Fecal Microbiota Transplantation promotes disease remission in a patient with active Crohn’s disease: A Case Report
Main Article Content
Abstract
Background: A woman aged 46 years presented with moderate to severe Crohn’s disease (CD). Sigmoidoscopy revealed proctitis, with Harvey-Bradshaw Index (HBI)=7. The patient experienced up to six bloody stools per day, stool samples showed increased fecal calprotectin, and low albumin.
Method: Case Report. Therapy with 1 portion fecal microbiota transplantation (FMT) capsules every day as add on treatment for 2 weeks was initiated. Hereafter, FMT therapy was tapered off for 21 weeks and stopped.
Results: The patient achieved disease remission at week 21. From 2020 till 2023, when the patient experienced disease relapses (up till twice a year), the patient was treated with FMT capsules (one portion per day) for 1-2 weeks, which promoted disease remission. At the age of 49, the patient was examined by colonoscopy, blood tests and fecal calprotectin. Endoscopy and biopsies (macroscopic and microscopic) showed mild inflammation around the appendix and anal regions and no inflammation in the remaining part of the colon and ileum. Blood samples and fecal calprotectin collected prior to colonoscopy were normal.
Conclusion: This study suggests that FMT may promote disease remission in CD patients with moderate to severe disease. Microbiome data shows that the patients gut microbiome changed to resemble the donor microbiome. FMT therapy was able to change the intestinal microbiome in a patient with CD accompanied by clinical disease remission.
Keywords:
Fecal Microbiota Transplantation, Crohn’s disease, Disease relapses, Disease remission, Colonoscopy, biopsy, Microbiome, probiotics
Article Details
How to Cite
LAURIDSE, Hengameh Chloe Mirsepasi et al.
Fecal Microbiota Transplantation promotes disease remission in a patient with active Crohn’s disease: A Case Report.
Medical Research Archives, [S.l.], v. 12, n. 5, may 2024.
ISSN 2375-1924.
Available at: <https://esmed.org/MRA/mra/article/view/5462>. Date accessed: 30 june 2024.
doi: https://doi.org/10.18103/mra.v12i5.5462.
Section
Case Reports
The Medical Research Archives grants authors the right to publish and reproduce the unrevised contribution in whole or in part at any time and in any form for any scholarly non-commercial purpose with the condition that all publications of the contribution include a full citation to the journal as published by the Medical Research Archives.
References
1. Baumgart DC, Sandborn WJ. Inflammatory bowel disease: clinical aspects and established and evolving therapies. Lancet. 2007;369 (9573):1641-1657. doi:DOI: 10.1016/S0140-6736(07)60751-X
2. Loftus CG, Loftus Jr. E V, Harmsen WS, et al. Update on the incidence and prevalence of Crohn’s disease and ulcerative colitis in Olmsted County, Minnesota, 1940-2000. InflammBowelDis. 2007;13(1078-0998):254-261. doi:10.1002/ibd.20029
3. Kappelman MD, Moore KR, Allen JK, Cook SF. Recent Trends in the Prevalence of Crohn ’ s Disease and Ulcerative Colitis in a Commercially Insured US Population. Dig Dis Sci. Published online 2013:519-525. doi:10.1007/s10620-012-2371-5
4. McGovern DPB, Kugathasan S, Cho JH. Genetics of Inflammatory Bowel Diseases. Gastroenterology. 2015;149(5):1163-1176. doi:10.1053/j.gastro.2015.08.001
5. Bouma G, Strober W. The immunological and genetic basis of inflammatory bowel disease. NatRevImmunol. 2003;3(1474-1733): 521-533. doi:10.1038/nri1132
6. Hebuterne X, Filippi J, Al Jaouni R, Schneider S. Nutritional consequences and nutrition therapy in Crohn’s disease. GastroenterolClin Biol. 2009;33 Suppl 3(2210-7401):S235-S244. doi:10.1016/S0399-8320(09)73159-8
7. Lauridsen, Hengameh Chloé; Vester-Andersen MS, Nikolaj; Krogfelt, Karen A.; Bendtsen, Flemming; Petersen AM. The intestinal dysbiosis found in IBD-patients are linked to colonization with Extra-intestinal Pathogenic Escherichia coli. In: Microbiome & Microbial Diseases in the Gastrointestinal Tract. Digestive disease Week 2017; 2017:2681280.
8. Mirsepasi-Lauridsen, Hengameh Chloé, Bruce Andrew Vallance, Karen Angeliki Krogfelt AMP. Escherichia coli Pathobionts Associated with Inflammatory Bowel Disease. Clin Microbiol Rev. 2019;32(2):1-16. doi:doi.org/10.1128/CMR .00060-18.
9. Khan RR, Lawson AD, Minnich LL, et al. Gastrointestinal norovirus infection associated with exacerbation of inflammatory bowel disease. J PediatrGastroenterolNutr. 2009;48(1536-4801):328-333.
10. Hume G, Radford-Smith GL. The pathogenesis of Crohn’s disease in the 21st century. Pathology. 2002;34(0031-3025):561-567. doi:10.1080/0031302021000035965
11. Hoffmann JC, Pawlowski NN, Kühl A a, Höhne W, Zeitz M. Animal models of inflammatory bowel disease: an overview. Pathobiol J. 2008;70(3):121-130. doi:10.1159/000068143
12. Mirsepasi-Lauridsen HC, Halkjaer SI, Mortensen EM, et al. Extraintestinal pathogenic Escherichia coli are associated with intestinal inflammation in patients with ulcerative colitis. Sci Rep. 2016;6(July):31152. doi:10.1038/srep31152
13. Khan KJ, Ullman TA, Ford AC, et al. Antibiotic therapy in inflammatory bowel disease: a systematic review and meta-analysis. Am J Gastroenterol. 2011;106 (4):661-673. doi:10.1038/ajg.2011.72
14. Baker PI, Love DR, Ferguson LR. Role of gut microbiota in Crohn’s disease. Expert Rev Gastroenterol Hepatol. 2009;3(5):535-546. doi:10.1586/egh.09.47
15. Hadas E, Bozek A, Cudak A, Ciuk A, Jarząb J. Examples of adverse effects after biological therapy. Postep Dermatologii i Alergol. 2020;37(5):712-718.
doi:10.5114/ada.2020.100482
16. Sood A, Midha V, Makharia GK, et al. The Probiotic Preparation, VSL#3 Induces Remission in Patients With Mild-to-Moderately Active Ulcerative Colitis. Clin Gastroenterol Hepatol. 2009;7(11):1202-1209.e1. doi:10.1016/j.cgh.2009.07.016
17. Mardini HE, Grigorian AY. Probiotic mix VSL#3 is effective adjunctive therapy for mild to moderately active ulcerative colitis: A meta-analysis. Inflamm Bowel Dis. 2014;20(9):1562-1567. doi:10.1097/MIB.0000000000000084
18. Cold F, Browne PD, Günther S, et al. Multidonor FMT capsules improve symptoms and decrease fecal calprotectin in ulcerative colitis patients while treated–an open-label pilot study. Scand J Gastroenterol. 2019;54(3):289-296. doi:10.1080/00365521.2019.1585939
19. Simon Mark Dahl Baunwall, Jens Frederik Dahlerup, Jørgen Harald Engberg, Christian Erikstrup, Morten Helms, Mie Agerbæk Juel, Jens Kjeldsen, Jens Nielsen, Hans Linde Nilsson, Anna Christine Rode, Anne Abildtrup, Lars Vinter-Jensen CLH. Danish national guideline for the treatment of Clostridioides difficile infection and use of faecal microbiota transplantation (FMT). Scand J Gastroenterol. 2021;56(9):1056-1077. doi:10.1080/00365521.2021.1922749
20. Cammarota G, Ianiro G, Tilg H, et al. European consensus conference on faecal microbiota transplantation in clinical practice. Gut. 2017;66(4):569-580. doi:10.1136/gutjnl-2016-313017
21. Hengameh Chloe Lauridsen. WO2021130182A1-FMT Capsule, International patent. Published online 2021.
22. Lauridsen HC. WO2021130181A1- Novel faecal composition, International patent. Published online 2021.
23. Vranckx K, Nielsen HV, Chloe H, et al. Substantial Intestinal Microbiota Differences Between Patients With Ulcerative Colitis From Ghana and Denmark. Front Cell a. 2022;12(March):1-11. doi:10.3389/fcimb.2022.832500
24. Hans Christian Ring, Jonathan Thorsen, Ditte M Saunte, Berit Lilje, Lene Bay, Peter Theut Riis, Niels Larsen, Lee O’Brien Andersen, Henrik V Nielsen, Iben M Miller, Thomas Bjarnsholt, Kurt Fuursted GBJ. The Follicular Skin Microbiome in Patients With Hidradenitis Suppurativa and Healthy Controls. JAMA Dermatol. 2017;153 (9)(SEP 9):897-905. doi:doi: 10.1001/jamadermatol.2017.0904
25. Vester-Andersen MK, Prosberg M V, Jess T, et al. Disease course and surgery rates in inflammatory bowel disease: a population-based, 7-year follow-up study in the era of immunomodulating therapy. Am J Gastroenterol. 2014;109(5):705-714. doi:10.1038/ajg.2014.45
26. Tian H, Ge X, Nie Y, et al. Fecal microbiota transplantation in patients with slow-transit constipation : A randomized , clinical trial. PLoS One. 2017;12(2): e01:1-10. doi:10.1371/journal.pone.0171308
27. Xu L, Zhang T, Cui B, et al. Clinical efficacy maintains patients’ positive attitudes toward fecal microbiota transplantation. Medicine (Baltimore). 2016;95(30):e4055. doi:10.1097/MD.0000000000004055
28. Runzhi Chen, Ying Xu, Peng Wu, Hao Zhou YL et al. transplantation of fecal microbiota rich in short chain fatty acids and butyric acid treat cerebral ischemic stroke by regulating gut microbiota. Pharmacol Res 148. 2019;148:1043-6618. doi:10.1016/j.phrs.2019.104403
29. Christian P, Goll R, Holger P, Hilp F. EBioMedicine The effect of fecal microbiota transplantation on IBS related quality of life and fatigue in moderate to severe non-constipated irritable bowel : Secondary endpoints of a double blind , randomized , placebo-controlled trial. 2019;51. doi:10.1016/j.ebiom.2019.11.023
30. Costello SP, Soo W, Bryant R V., Jairath V, Hart AL, Andrews JM. Systematic review with meta-analysis: faecal microbiota transplantation for the induction of remission for active ulcerative colitis. Aliment Pharmacol Ther. 2017;46(3):213-224. doi:10.1111/apt.14173
31. Mirsepasi-Lauridsen HC. Therapy Used to Promote Disease Remission Targeting Gut Dysbiosis, in UC Patients with Active Disease. J Clin Med. 2022;11(24). doi:10.3390/jcm11247472
32. Goyal A, Yeh A, Bush BR, et al. Safety, Clinical Response, and Microbiome Findings Following Fecal Microbiota Transplant in Children with Inflammatory Bowel Disease. Inflamm Bowel Dis. 2018;24(2):410-421. doi:10.1093/ibd/izx035
33. Rossen NG, Fuentes S, Spek MJ Van Der, et al. Findings From a Randomized Controlled Trial of Fecal Transplantation for Patients With Ulcerative Colitis. Gastroenterology. 2015; 149:110-118. doi:10.1053/j.gastro.2015.03.045
34. Costello SP, Hughes PA, Waters O, et al. Effect of Fecal Microbiota Transplantation on 8-Week Remission in Patients With Ulcerative Colitis. JAMA. 2019;321(2):156-164. doi:10.1001/jama.2018.20046
35. Zou B, Liu S, Li X, et al. Repeated and multiple fecal microbiota transplantations plus partial enteral nutrition as the first-line treatment in active pediatric Crohn’s disease. Front Cell Infect Microbiol. 2023;13(February):1-12. doi:10.3389/fcimb.2023.1083236
36. Cisek AA, Szymańska E, Wierzbicka-Rucińska A, Aleksandrzak-Piekarczyk T, Cukrowska B. Methanogenic Archaea in the Pediatric Inflammatory Bowel Disease in Relation to Disease Type and Activity. Int J Mol Sci. 2024;25(1). doi:10.3390/ijms25010673
37. Stojanov S, Berlec A, Štrukelj B. The influence of probiotics on the firmicutes/bacteroidetes ratio in the treatment of obesity and inflammatory bowel disease. Microorganisms. 2020;8(11):1-16. doi:10.3390/microorganisms8111715
38. Andreas Munk Petersen, Christen Rune Stensvold, Hengameh Mirsepasi, Jørgen Engberg, Alice Friis-Møller, Lone Jannok Porsbo, Anette M. Hammerum, Inge Nordgaard-Lassen, Henrik Vedel Nielsen KA krogfelt, Petersen AM, Stensvold CR, et al. Active ulcerative colitis associated with low prevalence of Blastocystis and Dientamoeba fra. Scand J Gastroenterol. 2013;48(5):638-639. doi:10.3109/00365521.2013.780094
39. Krogsgaard LR, Andersen LOB, Johannesen TB, et al. Characteristics of the bacterial microbiome in association with common intestinal parasites in irritable bowel syndrome. Clin Transl Gastroenterol. 2018;9(6):161. http://dx.doi.org/10.1038/s41424-018-0027-2
40. Kesuma Y, Firmansyah A, Bardosono S, Sari IP, Kurniawan A. Blastocystis ST-1 is associated with Irritable Bowel Syndrome-diarrhoea (IBS-D) in Indonesian adolescences. Parasite Epidemiol Control. 2019;6:e00112. doi:10.1016/j.parepi.2019.e00112
46. R Core Team (2021). R: A Language and Environment for Statistical Computing. R Found Stat Comput. Published online 2021.
47. Wickham H. 2016. ggplot2: elegant graphics for data analysis. Springer-Verlag, New York, NY. https://ggplot2.tidyverse.org.
2. Loftus CG, Loftus Jr. E V, Harmsen WS, et al. Update on the incidence and prevalence of Crohn’s disease and ulcerative colitis in Olmsted County, Minnesota, 1940-2000. InflammBowelDis. 2007;13(1078-0998):254-261. doi:10.1002/ibd.20029
3. Kappelman MD, Moore KR, Allen JK, Cook SF. Recent Trends in the Prevalence of Crohn ’ s Disease and Ulcerative Colitis in a Commercially Insured US Population. Dig Dis Sci. Published online 2013:519-525. doi:10.1007/s10620-012-2371-5
4. McGovern DPB, Kugathasan S, Cho JH. Genetics of Inflammatory Bowel Diseases. Gastroenterology. 2015;149(5):1163-1176. doi:10.1053/j.gastro.2015.08.001
5. Bouma G, Strober W. The immunological and genetic basis of inflammatory bowel disease. NatRevImmunol. 2003;3(1474-1733): 521-533. doi:10.1038/nri1132
6. Hebuterne X, Filippi J, Al Jaouni R, Schneider S. Nutritional consequences and nutrition therapy in Crohn’s disease. GastroenterolClin Biol. 2009;33 Suppl 3(2210-7401):S235-S244. doi:10.1016/S0399-8320(09)73159-8
7. Lauridsen, Hengameh Chloé; Vester-Andersen MS, Nikolaj; Krogfelt, Karen A.; Bendtsen, Flemming; Petersen AM. The intestinal dysbiosis found in IBD-patients are linked to colonization with Extra-intestinal Pathogenic Escherichia coli. In: Microbiome & Microbial Diseases in the Gastrointestinal Tract. Digestive disease Week 2017; 2017:2681280.
8. Mirsepasi-Lauridsen, Hengameh Chloé, Bruce Andrew Vallance, Karen Angeliki Krogfelt AMP. Escherichia coli Pathobionts Associated with Inflammatory Bowel Disease. Clin Microbiol Rev. 2019;32(2):1-16. doi:doi.org/10.1128/CMR .00060-18.
9. Khan RR, Lawson AD, Minnich LL, et al. Gastrointestinal norovirus infection associated with exacerbation of inflammatory bowel disease. J PediatrGastroenterolNutr. 2009;48(1536-4801):328-333.
10. Hume G, Radford-Smith GL. The pathogenesis of Crohn’s disease in the 21st century. Pathology. 2002;34(0031-3025):561-567. doi:10.1080/0031302021000035965
11. Hoffmann JC, Pawlowski NN, Kühl A a, Höhne W, Zeitz M. Animal models of inflammatory bowel disease: an overview. Pathobiol J. 2008;70(3):121-130. doi:10.1159/000068143
12. Mirsepasi-Lauridsen HC, Halkjaer SI, Mortensen EM, et al. Extraintestinal pathogenic Escherichia coli are associated with intestinal inflammation in patients with ulcerative colitis. Sci Rep. 2016;6(July):31152. doi:10.1038/srep31152
13. Khan KJ, Ullman TA, Ford AC, et al. Antibiotic therapy in inflammatory bowel disease: a systematic review and meta-analysis. Am J Gastroenterol. 2011;106 (4):661-673. doi:10.1038/ajg.2011.72
14. Baker PI, Love DR, Ferguson LR. Role of gut microbiota in Crohn’s disease. Expert Rev Gastroenterol Hepatol. 2009;3(5):535-546. doi:10.1586/egh.09.47
15. Hadas E, Bozek A, Cudak A, Ciuk A, Jarząb J. Examples of adverse effects after biological therapy. Postep Dermatologii i Alergol. 2020;37(5):712-718.
doi:10.5114/ada.2020.100482
16. Sood A, Midha V, Makharia GK, et al. The Probiotic Preparation, VSL#3 Induces Remission in Patients With Mild-to-Moderately Active Ulcerative Colitis. Clin Gastroenterol Hepatol. 2009;7(11):1202-1209.e1. doi:10.1016/j.cgh.2009.07.016
17. Mardini HE, Grigorian AY. Probiotic mix VSL#3 is effective adjunctive therapy for mild to moderately active ulcerative colitis: A meta-analysis. Inflamm Bowel Dis. 2014;20(9):1562-1567. doi:10.1097/MIB.0000000000000084
18. Cold F, Browne PD, Günther S, et al. Multidonor FMT capsules improve symptoms and decrease fecal calprotectin in ulcerative colitis patients while treated–an open-label pilot study. Scand J Gastroenterol. 2019;54(3):289-296. doi:10.1080/00365521.2019.1585939
19. Simon Mark Dahl Baunwall, Jens Frederik Dahlerup, Jørgen Harald Engberg, Christian Erikstrup, Morten Helms, Mie Agerbæk Juel, Jens Kjeldsen, Jens Nielsen, Hans Linde Nilsson, Anna Christine Rode, Anne Abildtrup, Lars Vinter-Jensen CLH. Danish national guideline for the treatment of Clostridioides difficile infection and use of faecal microbiota transplantation (FMT). Scand J Gastroenterol. 2021;56(9):1056-1077. doi:10.1080/00365521.2021.1922749
20. Cammarota G, Ianiro G, Tilg H, et al. European consensus conference on faecal microbiota transplantation in clinical practice. Gut. 2017;66(4):569-580. doi:10.1136/gutjnl-2016-313017
21. Hengameh Chloe Lauridsen. WO2021130182A1-FMT Capsule, International patent. Published online 2021.
22. Lauridsen HC. WO2021130181A1- Novel faecal composition, International patent. Published online 2021.
23. Vranckx K, Nielsen HV, Chloe H, et al. Substantial Intestinal Microbiota Differences Between Patients With Ulcerative Colitis From Ghana and Denmark. Front Cell a. 2022;12(March):1-11. doi:10.3389/fcimb.2022.832500
24. Hans Christian Ring, Jonathan Thorsen, Ditte M Saunte, Berit Lilje, Lene Bay, Peter Theut Riis, Niels Larsen, Lee O’Brien Andersen, Henrik V Nielsen, Iben M Miller, Thomas Bjarnsholt, Kurt Fuursted GBJ. The Follicular Skin Microbiome in Patients With Hidradenitis Suppurativa and Healthy Controls. JAMA Dermatol. 2017;153 (9)(SEP 9):897-905. doi:doi: 10.1001/jamadermatol.2017.0904
25. Vester-Andersen MK, Prosberg M V, Jess T, et al. Disease course and surgery rates in inflammatory bowel disease: a population-based, 7-year follow-up study in the era of immunomodulating therapy. Am J Gastroenterol. 2014;109(5):705-714. doi:10.1038/ajg.2014.45
26. Tian H, Ge X, Nie Y, et al. Fecal microbiota transplantation in patients with slow-transit constipation : A randomized , clinical trial. PLoS One. 2017;12(2): e01:1-10. doi:10.1371/journal.pone.0171308
27. Xu L, Zhang T, Cui B, et al. Clinical efficacy maintains patients’ positive attitudes toward fecal microbiota transplantation. Medicine (Baltimore). 2016;95(30):e4055. doi:10.1097/MD.0000000000004055
28. Runzhi Chen, Ying Xu, Peng Wu, Hao Zhou YL et al. transplantation of fecal microbiota rich in short chain fatty acids and butyric acid treat cerebral ischemic stroke by regulating gut microbiota. Pharmacol Res 148. 2019;148:1043-6618. doi:10.1016/j.phrs.2019.104403
29. Christian P, Goll R, Holger P, Hilp F. EBioMedicine The effect of fecal microbiota transplantation on IBS related quality of life and fatigue in moderate to severe non-constipated irritable bowel : Secondary endpoints of a double blind , randomized , placebo-controlled trial. 2019;51. doi:10.1016/j.ebiom.2019.11.023
30. Costello SP, Soo W, Bryant R V., Jairath V, Hart AL, Andrews JM. Systematic review with meta-analysis: faecal microbiota transplantation for the induction of remission for active ulcerative colitis. Aliment Pharmacol Ther. 2017;46(3):213-224. doi:10.1111/apt.14173
31. Mirsepasi-Lauridsen HC. Therapy Used to Promote Disease Remission Targeting Gut Dysbiosis, in UC Patients with Active Disease. J Clin Med. 2022;11(24). doi:10.3390/jcm11247472
32. Goyal A, Yeh A, Bush BR, et al. Safety, Clinical Response, and Microbiome Findings Following Fecal Microbiota Transplant in Children with Inflammatory Bowel Disease. Inflamm Bowel Dis. 2018;24(2):410-421. doi:10.1093/ibd/izx035
33. Rossen NG, Fuentes S, Spek MJ Van Der, et al. Findings From a Randomized Controlled Trial of Fecal Transplantation for Patients With Ulcerative Colitis. Gastroenterology. 2015; 149:110-118. doi:10.1053/j.gastro.2015.03.045
34. Costello SP, Hughes PA, Waters O, et al. Effect of Fecal Microbiota Transplantation on 8-Week Remission in Patients With Ulcerative Colitis. JAMA. 2019;321(2):156-164. doi:10.1001/jama.2018.20046
35. Zou B, Liu S, Li X, et al. Repeated and multiple fecal microbiota transplantations plus partial enteral nutrition as the first-line treatment in active pediatric Crohn’s disease. Front Cell Infect Microbiol. 2023;13(February):1-12. doi:10.3389/fcimb.2023.1083236
36. Cisek AA, Szymańska E, Wierzbicka-Rucińska A, Aleksandrzak-Piekarczyk T, Cukrowska B. Methanogenic Archaea in the Pediatric Inflammatory Bowel Disease in Relation to Disease Type and Activity. Int J Mol Sci. 2024;25(1). doi:10.3390/ijms25010673
37. Stojanov S, Berlec A, Štrukelj B. The influence of probiotics on the firmicutes/bacteroidetes ratio in the treatment of obesity and inflammatory bowel disease. Microorganisms. 2020;8(11):1-16. doi:10.3390/microorganisms8111715
38. Andreas Munk Petersen, Christen Rune Stensvold, Hengameh Mirsepasi, Jørgen Engberg, Alice Friis-Møller, Lone Jannok Porsbo, Anette M. Hammerum, Inge Nordgaard-Lassen, Henrik Vedel Nielsen KA krogfelt, Petersen AM, Stensvold CR, et al. Active ulcerative colitis associated with low prevalence of Blastocystis and Dientamoeba fra. Scand J Gastroenterol. 2013;48(5):638-639. doi:10.3109/00365521.2013.780094
39. Krogsgaard LR, Andersen LOB, Johannesen TB, et al. Characteristics of the bacterial microbiome in association with common intestinal parasites in irritable bowel syndrome. Clin Transl Gastroenterol. 2018;9(6):161. http://dx.doi.org/10.1038/s41424-018-0027-2
40. Kesuma Y, Firmansyah A, Bardosono S, Sari IP, Kurniawan A. Blastocystis ST-1 is associated with Irritable Bowel Syndrome-diarrhoea (IBS-D) in Indonesian adolescences. Parasite Epidemiol Control. 2019;6:e00112. doi:10.1016/j.parepi.2019.e00112
46. R Core Team (2021). R: A Language and Environment for Statistical Computing. R Found Stat Comput. Published online 2021.
47. Wickham H. 2016. ggplot2: elegant graphics for data analysis. Springer-Verlag, New York, NY. https://ggplot2.tidyverse.org.