Location via proxy:   [ UP ]  
[Report a bug]   [Manage cookies]                
Skip to main content

Advertisement

Distribution and phylogenetic analysis of Blastocystis sp. subtypes isolated from IBD patients and healthy individuals in Iran

  • Original Article
  • Published:
European Journal of Clinical Microbiology & Infectious Diseases Aims and scope Submit manuscript

Abstract

Blastocystis is a single-celled intestinal parasite commonly found in humans and a broad range of animals all over the world. In humans, its role in health and disease remains unsettled. The aim of our study was to investigate the distribution of Blastocystis and Blastocystis subtypes (ST) in patients with inflammatory bowel disease (IBD) and control subjects. A total of 71 stool samples were collected from IBD patients, 69 and 2 of whom had ulcerative colitis (UC) and Crohn’s Disease (CD), respectively. Moreover, 166 stool samples from healthy subjects were included as control samples. All stool samples were cultivated, and 550-bp fragments of the small subunit ribosomal RNA gene was amplified from Blastocystis-positive cultures. All PCR-positive samples were sequenced. Blastocystis was observed in 9 (12.67%) and 35 (21.1%) IBD patients and healthy controls, respectively. There was no statistically significant correlation between IBD and presence of Blastocystis (P = 0.147). There was a statistically significant correlation between age and Blastocystis colonization in the IBD group (P < 0.05), but not among healthy controls. No significant correlation between gender and colonization was observed. ST1 and ST3 were obtained from 1 (12.5%) and 7 (87.5%) IBD patients, respectively, while in the healthy control group, subtypes 1, 2, and 3 were found in 14 (40%), 12 (34.28%), and 9 (25.72%), respectively. Phylogenetic analysis showed no variation in the distribution of subtypes nor intra-subtype genetic diversity between samples acquired from IBD patients and healthy controls. This study showed a trend towards a lower prevalence of Blastocystis in IBD patients than in control subjects. ST3 sequences isolated from IBD patients and control individuals did not appear to differ genetically.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1

Similar content being viewed by others

References

  1. Clark CG, van der Giezen M, Alfellani MA, Stensvold CR (2013) Recent developments in Blastocystis research. Adv Parasitol 82:1–32. doi:10.1016/B978-0-12-407706-5.00001-0

    Article  PubMed  Google Scholar 

  2. Stensvold CR, Clark CG (2016) Current status of Blastocystis: a personal view. Parasitol Int 65(6 Pt B):763–771. doi:10.1016/j.parint.2016.05.015

  3. Duda A, Kosik-Bogacka D, Lanocha N, Szymanski S (2014) Blastocystis hominis—parasites or commensals? Ann Acad Med Stetin 60(1):23–28

    PubMed  Google Scholar 

  4. Tan KS (2008) New insights on classification, identification, and clinical relevance of Blastocystis spp. Clin Microbiol Rev 21(4):639–665. doi:10.1128/CMR.00022-08

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Elghareeb AS, Younis MS, El Fakahany AF, Nagaty IM, Nagib MM (2015) Laboratory diagnosis of Blastocystis spp. in diarrheic patients. Trop Parasitol 5(1):36–41. doi:10.4103/2229-5070.149919

    Article  PubMed  PubMed Central  Google Scholar 

  6. El Safadi D et al (2014) Children of Senegal River basin show the highest prevalence of Blastocystis sp. ever observed worldwide. BMC Infect Dis 14:164. doi:10.1186/1471-2334-14-164

    Article  PubMed  PubMed Central  Google Scholar 

  7. Scanlan PD, Knight R, Song SJ, Ackermann G, Cotter PD (2016) Prevalence and genetic diversity of Blastocystis in family units living in the United States. Infect Gen Evol 45:95–97. doi:10.1016/j.meegid.2016.08.018

    Article  Google Scholar 

  8. Poulsen CS, Efunshile AM, Nelson JA, Stensvold CR (2016) Epidemiological aspects of Blastocystis colonization in children in Ilero, Nigeria. Am J Trop Med Hyg 95(1):175–179. doi:10.4269/ajtmh.16-0074

  9. Lee LI, Chye TT, Karmacharya BM, Govind SK (2012) Blastocystis sp.: waterborne zoonotic organism, a possibility? Parasite Vectors 5:130. doi:10.1186/1756-3305-5-130

    Article  Google Scholar 

  10. Tan KS, Mirza H, Teo JD, Wu B, Macary PA (2010) Current views on the clinical relevance of Blastocystis spp. Curr Infect Dis Rep 12(1):28–35. doi:10.1007/s11908-009-0073-8

    Article  PubMed  Google Scholar 

  11. Balint A, Doczi I, Bereczki L et al (2014) Do not forget the stool examination!-cutaneous and gastrointestinal manifestations of Blastocystis sp. infection. Parasitol Res 113(4):1585–1590. doi:10.1007/s00436-014-3805-0

    Article  PubMed  Google Scholar 

  12. Fleta Zaragozano J, Clavel Parrilla A, Castillo Garcia FJ, Bueno Lozano M, Sarria Chueca A (1993) Blastocystis hominis and abdominal pain in childhood. An Esp Pediatr 38(1):13–16

    CAS  PubMed  Google Scholar 

  13. Gupta R, Parsi K (2006) Chronic urticaria due to Blastocystis hominis. Aust J Dermatol 47(2):117–119. doi:10.1111/j.1440-0960.2006.00244.x

    Article  Google Scholar 

  14. Hameed DM, Hassanin OM, Zuel-Fakkar NM (2011) Association of Blastocystis hominis genetic subtypes with urticaria. Parasitol Res 108(3):553–560. doi:10.1007/s00436-010-2097-2

    Article  PubMed  Google Scholar 

  15. Kick G, Rueff F, Przybilla B (2002) Palmoplantar pruritus subsiding after Blastocystis hominis eradication. Acta Dermatol Venereol 82(1):60

    Article  Google Scholar 

  16. El Deeb HK, Salah-Eldin H, Khodeer S (2012) Blastocystis hominis as a contributing risk factor for development of iron deficiency anemia in pregnant women. Parasitol Res 110(6):2167–2174. doi:10.1007/s00436-011-2743-3

    Article  PubMed  Google Scholar 

  17. Yavasoglu I, Kadikoylu G, Uysal H, Ertug S, Bolaman Z (2008) Is Blastocystis hominis a new etiologic factor or a coincidence in iron deficiency anemia? Eur J Haematol 81(1):47–50. doi:10.1111/j.1600-0609.2008.01080.x

    Article  CAS  PubMed  Google Scholar 

  18. Micheloud D, Jensen J, Fernandez-Cruz E, Carbone J (2007) Chronic angioedema and blastocystis hominis infection. Revist Gastroenterol del Peru [Article in Spanish] 27(2):191–193

    Google Scholar 

  19. Alfellani MA, Taner-Mulla D, Jacob AS et al (2013) Genetic diversity of Blastocystis in livestock and zoo animals. Protist 164(4):497–509. doi:10.1016/j.protis.2013.05.003

    Article  CAS  PubMed  Google Scholar 

  20. Alfellani MA, Stensvold CR, Vidal-Lapiedra A, Onuoha ES, Fagbenro-Beyioku AF, Clark CG (2013) Variable geographic distribution of Blastocystis subtypes and its potential implications. Acta Trop 126(1):11–18. doi:10.1016/j.actatropica.2012.12.011

    Article  PubMed  Google Scholar 

  21. Ko JK, Auyeung KK (2014) Inflammatory bowel disease: etiology, pathogenesis and current therapy. Curr Pharm Des 20(7):1082–1096

    Article  CAS  PubMed  Google Scholar 

  22. Hanauer SB (2006) Inflammatory bowel disease: epidemiology, pathogenesis, and therapeutic opportunities. Inflamm Bowel Dis 12(Suppl 1):S3–S9

    Article  PubMed  Google Scholar 

  23. Corridoni D, Arseneau KO, Cominelli F (2014) Inflammatory bowel disease. Immunol Lett 161(2):231–235. doi:10.1016/j.imlet.2014.04.004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Goyal N, Rana A, Ahlawat A, Bijjem KR, Kumar P (2014) Animal models of inflammatory bowel disease: a review. Inflammopharmacol 22(4):219–233. doi:10.1007/s10787-014-0207-y

    Article  Google Scholar 

  25. Boirivant M, Cossu A (2012) Inflammatory bowel disease. Oral Dis 18(1):1–15. doi:10.1111/j.1601-0825.2011.01811.x

    Article  CAS  PubMed  Google Scholar 

  26. Sartor RB, Mazmanian SK (2012) Intestinal microbes in inflammatory bowel diseases. Am J Gastroenterol Suppl 1(1):15–21

    Article  CAS  Google Scholar 

  27. Becker C, Neurath MF, Wirtz S (2015) The intestinal microbiota in inflammatory bowel disease. ILAR J 56(2):192–204. doi:10.1093/ilar/ilv030

    Article  CAS  PubMed  Google Scholar 

  28. Hold GL, Smith M, Grange C, Watt ER, El-Omar EM, Mukhopadhya I (2014) Role of the gut microbiota in inflammatory bowel disease pathogenesis: what have we learnt in the past 10 years? World J Gastroenterol 20(5):1192–1210. doi:10.3748/wjg.v20.i5.1192

    Article  PubMed  PubMed Central  Google Scholar 

  29. Marchesi JR, Adams DH, Fava F et al (2016) The gut microbiota and host health: a new clinical frontier. Gut 65(2):330–339. doi:10.1136/gutjnl-2015-309990

    Article  PubMed  Google Scholar 

  30. Ragavan ND, Kumar S, Chye TT, Mahadeva S, Shiaw-Hooi H (2015) Blastocystis sp. in irritable bowel syndrome (IBS) — detection in stool aspirates during colonoscopy. PLoS One 10(9):e0121173. doi:10.1371/journal.pone.0121173

    Article  PubMed  PubMed Central  Google Scholar 

  31. Mumcuoglu I, Coskun FA, Aksu N, Purnak T, Gungor C (2013) Role of Dientamoeba fragilis and Blastocystis spp. in irritable bowel syndrome. Turkiye Parazitol Derg (Turkish Society for Parasitology) 37(2):73–77. doi:10.5152/tpd.2013.19

    Article  Google Scholar 

  32. Surangsrirat S, Thamrongwittawatpong L, Piyaniran W et al (2010) Assessment of the association between Blastocystis infection and irritable bowel syndrome. J Med Assoc Thai 93(Suppl 6):S119–S124

    PubMed  Google Scholar 

  33. Dogruman-Al F, Simsek Z, Boorom K et al (2010) Comparison of methods for detection of Blastocystis infection in routinely submitted stool samples, and also in IBS/IBD patients in Ankara, Turkey. PLoS One 5(11):e15484. doi:10.1371/journal.pone.0015484

    Article  PubMed  PubMed Central  Google Scholar 

  34. Dogruman-Al F, Kustimur S, Yoshikawa H et al (2009) Blastocystis subtypes in irritable bowel syndrome and inflammatory bowel disease in Ankara, Turkey. Mem Inst Oswaldo Cruz 104(5):724–727

    Article  CAS  PubMed  Google Scholar 

  35. Petersen AM, Stensvold CR, Mirsepasi H et al (2013) Active ulcerative colitis associated with low prevalence of Blastocystis and Dientamoeba fragilis infection. Scand J Gastroenterol 48(5):638–639. doi:10.3109/00365521.2013.780094

    Article  PubMed  Google Scholar 

  36. Jalallou N, Iravani S, Rezaeian M, Alinaghizade A, Mirjalali H (2017) Subtypes distribution and frequency of Blastocystis sp., isolated from diarrheic and non-diarrheic patients. Iran J Parasitol 12(1):63–68

    PubMed  PubMed Central  Google Scholar 

  37. Alinaghizade A, Mirjalali H, Mohebali M, Stensvold CR, Rezaeian M (2017) Inter- and intra-subtype variation of Blastocystis subtypes isolated from diarrheic and non-diarrheic patients in Iran. Infect Gen Evol 50:77–82. doi:10.1016/j.meegid.2017.02.016

  38. Lanuza MD, Carbajal JA, Villar J, Borras R (1997) Description of an improved method for Blastocystis hominis culture and axenization. Parasitol Res 83(1):60–63

    Article  CAS  PubMed  Google Scholar 

  39. Bohm-Gloning B, Knobloch J, Walderich B (1997) Five subgroups of Blastocystis hominis from symptomatic and asymptomatic patients revealed by restriction site analysis of PCR-amplified 16S-like rDNA. Trop Med Int Health 2(8):771–778

    Article  CAS  PubMed  Google Scholar 

  40. Stensvold CR, Arendrup MC, Jespersgaard C, Molbak K, Nielsen HV (2007) Detecting Blastocystis using parasitologic and DNA-based methods: a comparative study. Diag Microbiol Infect Dis 59(3):303–307. doi:10.1016/j.diagmicrobio.2007.06.003

    Article  CAS  Google Scholar 

  41. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30(12):2725–2729. doi:10.1093/molbev/mst197

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Basak S, Rajurkar MN, Mallick SK (2014) Detection of Blastocystis hominis: a controversial human pathogen. Parasitol Res 113(1):261–265. doi:10.1007/s00436-013-3652-4

    Article  PubMed  Google Scholar 

  43. Engsbro AL, Stensvold CR (2012) Blastocystis: to treat or not to treat...But how? Clin Infect Dis : Off Publ Infect Dis Soc Am 55(10):1431–1432. doi:10.1093/cid/cis699

    Article  Google Scholar 

  44. Scanlan PD (2012) Blastocystis: past pitfalls and future perspectives. Trend Parasitol 28(8):327–334. doi:10.1016/j.pt.2012.05.001

    Article  Google Scholar 

  45. Scanlan PD, Stensvold CR (2013) Blastocystis: getting to grips with our guileful guest. Trend Parasitol 29(11):523–529. doi:10.1016/j.pt.2013.08.006

    Article  Google Scholar 

  46. Andersen LO, Stensvold CR (2016) Blastocystis in health and disease: are we moving from a clinical to a public health perspective? J Clin Microbiol 54(3):524–528. doi:10.1128/JCM.02520-15

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Jimenez-Gonzalez DE, Martinez-Flores WA, Reyes-Gordillo J et al (2012) Blastocystis infection is associated with irritable bowel syndrome in a Mexican patient population. Parasitol Res 110(3):1269–1275. doi:10.1007/s00436-011-2626-7

    Article  PubMed  Google Scholar 

  48. Nagel R, Traub RJ, Kwan MM, Bielefeldt-Ohmann H (2015) Blastocystis specific serum immunoglobulin in patients with irritable bowel syndrome (IBS) versus healthy controls. Parasite Vector 8(1):453. doi:10.1186/s13071-015-1069-x

    Article  Google Scholar 

  49. Yamamoto-Furusho JK, Torijano-Carrera E (2010) Intestinal protozoa infections among patients with ulcerative colitis: prevalence and impact on clinical disease course. Digestion 82(1):18–23. doi:10.1159/000273871

    Article  PubMed  Google Scholar 

  50. Cekin AH, Cekin Y, Adakan Y, Tasdemir E, Koclar FG, Yolcular BO (2012) Blastocystosis in patients with gastrointestinal symptoms: a case–control study. BMC Gastroenterol 12(1):122. doi:10.1186/1471-230x-12-122

    Article  PubMed  PubMed Central  Google Scholar 

  51. Rossen NG et al (2015) Low prevalence of Blastocystis sp. in active ulcerative colitis patients. Eur J Clin Microbiol Infect Dis 34(5):1039–1044. doi:10.1007/s10096-015-2312-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Moosavi A, Haghighi A, Mojarad EN et al (2012) Genetic variability of Blastocystis sp. isolated from symptomatic and asymptomatic individuals in Iran. Parasitol Res 111(6):2311–2315. doi:10.1007/s00436-012-3085-5

    Article  CAS  PubMed  Google Scholar 

  53. Badparva E, Sadraee J, Kheirandish F (2015) Genetic diversity of Blastocystis isolated from cattle in Khorramabad, Iran. Jundishapur J Microbiol 8(3):e14810. doi:10.5812/jjm.14810

    Article  PubMed  PubMed Central  Google Scholar 

  54. Badparva E, Sadraee J, Kheirandish F, Frouzandeh M (2014) Genetic diversity of human Blastocystis isolates in khorramabad, central iran. Iran J Parasitol 9(1):44–49

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Motazedian H, Ghasemi H, Sadjjadi SM (2008) Genomic diversity of Blastocystis hominis from patients in southern Iran. Ann Trop Med Parasitol 102(1):85–88. doi:10.1179/136485908X252197

    Article  CAS  PubMed  Google Scholar 

  56. Abu-Madi M, Aly M, Behnke JM, Clark CG, Balkhy H (2015) The distribution of Blastocystis subtypes in isolates from Qatar. Parasite Vector 8:465. doi:10.1186/s13071-015-1071-3

    Article  Google Scholar 

  57. Souppart L et al (2010) Subtype analysis of Blastocystis isolates from symptomatic patients in Egypt. Parasitol Res 106(2):505–511. doi:10.1007/s00436-009-1693-5

    Article  PubMed  Google Scholar 

  58. Pandey PK et al (2015) Prevalence and subtype analysis of Blastocystis in healthy Indian individuals. Infect Gen Evol 31:296–299. doi:10.1016/j.meegid.2015.02.012

    Article  CAS  Google Scholar 

  59. AbuOdeh R, Ezzedine S, Samie A, Stensvold CR, ElBakri A (2016) Prevalence and subtype distribution of Blastocystis in healthy individuals in Sharjah, United Arab Emirates. Infect Gen Evol 37:158–162. doi:10.1016/j.meegid.2015.11.021

    Article  Google Scholar 

  60. Das R, Khalil S, Mirdha BR, Makharia GK, Dattagupta S, Chaudhry R (2016) Molecular characterization and subtyping of Blastocystis species in irritable bowel syndrome patients from North India. PLoS One 11(1):e0147055. doi:10.1371/journal.pone.0147055

    Article  PubMed  PubMed Central  Google Scholar 

  61. Coskun A, Malatyali E, Ertabaklar H, Yasar MB, Karaoglu AO, Ertug S (2016) Blastocystis in ulcerative colitis patients: genetic diversity and analysis of laboratory findings. Asia Pac J Trop Med 9(9):916–919. doi:10.1016/j.apjtm.2016.07.018

    Article  CAS  Google Scholar 

  62. Mattiucci S, Crisafi B, Gabrielli S, Paoletti M, Cancrini G (2015) Molecular epidemiology and genetic diversity of Blastocystis infection in humans in Italy. Epidemiol Infect 1-12. doi:10.1017/S0950268815001697

  63. Mirza H, Tan KS (2009) Blastocystis exhibits inter- and intra-subtype variation in cysteine protease activity. Parasitol Res 104(2):355–361. doi:10.1007/s00436-008-1203-1

    Article  PubMed  Google Scholar 

  64. Wu Z, Mirza H, Tan KS (2014) Intra-subtype variation in enteroadhesion accounts for differences in epithelial barrier disruption and is associated with metronidazole resistance in Blastocystis subtype-7. PLoS Neglected Trop Dis 8(5):e2885. doi:10.1371/journal.pntd.0002885

    Article  Google Scholar 

  65. Sio SW, Puthia MK, Lee AS, Lu J, Tan KS (2006) Protease activity of Blastocystis hominis. Parasitol Res 99(2):126–130. doi:10.1007/s00436-006-0131-1

    Article  PubMed  Google Scholar 

  66. Puthia MK, Sio SW, Lu J, Tan KS (2006) Blastocystis ratti induces contact-independent apoptosis, F-actin rearrangement, and barrier function disruption in IEC-6 cells. Infect Immun 74(7):4114–4123. doi:10.1128/IAI.00328-06

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Puthia MK, Vaithilingam A, Lu J, Tan KS (2005) Degradation of human secretory immunoglobulin a by Blastocystis. Parasitol Res 97(5):386–389. doi:10.1007/s00436-005-1461-0

    Article  PubMed  Google Scholar 

  68. Poirier P, Wawrzyniak I, Vivares CP, Delbac F, El Alaoui H (2012) New insights into Blastocystis spp.: a potential link with irritable bowel syndrome. PLoS Path 8(3):e1002545. doi:10.1371/journal.ppat.1002545

    Article  CAS  Google Scholar 

  69. Boorom KF et al (2008) Oh my aching gut: irritable bowel syndrome, Blastocystis, and asymptomatic infection. Parasite Vector 1(1):40. doi:10.1186/1756-3305-1-40

    Article  Google Scholar 

  70. Abdel-Hameed DM, Hassanin OM (2011) Proteaese activity of Blastocystis hominis subtype3 in symptomatic and asymptomatic patients. Parasitol Res 109(2):321–327. doi:10.1007/s00436-011-2259-x

    Article  PubMed  Google Scholar 

  71. Audebert C, Even G, Cian A et al (2016) Colonization with the enteric protozoa Blastocystis is associated with increased diversity of human gut bacterial microbiota. Sci Rep 6:25255. doi:10.1038/srep25255

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Nourrisson C, Scanzi J, Pereira B et al (2014) Blastocystis is associated with decrease of fecal microbiota protective bacteria: comparative analysis between patients with irritable bowel syndrome and control subjects. PLoS One 9(11):e111868. doi:10.1371/journal.pone.0111868

    Article  PubMed  PubMed Central  Google Scholar 

  73. Scanlan PD, Stensvold CR, Rajilic-Stojanovic M et al (2014) The microbial eukaryote Blastocystis is a prevalent and diverse member of the healthy human gut microbiota. FEMS Microbiol Ecol 90(1):326–330. doi:10.1111/1574-6941.12396

    Article  CAS  PubMed  Google Scholar 

  74. Andersen LO, Bonde I, Nielsen HB, Stensvold CR (2015) A retrospective metagenomics approach to studying Blastocystis. FEMS Microbiol Ecol 91(7). doi:10.1093/femsec/fiv072

  75. Rook GA (2010) 99th Dahlem conference on infection, inflammation and chronic inflammatory disorders: Darwinian medicine and the ‘hygiene’ or‘old friends’ hypothesis. Clin Exp Immunol 160(1):70–79

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Rook GA (2011) Hygiene and other early childhood influences on the subsequent function of the immune system. Dig Dis (Basel, Switzerland) 29(2):144–153. doi:10.1159/000323877

    Article  Google Scholar 

Download references

Acknowledgements

The authors would like to thank all of the colleagues of the Foodborne and Waterborne Diseases Research Center and Gastroenterology and Liver diseases Research Institute for their laboratory cooperation.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to H. Asadzadeh Aghdaei.

Ethics declarations

Funding

This project was financially supported by the Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran with grant number: RIGLD 832.

Ethical approval

The present study was approved by the Ethics Committee of Shahid Beheshti University of Medical Sciences.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Conflict of interest

The authors declare that they have no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mirjalali, H., Abbasi, M.R., Naderi, N. et al. Distribution and phylogenetic analysis of Blastocystis sp. subtypes isolated from IBD patients and healthy individuals in Iran. Eur J Clin Microbiol Infect Dis 36, 2335–2342 (2017). https://doi.org/10.1007/s10096-017-3065-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10096-017-3065-x

Keywords