSA; 23.1% were methicillin-sensitive S. aureus. 91.7% of CoNS isolates were methicillin-resistant CoNS and 8.3% were methicillin-sensitive CoNS. The sensitivity and specificity of the oxacillin disk diffusion method in relation to mecA PCR were 95.2% and 87.5%. The total time required to perform the multiplex PCR assay directly from positive blood cultures was 3 h.
Conclusion
Current standard laboratory methods for detection of oxacillin resistance require at least 48–72 h for isolation, identification, and susceptibility testing. The multiplex PCR assay is a rapid, sensitive, and specific method for the detection of MRSA colonies and MRSA-positive blood culture bottles.

Keywords: methicillin-resistant Staphylococcus aureus, multiplex polymerase chain reaction, oxacillin disk diffusion


How to cite this article:
Mohamed MM, Bialy AA, Ahmed AA. Methicillin-resistant Staphylococcus aureus: A challenge for infection control. Menoufia Med J 2016;29:812-7

How to cite this URL:
Mohamed MM, Bialy AA, Ahmed AA. Methicillin-resistant Staphylococcus aureus: A challenge for infection control. Menoufia Med J [serial online] 2016 [cited 2020 Feb 28];29:812-7. Available from: http://www.mmj.eg.net/text.asp?2016/29/4/812/202519


  Introduction Top


Methicillin-resistant Staphylococcus aureus (MRSA) infections are associated with significant morbidity and mortality, especially in patients with bacteremia. Thus, rapid detection of methicillin resistance in staphylococci from bacteremia patients is essential for prompt institution of effective antimicrobial therapy [1].

Several investigations have described PCR-based assays for the detection of methicillin-resistant staphylococci from clinical samples. Some reports have shown that a pure culture of the organism is required, whereas other methods involving direct detection from clinical samples require long DNA extraction protocols [2].

A rapid method for extraction of DNA from blood cultures has been reported, but it required several individual PCRs to detect all necessary products for identification of staphylococci [3].

A rapid and simple method was described, followed by a multiplex PCR assay designed to detect the bacterial 16S rRNA (universal gene for the bacteria), nuc (gene encoding thermostable nuclease), and mecA (gene encoding methicillin resistance) genes to detect methicillin-resistant staphylococci directly from positive blood culture bottles, markedly reducing the time for identification from 24 to 48 h to ˜3 h.


  Patients and Methods Top


This study was carried out at the Infection Control Unit, Microbiology and Immunology Department, and the Surgery ICU of Zagazig University Hospital, Faculty of Medicine, Zagazig University, in the period from April 2012 to April 2013.

The patient group included 51 males and 49 females ranging in age from 16 to 75 years.

Clinical data were collected. These data included

  1. Age and sex.
  2. Acute physiology and chronic health evaluation (APACHE) II score: this is a classification system for severity of disease. After admission of a patient to ICU, a score from 0 to 71 is computed on the basis of several measurements; higher scores imply a more severe disease and a higher risk of death [4].


Methods

Specimen collection

Blood for culture was collected and dispensed with great care as indicated to avoid contamination of the specimen and culture medium. Using gloves, the venepuncture site was thoroughly disinfected as follows:

  1. Using 70% ethanol, an area about 50 mm in diameter was cleansed and allowed to air dry.
  2. Using 2% tincture of iodine and a circular action, the area beginning at the point where the needle enters the vein was swabbed. The iodine was allowed to dry on the skin for at least 1 min. Using a sterile syringe, about 5 ml of blood was withdrawn and inoculated immediately in the blood culture bottle [5].


Blood culture

  1. The foil cap of the blood culture bottle was held out and then the rubber cap was wiped using an ethanol swab and then perforated with the syringe containing the collected blood.
  2. The top of the culture bottle was wiped again with ethanol swab and the foil cap was replaced.
  3. The blood was mixed with the broth without delay.
  4. The bottles were labeled with the name, the number of the case, and the date of collection.
  5. The blood culture bottles were incubated at 37°C.
  6. All positive blood culture bottles were aliquoted into 1.5 ml microcentrifuge tubes as soon as gas production was noted and Gram stain showed the presence of bacteria. The microcentrifuge tubes were frozen at −20°C until tested for multiplex PCR.
  7. Subculture was performed after the first overnight incubation on blood agar that was aerobically incubated at 37°C for 24 h.
  8. The plates were examined after the incubation period for growth. Negative blood culture bottles were checked daily for evidence of microbial growth. If there was no evidence of microbial growth after 10 days of incubation, Gram stains and subcultures were performed before considering the culture negative [6].


Identification of staphylococcal isolates

  1. Colony morphology: S. aureus colonies are round, smooth, and glistening, with a gray to deep yellow endopigment. S. aureus produces complete hemolysis on blood agar, whereas coagulase-negative staphylococci (CoNS) do not.
  2. Gram stain: only Gram-positive cocci in clusters were further examined by the following tests:
    1. Catalase test: Gram-positive cocci were subjected to a catalase test to differentiate staphylococci and micrococci (positive) from streptococci (negative). One milliliter of 3% hydrogen peroxide solution, H2O2, was poured over an overnight nutrient agar slope culture of the test organism and the tube was held in a slanting position. The slant was observed for immediate production of gas bubbles [7].
    2. Bacitracin susceptibility: Catalase-positive Gram-positive cocci were subjected to a bacitracin susceptibility test. A suspension of 0.5 McFarland of the bacterium was spread over a plate of Muller–Hinton agar. Then, a bacitracin disc containing 0.04 U was applied and incubated overnight and observed for any zone of inhibition around the disk. The micrococci should yield a zone of inhibition around the disk, usually greater than 10 mm, whereas the staphylococci should grow up to the disk [8].


Phenotypic detection of methicillin resistance among staphylococcal isolates (oxacillin disk diffusion method)

Four to five isolated colonies of similar morphology from each isolated strain were obtained using a loop, inoculated into 5 ml sterile nutrient broth, and incubated for 2–5 h at 35°C until turbidity was obtained. This turbidity was then adjusted with saline to 0.5 McFarland standards. This equals a cell density of 108 cells/ml approximately. From these broth suspensions, inocula were applied by sterile swabs on Muller–Hinton agar without added salts. The oxacillin (1 g) disks were applied to the center of the plates. Plates were incubated at 33–35°C for 24 h [9]. Irrespective of zone size, any discernable growth within the zone of inhibition is indicative of resistance as shown in [Table 1].
Table 1 Zone diameter interpretative standards

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Multiplex polymerase chain reaction

Multiplex PCR targeting 16S rRNA, nuc, and mecA genes was performed for (i) the aliquots prepared from blood culture bottles and (ii) all bacterial isolates on culture plates.

  1. DNA extraction:
    1. DNA extraction directly from positive blood culture bottles: One milliliter of sterile, distilled water was added to a 100 µl aliquot of each sample, mixed by inversion, and incubated at room temperature for 5 min. After centrifugation at 16 000g for 1 min, the supernatant was discarded and the pellet was suspended in 100 µl of Triton X-100 lysis buffer (100 mmol/l NaCl, 10 mmol/l Tris-HCl, 1 mmol/l EDTA, and 1% Triton X-100). Five microliters of 1 mg/ml lysostaphin solution was added, mixed, and incubated at 37°C for 10 min. This suspension was then boiled for 10 min. After cooling to room temperature for 5 min, the sample was centrifuged at 16 000g for 1 min and 1 µl of the supernatant was used for PCR.
    2. DNA extraction from bacterial colonies: A loopful of organism was inoculated into a 100 µl aliquot of Triton X-100 lysis buffer containing 2 µl of a 1 mg/solution of lysostaphin. The suspension was then incubated in a 37°C water bath for 10 min and boiled for an additional 10 min. The suspension was cooled at room temperature for 5 min and centrifuged at 16 000g for 1 min. A 1 µl aliquot of the supernatant was used for PCR testing.
    3. Seeding experiments: A suspension of S. aureus ATCC 43300 (MRSA) (>108 CFU/ml) was prepared in 1 ml of TE buffer. This suspension was serially diluted. Each dilution was then centrifuged and the pellet was suspended in 100 µl of a negative blood culture. The sample was treated and processed as above. One microliter from each dilution was then used for PCR. Original concentrations of organisms were calculated from plating on 5% blood agar.
  2. Amplification: Preparation of the amplification reaction was carried out under the biosafety cabinet (Micro Flow; Fernbrook Lane, Plymouth, MN USA). Steps were followed according to the manufacturer's instructions.
    1. Primer solutions were allowed to thaw, kept on ice after complete thawing, and mixed well before use as shown in [Table 2].
    2. Taq PCR Master Mix was mixed by brief vortexing. 50 µl was dispensed into each 0.5 ml PCR tube. It is important to mix the Taq PCR Master Mix before use to avoid localized differences in salt concentration. Taq PCR Master Mix is provided as a 2× concentrate (i.e. a 50 µl volume of the Taq PCR Master Mix is required for amplification reactions with a final volume of 100 µl). For volumes smaller than 100 µl, the 1 : 1 ratio of Taq PCR Master Mix to diluted primer mix and template should be maintained. A negative control (without template DNA) should be included in every experiment. PCR tubes should be kept on ice until they are placed in the thermal cycler.
    3. The appropriate volume of diluted primer mix was added to the PCR tubes containing the Master Mix. The primer concentration in the reaction should be 0.2 µmol/l for each the 16S rRNA and nuc primers and 0.5 µmol/l for the mecA primer.
    4. Six microliter of 25 mmol/l MgCl2 was added to achieve a final concentration of 3 mmol/l MgCl2 in the reaction.
    5. Template DNA (1 µl of the DNA extract) was then added to the individual PCR tubes.
    6. The final volume was completed to 100 µl with distilled water.
    7. One hundred microliter mineral oil was the last to be added to the PCR tubes.
    8. Thermal cycler was programmed. For multiplex PCR performed directly on blood cultures, initial denaturation was adjusted to 94°C for 2 min, followed by 25 cycles of 94°C for 15 s, 55°C for 30 s, and 72°C for 30 s, followed by a final extension at 72°C for 10 min. For multiplex PCR performed on bacterial isolates on culture plates, initial denaturation was adjusted to 94°C for 2 min, followed by 30 cycles of 94°C for 1 s and 55°C for 15 s, followed by a final extension at 72°C for 10 min.
    9. After amplification, samples can be stored overnight at 2–8°C or at –20°C for longer storage.
  3. Detection of the amplification product:
    1. A 1% agarose solution was prepared by adding 1 g agarose to 100 ml of 1× electrophoresis buffer in a 250 ml flask. The agarose was then dissolved by heating for 2 min. Boiling was avoided.
    2. The agarose was cooled to 50°C and then 5 µl of 10 mg/ml ethidium bromide was added to the gel to obtain a final concentration of 0.5 µg/ml.
    3. A comb was inserted into the electrophoretic bed and the agarose was poured into it. Great care was taken during pouring of the agarose to avoid the formation of bubbles. The gel became solidified within 15 min. The electrophoresis apparatus was filled with the electrophoresis buffer. The comb was removed, creating eight wells for sample application, and then the electrophoretic bed was relocated within the apparatus with the sample wells closest to the negative electrode.
    4. To each 2 µl of marker, 10 µl negative control, and 10 µl of each sample, 2 µl of loading dye was added in different Eppendorf tubes, and then the contents of each Eppendorf tube were loaded slowly into a separate well using a 20 µl pipette. After loading all samples, the apparatus was covered and the electrodes were connected to the power supply and the latter was turned on. It was adjusted at 100 V for 30 min.
    5. The gel was removed for examination.
  4. Interpretation:
    1. The gel was examined under an ultraviolet light transilluminator as ethidium bromide intercalate between the bases of the DNA and fluoresces.
    2. The molecular size marker yielded different bands ranging from 100 to 1000 bp.
    3. MRSA cases yielded three bands located at 798, 533, and 270 bp corresponding to bacterial 16S rRNA, nuc, and mecA genes, respectively. Methicillin-resistant coagulase-negative staphylococci (MRCoNS) yielded two bands located at 798 and 533 bp, corresponding to bacterial 16S rRNA,nuc and mecA genes, respectively. Methicillin-sensitive S. aureus yielded two bands located at 798 and 270 bp, corresponding to bacterial 1S rRNA,nuc and mecA genes, respectively. Methicillin-sensitive CoNS yielded one band located at 798 bp, corresponding to the bacterial 16S rRNA gene. Bacteria other than staphylococci yielded one band located at 798 bp, corresponding to the bacterial 16S rRNA gene.
    4. The negative control and negative cases yielded no bands [10].



  Results Top


This study included 100 patients with a picture of suspected bacteremia in the Surgery ICU of Zagazig University Hospital in the period from April 2012 to April 2013. Blood samples were obtained from patients for blood culture. The results of blood culture are presented in [Table 3]. This table shows that 85 of the 100 (85%) patients had positive blood cultures. This table also shows that staphylococci were isolated from 50 of the 85 (58.8%) positive cases, whereas Gram-negative bacilli were isolated from 35 (41.2%) patients. No mixed infection was found.
Table 2 Primers used for multiplex PCR detection of methicillin-resistant staphylococci

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Table 3 Results of blood culture of the studied groups

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The 50 staphylococcal isolates were tested for methicillin susceptibility. Testing for methicillin susceptibility was performed by oxacillin disk diffusion and mecA gene detection by PCR. The results of both tests are shown in [Table 4]. Our results showed that 40 isolates were identified as methicillin resistant by both tests and seven isolates were shown to be susceptible by both methods. However, two staphylococcal isolates that had the mecA gene yielded a susceptible pattern by the disk diffusion method. Also, one S. aureus isolate that had no mecA gene showed a resistant pattern by the disk diffusion method. This table also shows the sensitivity, specificity, and positive and negative predictive values of the oxacillin disk diffusion method in relation to mecA gene detection by PCR, which were found to be 95.2, 87.5, 97.6, and 77.8%, respectively, with an accuracy of 94%. The results of oxacillin disk diffusion are shown in [Figure 1].
Table 4 Validity of oxacillin disk diffusion in relation to the mecA gene PCR for the detection of methicillin resistance

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Figure 1: Oxacillin disk diffusion. (a) Methicillin-susceptible staphylococci. (b) Methicillin-resistant staphylococci.

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All positive blood cultures were subjected to multiplex PCR targeting 16S rRNA, nuc, and mecA genes. [Figure 2] shows the results of the multiplex PCR with the 16S rRNA, nuc, and mecA gene products after PCR amplification with different organisms.
Figure 2: Agarose gel showing PCR amplified products of the 16S rRNA, mecA, and nuc genes. Lanes 1 and 8 show a 100-bp DNA ladder. Lanes 2 and 5 show methicillin-resistant Staphylococcus aureus with all three amplification products (16S at 798 bp, mecA at 533 bp, and nuc at 270 bp). Lane 3 shows methicillin-sensitive S. aureus with only the 16S and nuc products. Lane 4 shows methicillin-sensitive coagulase-negative staphylococci with only the 16S product. Lane 6 shows methicillin-resistant coagulase-negative staphylococci with the 16S and mecA products. Lane 7 shows the reagent control with no band.

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The results of multiplex PCR direct testing on blood cultures and on bacterial isolates are summarized in [Table 5]. Direct multiplex PCR testing with DNA extracted from positive blood cultures did not misidentify any isolate compared with the PCR test results with bacterial growth on solid plates.
Table 5 Multiplex PCR identification of 85 positive blood cultures by comparison of PCR on bacterial isolates with direct PCR testing on blood cultures

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Thirty-five positive blood cultures with non-staphylococcal organisms and 15 negative blood cultures were used to assess the specificity of the assay. None of the positive blood cultures that grew organisms other than staphylococci showed amplification products corresponding to either the nuc or mecA product. When these data were studied statistically, a significant agreement was found between direct multiplex PCR testing on blood cultures and PCR testing on bacterial isolates.

However, a few discrepant results were obtained. The 16S rRNA gene failed to amplify in one blood culture, yielding MRCoNS, and two blood cultures yielded Gram-negative bacilli; however, the correct results were obtained for both nuc and mecA genes.


  Discussion Top


This study included one hundred patients with a picture of suspected bacteremia in the Surgery ICU of Zagazig University Hospital in the period from April 2012 to April 2013.

The blood cultures were positive for 85% of the patients. Staphylococci accounted for 58.8% of the positive cases. In total, 52% of the isolated staphylococci were S. aureus. These results are in agreement with that of other workers [11], who carried out a 2-year retrospective study between 1999 and 2001 in Imam Khomeini Hospital to study cases with nosocomial bloodstream infections. The authors found that Gram-positive cocci were the most commonly isolated organisms and accounted for 42.3% of bloodstream infections. They also found that staphylococci were the main Gram-positive cocci isolated and accounted for 86.9% of them.

In this work, [Table 4] shows some discrepant results between the disk diffusion and PCR manifested by variation of resistance to methicillin genotypically and phenotypically. Two staphylococcal isolates that had the mecA gene were susceptible by disk diffusion and one isolate that had no mecA gene was resistant by the disk diffusion method. The sensitivity and specificity of oxacillin disk diffusion in relation to PCR were calculated to be 95.2 and 87.5%, respectively.

These values are in agreement with those of other workers [12], who found that eight strains of S. aureus were identified to be methicillin susceptible and 20 strains were identified to be resistant by both methods. However, one strain that was resistant by disk diffusion contained no mecA gene and two strains that were sensitive by disk diffusion proved to have the mecA gene. El-Behedy and colleagues calculated the sensitivity and specificity of the disk diffusion to be 90 and 87.9% in relation to PCR.

In this study ([Table 5]), significant agreement was found between direct multiplex PCR testing on blood cultures and PCR testing on bacterial isolates. However, in one sample that grew MRCoNS, the 16S rRNA gene failed to amplify, but PCR yielded the correct mecA result. The 16S rRNA gene was then amplified when PCR was repeated with the sample diluted 1 : 10. The assay was also successful in amplifying the 16S rRNA gene from other bacteria when present. Only two such specimens initially failed to show a product after PCR amplification. In these samples, the multiplex PCR assay could detect the 16S rRNA gene product after a 1 : 10 dilution of the template.

Our results are in agreement with those of other workers [10] who carried out a study on 236 blood cultures positive for staphylococci. Direct multiplex PCR testing on these blood cultures, targeting the same genes in our study, correctly identified 223 of them compared with the multiplex PCR of the corresponding bacterial isolates. However, in 13 samples, the 16S rRNA gene failed to amplify, but PCR yielded the correct mecA and nuc results. These 13 samples were diluted 1 : 10 and the multiplex PCR assay was repeated. PCR could now amplify not only the mecA and/or nuc products but also the 16S rRNA product.

This study concludes that the multiplex PCR assay is a rapid, sensitive, and specific method for the detection of MRSA colonies and MRSA-positive blood culture bottles.


  Acknowledgements Top


Conflicts of interest

There are no conflicts of interest.

 
  References Top

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Whitby M, McLaws ML, Berry G. Risk of death from methicillin resistant Staphylococcus aureus bacteremia. A meta-analysis. Med J Aust 2001; 175:262–267.  Back to cited text no. 1
    
2.
Mason WJ, Blevins JS, Beenken K, et al. Multiplex PCR protocol for the diagnosis of staphylococcal infection. J Clin Microbiol 2001; 39:3332–3338.  Back to cited text no. 2
    
3.
Jaffe RI, Lane JD, Albury V, Niemeyer DM. Rapid extraction from and direct identification in clinical samples of methicillin resistant staphylococci with the PCR. J Clin Microbiol 2000; 38:3407–3412.  Back to cited text no. 3
    
4.
Knaus WA, Draper EA, Wagner DP, Zimmerman JE. APACHE II: a severity of disease classification system. Crit Care Med 1985; 13:818–829.  Back to cited text no. 4
    
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Cheesbrough, M. Preparation of reagents and culture media. In: District laboratory practice in tropical countries Part 2. UK: Cambridge University Press; 2000. 382–407.  Back to cited text no. 5
    
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Raul RM, Melvin PW. Laboratory diagnosis of bacteremia and fungemia. Infect Dis Clin North Am 2001; 15:20.  Back to cited text no. 6
    
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Lauderdale TL, Kimberle C, Murray PR. Reagents, stains and media. In: Murray PR, Boron EJ, Pfaller MA, Tenover FC, Yolken RH, editors. Manual of clinical microbiology. 7th ed. Washington, DC: American Society for Microbiology; 1999. 1663–1707.  Back to cited text no. 7
    
8.
Baron EJ, Peterson LR, Finegold SM. Micrococcaceae: staphylococci, micrococci, and stomatococci. In: Mosby, St. Louis, editor. Bailey and Scott's Diagnostic Microbiology. 8th ed. St. Louis: CV Mosby Co.; 1990. 323–332.  Back to cited text no. 8
    
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National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial disc susceptibility testing. Fourteenth informational supplement M100-S14.NCCLS. PA, USA: Wayne; 2004.  Back to cited text no. 9
    
10.
Louie, L, Good fellow J, Mathieu P, et al. Rapid detection of methicillin resistant staphylococci from blood culture bottles by using a multiplex PCR assay. J Clin Microbiol 2002; 40:2786–2790.  Back to cited text no. 10
    
11.
Rahbar M, Gra-Agaji R, Hashemi S. Nosocomial blood stream infections in Imam Khomeini Hospital, Urmia, Islamic Republic of Iran, 1999–2001. East Mediterr Health J 2005; 11:478–484.  Back to cited text no. 11
    
12.
El-Behedy EM, Shaheen AA, Amer FA, Fathy ZM. Comparison of phenotypic and genotypic methods for detection of methicillin resistant Staphylococcus aureus [MD Thesis]. Microbiology and Immunology, Faculty of Medicine, Zagazig University; 2000.  Back to cited text no. 12
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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