Review Article | Published:

Intestinal dysbiosis and necrotizing enterocolitis: assessment for causality using Bradford Hill criteria

Abstract

In recent years, several studies have shown that premature infants who develop NEC frequently display enteric dysbiosis with increased Gram-negative bacteria for several days to weeks prior to NEC onset. The importance of these findings, for the possibility of a causal role of these bacteria in NEC pathogenesis, and for potential value of gut dysbiosis as a biomarker of NEC, is well-recognized. In this review, we present current evidence supporting the association between NEC in premature infants and enteric dysbiosis, and its evaluation using the Bradford Hill criteria for causality. To provide an objective appraisal, we developed a novel scoring system for causal inference. Despite important methodological and statistical limitations, there is support for the association from several large studies and a meta-analysis. The association draws strength from strong biological plausibility of a role of Gram-negative bacteria in NEC and from evidence for temporality, that dysbiosis may antedate NEC onset. The weakness of the association is in the low level of consistency across studies, and the lack of specificity of effect. There is a need for an improved definition of dysbiosis, either based on a critical threshold of relative abundances or at higher levels of taxonomic resolution.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    Pammi, M. et al. Intestinal dysbiosis in preterm infants preceding necrotizing enterocolitis: a systematic review and meta-analysis. Microbiome 5, 31 (2017).

  2. 2.

    Wang, Y. et al. 16S rRNA gene-based analysis of fecal microbiota from preterm infants with and without necrotizing enterocolitis. ISME J. 3, 944–954 (2009).

  3. 3.

    Millar, M. R. et al. Application of 16S rRNA gene PCR to study bowel flora of preterm infants with and without necrotizing enterocolitis. J. Clin. Microbiol. 34, 2506–2510 (1996).

  4. 4.

    Groer, M. W. et al. Development of the preterm infant gut microbiome: a research priority. Microbiome 2, 38 (2014).

  5. 5.

    Mshvildadze, M. et al. Intestinal microbial ecology in premature infants assessed with non-culture-based techniques. J. Pediatr. 156, 20–25 (2010).

  6. 6.

    Mai, V. et al. Fecal microbiota in premature infants prior to necrotizing enterocolitis. PLoS ONE 6, e20647 (2011).

  7. 7.

    Zhou, Y. et al. Longitudinal analysis of the premature infant intestinal microbiome prior to necrotizing enterocolitis: a case-control study. PLoS ONE 10, e0118632 (2015).

  8. 8.

    Warner B. B., et al. Gut bacteria dysbiosis and necrotising enterocolitis in very low birthweight infants: a prospective case-control study. Lancet 387, 1928–36 (2016).

  9. 9.

    Lindberg T. P., et al. Preterm infant gut microbial patterns related to the development of necrotizing enterocolitis. J. Matern. Fetal Neonatal Med. 9, 1–10 (2018).

  10. 10.

    Hill, A. B. The environment and disease: association or causation? Proc. R Soc. Med. 58, 295–300 (1965).

  11. 11.

    Fedak, K. M., Bernal, A., Capshaw, Z. A. & Gross, S. Applying the Bradford Hill criteria in the 21st century: how data integration has changed causal inference in molecular epidemiology. Emerg. Themes Epidemiol. 12, 14 (2015).

  12. 12.

    Polin, R. A. et al. Necrotizing enterocolitis in term infants. J. Pediatr. 89, 460–462 (1976).

  13. 13.

    Ballance, W. A., Dahms, B. B., Shenker, N. & Kliegman, R. M. Pathology of neonatal necrotizing enterocolitis: a ten-year experience. J. Pediatr. 117, S6–S13 (1990).

  14. 14.

    Tait, R. A. & Kealy, W. F. Neonatal necrotising enterocolitis. J. Clin. Pathol. 32, 1090–1099 (1979).

  15. 15.

    Berdon, W. E. et al. Necrotizing enterocolitis in the premature infant. Radiology 83, 879–887 (1964).

  16. 16.

    Remon, J. I. et al. Depth of bacterial invasion in resected intestinal tissue predicts mortality in surgical necrotizing enterocolitis. J. Perinatol. 35, 755–762 (2015).

  17. 17.

    Pear, B. L. Pneumatosis intestinalis: a review. Radiology 207, 13–19 (1998).

  18. 18.

    Bury R. G., Tudehope D. 2001 Enteral antibiotics for preventing necrotizing enterocolitis in low birthweight or preterm infants. Cochrane Database Syst. Rev. CD000405.

  19. 19.

    Maheshwari, A. Immunologic and hematological abnormalities in necrotizing enterocolitis. Clin. Perinatol. 42, 567–585 (2015).

  20. 20.

    Gephart, S. M. et al. Changing the paradigm of defining, detecting, and diagnosing NEC: Perspectives on Bell’s stages and biomarkers for NEC. Semin. Pediatr. Surg. 27, 3–10 (2018).

  21. 21.

    Coggins, S. A., Wynn, J. L. & Weitkamp, J. H. Infectious causes of necrotizing enterocolitis. Clin. Perinatol. 42, 133–154 (2015), ix.

  22. 22.

    Nanthakumar, N. N., Fusunyan, R. D., Sanderson, I. & Walker, W. A. Inflammation in the developing human intestine: a possible pathophysiologic contribution to necrotizing enterocolitis. Proc. Natl Acad. Sci. USA 97, 6043–6048 (2000).

  23. 23.

    Mollitt, D. L., Tepas, J. J. 3rd & Talbert, J. L. The microbiology of neonatal peritonitis. Arch. Surg. 123, 176–179 (1988).

  24. 24.

    Boccia, D., Stolfi, I., Lana, S. & Moro, M. L. Nosocomial necrotising enterocolitis outbreaks: epidemiology and control measures. Eur. J. Pediatr. 160, 385–391 (2001).

  25. 25.

    Gregersen, N. et al. Klebsiella pneumoniae with extended spectrum beta-lactamase activity associated with a necrotizing enterocolitis outbreak. Pediatr. Infect. Dis. J. 18, 963–967 (1999).

  26. 26.

    Hill, H. R., Hunt, C. E. & Matsen, J. M. Nosocomial colonization with Klebsiella, type 26, in a neonatal intensive-care unit associated with an outbreak of sepsis, meningitis, and necrotizing enterocolitis. J. Pediatr. 85, 415–419 (1974).

  27. 27.

    van Acker, J. et al. Outbreak of necrotizing enterocolitis associated with Enterobacter sakazakii in powdered milk formula. J. Clin. Microbiol. 39, 293–297 (2001).

  28. 28.

    Torrazza, R. M. et al. Intestinal microbial ecology and environmental factors affecting necrotizing enterocolitis. PLoS ONE 8, e83304 (2013).

  29. 29.

    Morrow, A. L. et al. Early microbial and metabolomic signatures predict later onset of necrotizing enterocolitis in preterm infants. Microbiome 1, 13 (2013).

  30. 30.

    Hofler, M. The Bradford Hill considerations on causality: a counterfactual perspective. Emerg. Themes Epidemiol. 2, 11 (2005).

  31. 31.

    McMurtry, V. E. et al. Bacterial diversity and Clostridia abundance decrease with increasing severity of necrotizing enterocolitis. Microbiome 3, 11 (2015).

  32. 32.

    Smith, B. et al. Investigation of the early intestinal microflora in premature infants with/without necrotizing enterocolitis using two different methods. Pediatr. Res. 71, 115–120 (2012).

  33. 33.

    Sim, K. et al. Dysbiosis anticipating necrotizing enterocolitis in very premature infants. Clin. Infect. Dis. 60, 389–397 (2015).

  34. 34.

    de la Cochetiere, M. F. et al. Early intestinal bacterial colonization and necrotizing enterocolitis in premature infants: the putative role of Clostridium. Pediatr Res 56, 366–370 (2004).

  35. 35.

    Romano-Keeler, J. et al. Distinct mucosal microbial communities in infants with surgical necrotizing enterocolitis correlate with age and antibiotic exposure. PLoS One 13, e0206366 (2018).

  36. 36.

    Stewart, C. J. et al. The preterm gut microbiota: changes associated with necrotizing enterocolitis and infection. Acta Paediatr 101, 1121–1127 (2012).

  37. 37.

    Normann, E., Fahlen, A., Engstrand, L. & Lilja, H. E. Intestinal microbial profiles in extremely preterm infants with and without necrotizing enterocolitis. Acta Paediatr 102, 129–136 (2013).

  38. 38.

    Wandro S., et al. The Microbiome and Metabolome of Preterm Infant Stool Are Personalized and Not Driven by Health Outcomes, Including Necrotizing Enterocolitis and Late-Onset Sepsis. mSphere 3 (2018).

  39. 39.

    Itani, T. et al. Preterm infants with necrotising enterocolitis demonstrate an unbalanced gut microbiota. Acta Paediatr 107, 40–47 (2018).

  40. 40.

    Barron, L. K. et al. Independence of gut bacterial content and neonatal necrotizing enterocolitis severity. J Pediatr Surg 52, 993–998 (2017).

  41. 41.

    Brown, C. T. et al. Hospitalized Premature Infants Are Colonized by Related Bacterial Strains with Distinct Proteomic Profiles. MBio 9, e00441–18 (2018).

  42. 42.

    Ravi, A. et al. Association of the gut microbiota mobilome with hospital location and birth weight in preterm infants. Pediatr Res 82, 829–838 (2017).

  43. 43.

    Stewart, C. J. et al. Temporal bacterial and metabolic development of the preterm gut reveals specific signatures in health and disease. Microbiome 4, 67 (2016).

  44. 44.

    Heida, F. H. et al. A Necrotizing Enterocolitis-Associated Gut Microbiota Is Present in the Meconium: Results of a Prospective Study. Clin Infect Dis 62, 863–870 (2016).

  45. 45.

    La Rosa, P. S. et al. Patterned progression of bacterial populations in the premature infant gut. Proc Natl Acad Sci U S A 111, 12522–12527 (2014).

  46. 46.

    Itani, T. et al. Establishment and development of the intestinal microbiota of preterm infants in a Lebanese tertiary hospital. Anaerobe 43, 4–14 (2017).

  47. 47.

    Gregory, K. E. et al. Influence of maternal breast milk ingestion on acquisition of the intestinal microbiome in preterm infants. Microbiome 4, 68 (2016).

  48. 48.

    Cong, X. et al. Gut microbiome developmental patterns in early life of preterm infants: impacts of feeding and gender. PLoS ONE 11, e0152751 (2016).

  49. 49.

    Parm, U., Metsvaht, T., Ilmoja, M. L. & Lutsar, I. Gut colonization by aerobic microorganisms is associated with route and type of nutrition in premature neonates. Nutr. Res. 35, 496–503 (2015).

  50. 50.

    Raveh-Sadka T., et al. Gut bacteria are rarely shared by co-hospitalized premature infants, regardless of necrotizing enterocolitis development. Elife 4, e05477 (2015).

  51. 51.

    Arboleya, S. et al. Intestinal microbiota development in preterm neonates and effect of perinatal antibiotics. J. Pediatr. 166, 538–544 (2015).

  52. 52.

    Arboleya, S. et al. Establishment and development of intestinal microbiota in preterm neonates. FEMS Microbiol. Ecol. 79, 763–772 (2012).

  53. 53.

    Mai, V. et al. Distortions in development of intestinal microbiota associated with late onset sepsis in preterm infants. PLoS ONE 8, e52876 (2013).

  54. 54.

    Schwiertz, A. et al. Development of the intestinal bacterial composition in hospitalized preterm infants in comparison with breast-fed, full-term infants. Pediatr. Res. 54, 393–399 (2003).

  55. 55.

    Ho, T. B. T. et al. Dichotomous development of the gut microbiome in preterm infants. Microbiome 6, 157 (2018).

  56. 56.

    Gibson, M. K. et al. Developmental dynamics of the preterm infant gut microbiota and antibiotic resistome. Nat. Microbiol. 1, 16024 (2016).

  57. 57.

    Morowitz, M. J. et al. Strain-resolved community genomic analysis of gut microbial colonization in a premature infant. Proc. Natl Acad. Sci. USA 108, 1128–1133 (2011).

  58. 58.

    Brooks, B. et al. Microbes in the neonatal intensive care unit resemble those found in the gut of premature infants. Microbiome 2, 1 (2014).

  59. 59.

    Greenwood, C. et al. Early empiric antibiotic use in preterm infants is associated with lower bacterial diversity and higher relative abundance of Enterobacter. J. Pediatr. 165, 23–29 (2014).

  60. 60.

    Aagaard, K. et al. The placenta harbors a unique microbiome. Sci. Transl. Med. 6, 237ra265 (2014).

  61. 61.

    Palmer, C., Bik, E. M., DiGiulio, D. B., Relman, D. A. & Brown, P. O. Development of the human infant intestinal microbiota. PLoS Biol. 5, e177 (2007).

  62. 62.

    Dominguez-Bello, M. G. et al. Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns. Proc. Natl Acad. Sci. USA 107, 11971–11975 (2010).

  63. 63.

    MacQueen, B. C. et al. Elevated fecal calprotectin levels during necrotizing enterocolitis are associated with activated neutrophils extruding neutrophil extracellular traps. J. Perinatol. 36, 862–869 (2016).

  64. 64.

    Ho T. B. T., et al. Enteric dysbiosis and fecal calprotectin expression in premature infants. Pediatr. Res. 85, 361–368 2018. in press.

  65. 65.

    Zhang, M., Zhang, X. & Zhang, J. Diagnostic value of fecal calprotectin in preterm infants with necrotizing enterocolitis. Clin. Lab 62, 863–869 (2016).

  66. 66.

    Lu, M. C. et al. Colibactin contributes to the hypervirulence of pks( + ) K1 CC23 Klebsiella pneumoniae in mouse meningitis infections. Front. Cell. Infect. Microbiol. 7, 103 (2017).

  67. 67.

    Straus, D. C., Lonon, M. K., Woods, D. E. & Garner, C. W. Production of an extracellular toxic complex by various strains of Pseudomonas cepacia. J. Med. Microbiol. 30, 17–22 (1989).

  68. 68.

    Stone, H. H., Kolb, L. D. & Geheber, C. E. Bacteriologic considerations in perforated necrotizing enterocolitis. South Med. J. 72, 1540–1544 (1979).

  69. 69.

    Canioni, D. et al. Histopathology and microbiology of isolated rectal bleeding in neonates: the so-called ‘ecchymotic colitis’. Histopathology 30, 472–477 (1997).

  70. 70.

    Rhoads, J. M. et al. Altered fecal microflora and increased fecal calprotectin in infants with colic. J. Pediatr. 155, 823–828 e821 (2009).

  71. 71.

    Arrieta, M. C., Stiemsma, L. T., Amenyogbe, N., Brown, E. M. & Finlay, B. The intestinal microbiome in early life: health and disease. Front. Immunol. 5, 427 (2014).

  72. 72.

    Koenig, J. E. et al. Succession of microbial consortia in the developing infant gut microbiome. Proc. Natl Acad. Sci. USA 108(Suppl 1), 4578–4585 (2011).

  73. 73.

    Fallani, M. et al. Determinants of the human infant intestinal microbiota after the introduction of first complementary foods in infant samples from five European centres. Microbiology 157, 1385–1392 (2011).

  74. 74.

    MohanKumar, K. et al. Trinitrobenzene sulfonic acid-induced intestinal injury in neonatal mice activates transcriptional networks similar to those seen in human necrotizing enterocolitis. Pediatr. Res. 81, 99–112 (2016).

  75. 75.

    MohanKumar, K. et al. Cytokines and growth factors in the developing intestine and during necrotizing enterocolitis. Semin. Perinatol. 41, 52–60 (2016).

  76. 76.

    Good, M. et al. Amniotic fluid inhibits Toll-like receptor 4 signaling in the fetal and neonatal intestinal epithelium. Proc. Natl Acad. Sci. USA 109, 11330–11335 (2012).

  77. 77.

    Hackam, D. J., Upperman, J. S., Grishin, A. & Ford, H. R. Disordered enterocyte signaling and intestinal barrier dysfunction in the pathogenesis of necrotizing enterocolitis. Semin. Pediatr. Surg. 14, 49–57 (2005).

  78. 78.

    Leaphart, C. L. et al. A critical role for TLR4 in the pathogenesis of necrotizing enterocolitis by modulating intestinal injury and repair. J. Immunol. 179, 4808–4820 (2007).

  79. 79.

    Smythies, L. E. et al. Human intestinal macrophages display profound inflammatory anergy despite avid phagocytic and bacteriocidal activity. J. Clin. Invest. 115, 66–75 (2005).

  80. 80.

    Maheshwari, A. et al. Cytomegalovirus blocks intestinal stroma-induced down-regulation of macrophage HIV-1 infection. J. Leukoc. Biol. 80, 1111–1117 (2006).

  81. 81.

    Maheshwari, A. et al. TGF-β2 suppresses macrophage cytokine production and mucosal inflammatory responses in the developing intestine. Gastroenterology 140, 242–253 (2011).

  82. 82.

    Namachivayam, K. et al. Smad7 inhibits autocrine expression of TGF-beta2 in intestinal epithelial cells in baboon necrotizing enterocolitis. Am. J. Physiol. Gastrointest. Liver Physiol. 304, G167–G180 (2013).

  83. 83.

    MohanKumar, K. et al. Smad7 interrupts TGF-β signaling in intestinal macrophages and promotes inflammatory activation of these cells during necrotizing enterocolitis. Pediatr. Res. 79, 951–961 (2016).

  84. 84.

    Nanthakumar, N. et al. The mechanism of excessive intestinal inflammation in necrotizing enterocolitis: an immature innate immune response. PLoS ONE 6, e17776 (2011).

  85. 85.

    Montgomery, R. K., Mulberg, A. E. & Grand, R. J. Development of the human gastrointestinal tract: twenty years of progress. Gastroenterology 116, 702–731 (1999).

  86. 86.

    Martin, N. A. et al. Active transport of bile acids decreases mucin 2 in neonatal ileum: implications for development of necrotizing enterocolitis. PLoS ONE 6, e27191 (2011).

  87. 87.

    Zhang, C. et al. Paneth cell ablation in the presence of Klebsiella pneumoniae induces necrotizing enterocolitis (NEC)-like injury in the small intestine of immature mice. Dis. Model Mech. 5, 522–532 (2012).

  88. 88.

    Haworth, J. C. & Dilling, L. Concentration of gamma-A-globulin in serum, saliva, and nasopharyngeal secretions of infants and children. J. Lab Clin. Med. 67, 922–933 (1966).

  89. 89.

    Brandtzaeg, P., Nilssen, D. E., Rognum, T. O. & Thrane, P. S. Ontogeny of the mucosal immune system and IgA deficiency. Gastroenterol. Clin. North Am. 20, 397–439 (1991).

  90. 90.

    Gleeson, M. et al. Ontogeny of the secretory immune system in man. Aust. N. Z. J. Med. 12, 255–258 (1982).

  91. 91.

    Mellander, L., Carlsson, B. & Hanson, L. A. Appearance of secretory IgM and IgA antibodies to Escherichia coli in saliva during early infancy and childhood. J. Pediatr. 104, 564–568 (1984).

  92. 92.

    Cripps, A. W., Gleeson, M. & Clancy, R. L. Ontogeny of the mucosal immune response in children. Adv. Exp. Med. Biol. 310, 87–92 (1991).

  93. 93.

    Weemaes, C. et al. Development of immunoglobulin A in infancy and childhood. Scand. J. Immunol. 58, 642–648 (2003).

  94. 94.

    Fitzsimmons, S. P. et al. Immunoglobulin A subclasses in infants’ saliva and in saliva and milk from their mothers. J. Pediatr. 124, 566–573 (1994).

  95. 95.

    Bhat, N. M. et al. The ontogeny and functional characteristics of human B-1 (CD5 + B) cells. Int. Immunol. 4, 243–252 (1992).

  96. 96.

    Chen, Z. J. et al. Polyreactive antigen-binding B cells are the predominant cell type in the newborn B cell repertoire. Eur. J. Immunol. 28, 989–994 (1998).

  97. 97.

    Bauer, K. et al. Diversification of Ig heavy chain genes in human preterm neonates prematurely exposed to environmental antigens. J. Immunol. 169, 1349–1356 (2002).

  98. 98.

    Fouhy, F. et al. High-throughput sequencing reveals the incomplete, short-term recovery of infant gut microbiota following parenteral antibiotic treatment with ampicillin and gentamicin. Antimicrob. Agents Chemother. 56, 5811–5820 (2012).

  99. 99.

    Cotten, C. M. et al. Prolonged duration of initial empirical antibiotic treatment is associated with increased rates of necrotizing enterocolitis and death for extremely low birth weight infants. Pediatrics 123, 58–66 (2009).

  100. 100.

    Alexander, V. N., Northrup, V. & Bizzarro, M. J. Antibiotic exposure in the newborn intensive care unit and the risk of necrotizing enterocolitis. J. Pediatr. 159, 392–397 (2011).

  101. 101.

    Esmaeilizand, R. et al. Antibiotic exposure and development of necrotizing enterocolitis in very preterm neonates. Paediatr. Child Health 23, e56–e61 (2018).

  102. 102.

    Abdel Ghany, E. A. & Ali, A. A. Empirical antibiotic treatment and the risk of necrotizing enterocolitis and death in very low birth weight neonates. Ann. Saudi Med. 32, 521–526 (2012).

  103. 103.

    Kuppala, V. S., Meinzen-Derr, J., Morrow, A. L. & Schibler, K. R. Prolonged initial empirical antibiotic treatment is associated with adverse outcomes in premature infants. J. Pediatr. 159, 720–725 (2011).

  104. 104.

    Cantey, J. B., Pyle, A. K., Wozniak, P. S., Hynan, L. S. & Sanchez, P. J. Early antibiotic exposure and adverse outcomes in preterm, very low birth weight infants. J. Pediatr. 203, 62–67 (2018).

  105. 105.

    Weintraub, A. S. et al. Antenatal antibiotic exposure in preterm infants with necrotizing enterocolitis. J. Perinatol. 32, 705–709 (2012).

  106. 106.

    Greenberg R. G., et al., Eunice Kennedy Shriver National Institute of Child H, Human Development Neonatal Research N Prolonged duration of early antibiotic therapy in extremely premature infants. Pediatr. Res. 85, 994–1000 (2019).

  107. 107.

    Gupta, R. W. et al. Histamine-2 receptor blockers alter the fecal microbiota in premature infants. J. Pediatr. Gastroenterol. Nutr. 56, 397–400 (2013).

  108. 108.

    Guillet, R. et al. Association of H2-blocker therapy and higher incidence of necrotizing enterocolitis in very low birth weight infants. Pediatrics 117, e137–e142 (2006).

  109. 109.

    More, K., Athalye-Jape, G., Rao, S. & Patole, S. Association of inhibitors of gastric acid secretion and higher incidence of necrotizing enterocolitis in preterm very low-birth-weight infants. Am. J. Perinatol. 30, 849–856 (2013).

  110. 110.

    Winter, S. E. & Baumler, A. J. Dysbiosis in the inflamed intestine: chance favors the prepared microbe. Gut Microbes 5, 71–73 (2014).

  111. 111.

    Atarashi, K. et al. Ectopic colonization of oral bacteria in the intestine drives TH1 cell induction and inflammation. Science 358, 359–365 (2017).

  112. 112.

    Larmonier, C. B., Shehab, K. W., Ghishan, F. K. & Kiela, P. R. T lymphocyte dynamics in inflammatory bowel diseases: role of the microbiome. Biomed. Res. Int. 2015, 504638 (2015).

  113. 113.

    Zeng, M. Y., Inohara, N. & Nunez, G. Mechanisms of inflammation-driven bacterial dysbiosis in the gut. Mucosal Immunol. 10, 18–26 (2017).

  114. 114.

    Friede, T., Rover, C., Wandel, S. & Neuenschwander, B. Meta-analysis of few small studies in orphan diseases. Res. Synth. Methods 8, 79–91 (2017).

  115. 115.

    Been, J. V., Lievense, S., Zimmermann, L. J., Kramer, B. W. & Wolfs, T. G. Chorioamnionitis as a risk factor for necrotizing enterocolitis: a systematic review and meta-analysis. J. Pediatr. 162, 236–242 e232 (2013).

Download references

Acknowledgements

NIH awards HL124078 and HL133022 (to A.M.).

Author information

J.B.F., P.G., D.R.S., M.P., and A.M. reviewed the literature and contributed to the manuscript. A.M. developed the Bradford Hill Causality Score. All the authors reviewed and approved the manuscript.

Correspondence to Akhil Maheshwari.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark