Molecular and serological diversity of Neisseria meningitidis carrier strains isolated from Italian students aged 14 to 22 years

Journal of Clinical Microbiology

Volumn 52, Issue 6, 2014, Pages 1901-1910

  Gasparini, Roberto ^a   Comanducci, Maurizio ^b,c   Amicizia, Daniela ^a   Ansaldi, Filippo ^a   Canepa, Paola ^a   Orsi, Andrea ^a   Icardi, Giancarlo ^a   Rizzitelli, Emanuela ^a   De Angelis, Gabriella ^b   Bambini, Stefania ^b   Moschioni, Monica ^b   Comandi, Sara ^b   Simmini, Isabella ^b   Boccadifuoco, Giueseppe ^b   Brunelli, Brunella ^b   Giuliani, Marzia Monica ^b   Pizza, Mariagrazia ^b   Panatto, Donatella ^a  

^a UNIVERSITY OF GENOA   (Italy)

^b NOVARTIS VACCINES AND DIAGNOSTICS   (Italy)

^c CRUCELL HOLLAND BV   (Netherlands)

Author keywords

[No Author keywords available]

Indexed keywords

BACTERIAL ANTIGEN; GENOMIC DNA; PORIN A; BACTERIAL DNA;

ADOLESCENT; ADULT; ANTIGEN EXPRESSION; ANTIGENIC VARIATION; ARTICLE; BACTERIAL CAPSULE; BACTERIAL COLONIZATION; BACTERIAL GENE; BACTERIAL STRAIN; BACTERIUM CARRIER; BACTERIUM IDENTIFICATION; BACTERIUM ISOLATE; CONTROLLED STUDY; ETHNIC GROUP; FEMALE; FHBP GENE; GENETIC VARIABILITY; HUMAN; ITALIAN; LONGITUDINAL STUDY; MALE; MICROBIAL DIVERSITY; MULTILOCUS SEQUENCE TYPING; NADA GENE; NASOPHARYNX; NEISSERIA MENINGITIDIS; NHBA GENE; NONHUMAN; OROPHARYNX; POLYMERASE CHAIN REACTION; PRIORITY JOURNAL; YOUNG ADULT; CLASSIFICATION; GENETICS; GENOTYPE; HETEROZYGOTE; IMMUNOLOGY; ISOLATION AND PURIFICATION; ITALY; MENINGOCOCCAL INFECTIONS; MICROBIOLOGY; MOLECULAR TYPING; SEROTYPING;

ADOLESCENT; ANTIGENS, BACTERIAL; BACTERIAL TYPING TECHNIQUES; CARRIER STATE; DNA, BACTERIAL; FEMALE; GENETIC VARIATION; GENOTYPE; HUMANS; ITALY; LONGITUDINAL STUDIES; MALE; MENINGOCOCCAL INFECTIONS; MOLECULAR TYPING; NEISSERIA MENINGITIDIS; OROPHARYNX; SEROTYPING; YOUNG ADULT;

EID: 84901673710     PISSN: 00951137     EISSN: 1098660X     Source Type: Journal    
DOI: 10.1128/JCM.03584-13     Document Type: Article

References (60)

1. Hill DJ, Griffiths NJ, Borodina E, Virji M. 2010. Cellular and molecular biology of Neisseria meningitidis colonization and invasive disease. Clin. Sci. (Lond.) 118:547-564
2. Gasparini R, Amicizia D, Lai PL, Panatto D. 2012. Neisseria meningitidis, pathogenetic mechanisms to overcome the human immune defences. J. Prev. Med. Hyg. 53:50-55
3. Muzzi A, Mora M, Pizza M, Rappuoli R, Donati C. 2013. Conservation of meningococcal antigens in the genus Neisseria. mBio 4(3):e00163-13. http://dx.doi.org/10.1128/mBio.00163-13
4. Yazdankhah SP, Caugant DA. 2004. Neisseria meningitidis: an overview of the carriage state. J. Med. Microbiol. 53:821-832. http://dx.doi.org/10.1099/ jmm.0.45529-0 (Pubitemid 39238387)
5. Goldschneider I, Gotschlich EC, Artenstein MS. 1969. Human immunity to the meningococcus. I. The role of humoral antibodies. J. Exp. Med. 129:1307-1326
6. Goldschneider I, Gotschlich EC, Artenstein MS. 1969 1969. Human immunity to the meningococcus. II. Development of natural immunity. J. Exp. Med. 129:1327-1348
7. Christensen H, May M, Bowen L, Hickman M, Trotter CL. 2010. Meningococcal carriage by age: a systematic review and meta-analysis. Lancet Infect. Dis. 10:853-861. http://dx.doi.org/10.1016/S1473-3099(10)70251-6
8. Maiden MC, Ibarz-Pavòn AB, Urwin R, Gray SJ, Andrews NJ, Clarke SC, Walker AM, Evans MR, Kroll JS, Neal KR, Ala?aldeen DA, Crook DW, Cann K, Harrison S, Cunningham R, Baxter D, Kaczmarski E, Maclennan J, Cameron JC, Stuart JM. 2008. Impact of meningococcal serogroup C conjugate vaccines on carriage and herd immunity. J. Infect. Dis. 197:737-743. http://dx.doi.org/10. 1086/527401 (Pubitemid 351364476)
9. Guzzetta G, Manfredi P, Gasparini R, Panatto D, Edmunds WJ. 2009. On the relationship between meningococcal transmission dynamics and disease: remarks on humoral immunity. Vaccine 27:3429-3434. http://dx.doi.org/10.1016/j.vaccine. 2009.01.092
10. Glitza IC, Ehrhard I, Müller-Pebody B, Reintjes R, Breuer T, Ammon A, Sonntag HG. 2008. Longitudinal study of meningococcal carrier rates in teenagers. Int. J. Hyg. Environ. Health 211:263-272. http://dx.doi.org/10.1016/ j.ijheh.2007.05.006 (Pubitemid 351847858)
11. Caugant DA, Fogg C, Bajunirwe F, Piola P, Twesigye R, Mutebi F, Fr?holm LO, Rosenqvist E, Batwala V, Aaberge IS, Rottingen JA, Guerin PJ. 2006. Pharyngeal carriage of Neisseria meningitidis in 2-19-year-old individuals in Uganda. Trans. R. Soc. Trop. Med. Hyg. 100:1159-1163. http://dx.doi.org/10.1016/ j.trstmh.2006.01.004 (Pubitemid 44464012)
12. Yazdankhah SP, Kriz P, Tzanakaki G, Kremastinou J, Kalmusova J, Musilek M, Alvestad T, Jolley KA, Wilson DJ, McCarthy ND, Caugant DA, Maiden MC. 2010. Distribution of serogroups and genotypes among disease-associated and carried isolates of Neisseria meningitidis from the Czech Republic, Greece, and Norway. J. Clin. Microbiol. 42:5146-5153. http://dx.doi.org/10.1128/JCM.42.11.5146-5153. 2004
13. Maiden MC, Bygraves JA, Feil E, Morelli G, Russell JE, Urwin R, Zhang Q, Zhou J, Zurth K, Caugant DA, Feavers IM, Achtman M, Spratt BG. 1998. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc. Natl. Acad. Sci. U. S. A. 95:3140-3145. http://dx.doi.org/10.1073/pnas.95.6.3140 (Pubitemid 28135868)
14. Caugant DA, Tzanakaki G, Kriz P. 2007. Lessons from meningococcal carriage studies. FEMS Microbiol. Rev. 31:52-63. http://dx.doi.org/10.1111/j. 1574-6976.2006.00052.x. (Pubitemid 46058820)
15. Urwin R, Russell JE, Thompson EA, Holmes EC, Feavers IM, Maiden MC. 2004. Distribution of surface protein variants among hyperinvasive meningococci: implications for vaccine design. Infect. Immun. 72:5955-5962. http://dx.doi.org/10.1128/IAI.72.10.5955-5962.2004 (Pubitemid 39303710)
16. Bambini S, Muzzi A, Olcen P, Rappuoli R, Pizza M, Comanducci M. 2009. Distribution and genetic variability of three vaccine components in a panel of strains representative of the diversity of serogroup B meningococcus. Vaccine 27:2794-2803. http://dx.doi.org/10.1016/j.vaccine.2009.02.098
17. Hammerschmidt S, Müller A, Sillmann H, Mühlenhoff M, Borrow R, Fox A, van Putten J, Zollinger WD, Gerardy-Schahn R, Frosch M. 1996. Capsule phase variation in Neisseria meningitidis serogroup B by slippedstrand mispairing in the polysialyltransferase gene (siaD): correlation with bacterial invasion and the outbreak of meningococcal disease. Mol. Microbiol. 20:1211-1220. http://dx.doi.org/10.1111/j.1365-2958.1996 .tb02641.x. (Pubitemid 26225429)
18. Dolan-Livengood JM, Miller YK, Martin LE, Urwin R, Stephens DS. 2003. Genetic basis for nongroupable Neisseria meningitidis. J. Infect. Dis. 187:1616-1628. http://dx.doi.org/10.1086/374740 (Pubitemid 36578968)
19. Claus H, Maiden MC, Maag R, Frosch M, Vogel U. 2002. Many carried meningococci lack the genes required for capsule synthesis and transport. Microbiology 148:1813-1819 (Pubitemid 34679035)
20. Harrison LH, Trotter CL, Ramsay ME. 2009. Global epidemiology of meningococcal disease. Vaccine 27:B51-63. http://dx.doi.org/10.1016/j.vaccine. 2009.04.063
21. Boisier P, Nicolas P, Djibo S, Taha MK, Jeanne I, Ma?nassara HB, Tenebray B, Kairo KK, Giorgini D, Chanteau S. 2007. Meningococcal meningitis: unprecedented incidence of serogroup X-related cases in 2006 in Niger. Clin. Infect. Dis. 44:657-663. http://dx.doi.org/10.1086/511646
22. Finne J, Leinonen M, Makela PH. 1983. Antigenic similarities between brain components and bacteria causing meningitis. Implications for vaccine development and pathogenesis. Lancet 2:355-357 (Pubitemid 13028927)
23. Martin NG, Snape MD. 2013. A multicomponent serogroup B meningococcal vaccine is licensed for use in Europe: what do we know, and what are yet to learn? Expert Rev. Vaccines 12:837-858. http://dx.doi.org/10.1586/14760584.2013. 814862
24. Toneatto D, Ismaili S, Ypma E, Vienken K, Oster P, Dull P. 2011. The first use of an investigational multicomponent meningococcal serogroup B vaccine (4CMenB) in human. Hum. Vaccines 7:646-653. http://dx.doi.org/10.4161/hv.7.6. 15482
25. Findlow J, Borrow R, Snape MD, Dawson T, Holland A, John TM, Evans A, Telford KL, Ypma E, Toneatto D, Oster P, Miller E, Pollard AJ. 2010. Multicenter, open-label, randomized phase II controlled trial of an investigational recombinant meningococcal serogroup B vaccine with and without outer membrane vesicles, administered in infant. Clin. Infect. Dis. 51:1127-1137. http://dx.doi.org/10.1086/656741
26. Dull PM, McIntosh ED. 2012. Meningococcal vaccine development-from glycoconjugates against MenACWY to proteins against MenB-potential for broad protection against meningococcal disease. Vaccine 30S:B18-B25. http://dx.doi.org/10.1016/j.vaccine.2012.01.062
27. Martin DR, Ruijne N, McCallum L, O?Hallahan J, Oster P. 2006. The VR2 epitope on the PorA P1.7-2,4 protein is the major target for the immune response elicited by the strain-specific group B meningococcal vaccine MeNZB. Clin. Vaccine Immunol. 13:486-491. http://dx.doi.org/10.1128/CVI.13.4.486-491.2006 (Pubitemid 44044834)
28. Masignani V, Comanducci M, Giuliani MM, Bambini S, Adu-Bobie J, Arico B, Brunelli B, Pieri A, Santini L, Savino S, Serruto D, Litt D, Kroll S, Welsch JA, Granoff DM, Rappuoli R, Pizza M. 2003. Vaccination against Neisseria meningitidis using three variants of the lipoprotein GNA1870. J. Exp. Med. 197:789-799. http://dx.doi.org/10.1084/jem.20021911 (Pubitemid 36350873)
29. Serruto D, Spadafina T, Ciucchi L, Lewis LA, Ram S, Tontini M, Santini L, Biolchi A, Seib KL, Giuliani MM, Donnelly JJ, Berti F, Savino S, Scarselli M, Costantino P, Kroll JS, O?Dwyer C, Qiu J, Plaut AG, Moxon R, Rappuoli R, Pizza M, Aricò B. 2010. Neisseria meningitidis GNA2132, a heparin-binding protein that induces protective immunity in humans. Proc. Natl. Acad. Sci. U. S. A. 107:3770-3775. http://dx.doi.org/10.1073/pnas.0915162107
30. Comanducci M, Bambini S, Brunelli B, Adu-Bobie J, Aricò B, Capecchi B, Giuliani MM, Masignani V, Santini L, Savino S, Granoff DM, Caugant DA, Pizza M, Rappuoli R, Mora M. 2002. NadA, a novel vaccine candidate of Neisseria meningitidis. J. Exp. Med. 195:1445-1454. http://dx.doi.org/10.1084/ jem.20020407 (Pubitemid 34602821)
31. Donnelly J, Medini D, Boccadifuoco G, Biolchi A, Ward J, Frasch C, Moxon ER, Stella M, Comanducci M, Bambini S, Muzzi A, Andrews W, Chen J, Santos G, Santini L, Boucher P, Serruto D, Pizza M, Rappuoli R, Giuliani MM. 2010. Qualitative and quantitative assessment of meningococcal antigens to evaluate the potential strain coverage of proteinbased vaccines. Proc. Natl. Acad. Sci. U. S. A. 107:19490-19495. http://dx.doi.org/10.1073/pnas.1013758107
32. Taha MK. 2000. Simultaneous approach for nonculture PCR-based identification and serogroup prediction of Neisseria meningitidis. J. Clin. Microbiol. 38:855-857
33. Lewis C, Clarke SC. 2003. Identification of Neisseria meningitidis serogroups Y and W135 by siaD nucleotide sequence analysis. J. Clin. Microbiol. 41:2697-2699. http://dx.doi.org/10.1128/JCM.41.6.2697-2699.2003 (Pubitemid 36712495)
34. Bennett DE, Mulhall RM, Cafferkey MT. 2004. PCR-based assay for detection of Neisseria meningitidis capsular serogroups 29E, X, and Z. J. Clin. Microbiol. 42:1764-1765. http://dx.doi.org/10.1128/JCM.42.4.1764-1765.2004 (Pubitemid 38500834)
35. Mölling P, Unemo M, Bäckman A, Olcén P. 2000. Genosubtyping by sequencing group A, B and C meningococci; a tool for epidemiological studies of epidemics, clusters and sporadic cases. APMIS 108:509-516. http://dx.doi.org/10.1034/j.1600-0463.2000.d01-90.x.
36. Clarke SC, Diggle MA, Mölling P, Unemo M, Olcén P. 2003. Analysis of PorA variable region 3 in meningococci: implications for vaccine policy? Vaccine 21:2468-2473. http://dx.doi.org/10.1016/S0264-410X(03)00033-1 (Pubitemid 36561338)
37. Sacchi CT, Whitney AM, Reeves MW, Mayer LW, Popovic T. 2002. Sequence diversity of Neisseria meningitidis 16S rRNA genes and use of 16S rRNA gene sequencing as a molecular subtyping tool. J. Clin. Microbiol. 40:4520-4527. http://dx.doi.org/10.1128/JCM.40.12.4520-4527.2002 (Pubitemid 35434940)
38. Lucidarme J, Comanducci M, Findlow J, Gray SJ, Kaczmarski EB, Guiver M, Kugelberg E, Vallely PJ, Oster P, Pizza M, Bambini S, Muzzi A, Tang CM, Borrow R. 2009. Characterization of fHbp, nhba (gna2132), nadA, porA, sequence type (ST), and genomic presence of IS1301 in group B meningococcal ST269 clonal complex isolates from England and Wales. J. Clin. Microbiol. 47:3577-3585. http://dx.doi.org/10.1128/JCM.00936-09
39. Lucidarme J, Newbold LS, Findlow J, Gilchrist S, Gray SJ, Carr AD, Hewitt J, Kaczmarski EB, Borrow R. 2011. Molecular targets in meningococci: efficient routine characterization and optimal outbreak investigation in conjunction with routine surveillance of the meningococcal group B vaccine candidate, fHBP. Clin. Vaccine Immunol. 18:194-202. http://dx.doi.org/10.1128/CVI.00401-10
40. Yazdankhah SP, Caugant DA. 2004. Neisseria meningitidis: an overview of the carriage state. J. Med. Microbiol. 53:821-832. http://dx.doi.org/10.1099/ jmm.0.45529-0 (Pubitemid 39238387)
41. Halperin SA, Bettinger JA, Greenwood B, Harrison LH, Jelfs J, Ladhani SN, McIntyre P, Ramsay ME, Sáfadi MA. 2012. The changing and dynamic epidemiology of meningococcal disease. Vaccine 30:B26-B36. http://dx.doi.org/10. 1016/j.vaccine.2011.12.032
42. Centers for Disease Control and Prevention (CDC). 2013. Infant meningococcal vaccination: Advisory Committee on Immunization Practices (ACIP) recommendations and rationale. MMWR Morb. Mortal. Wkly. Rep. 62:52-54
43. R?nne T, Berthelsen L, Buhl LH, Lind I. 1993. Comparative studies on pharyngeal carriage of Neisseria meningitidis during a localized outbreak of serogroup C meningococcal disease. Scand. J. Infect. Dis. 25:331-339. http://dx.doi.org/10.3109/00365549309008507 (Pubitemid 23212321)
44. Fraser PK, Bailey GK, Abbott JD, Gill JB, Walker DJC. 1973. The meningococcal carrier-rate. Lancet 301:1235-1237. http://dx.doi.org/10.1016/ S0140-6736(73)90541-2
45. MacLennan J, Kafatos G, Neal K, Andrews N, Cameron JC, Roberts R, Evans MR, Cann K, Baxter DN, Maiden MC, Stuart JM, United Kingdom Meningococcal Carriage Group. 2006. Social behavior and meningococcal carriage in British teenagers. Emerg. Infect. Dis. 12:950-957. http://dx.doi.org/10.3201/eid1206. 051297 (Pubitemid 43848417)
46. Aguilera JF, Perrocheau A, Meffre C, Hahné S, W135 Working Group. 2002. Outbreak of serogroup W135 meningococcal disease after the Hajj pilgrimage, Europe, 2000. Emerg. Infect. Dis. 8:761-767. http://dx.doi.org/10. 3201/eid0808.010422
47. Fazio C, Starnino S, Dal Soldà M, Sofia T, Neri A, Mastrantonio P, Stefanelli P. 2010. Neisseria meningitidis serogroup X sequence type 2888, Italy. Emerg. Infect. Dis. 16:359-360. http://dx.doi.org/10.3201/eid1602.091553
48. Germinario C, Tafuri S, Napoli C, Montagna MT, Balducci MT, Fortunato F, Martinelli D, Prato R. 2010. Young-adult carriers of Neisseria meningitidis in Puglia (Italy): will the pattern of circulating meningococci change following the introduction of meningococcal serogroup C conjugate vaccines? Hum. Vaccin. 6:1025-1027. http://dx.doi.org/10.4161/hv.6.12.13145
49. Trotter CL, Maiden MC. 2009. Meningococcal vaccines and herd immunity: lessons learned from serogroup C conjugate vaccination programs. Expert Rev. Vaccines 8:851-861. http://dx.doi.org/10.1586/erv.09.48
50. Soriano-Gabarrò M, Wolter J, Hogea C, Vyse A. 2011. Carriage of Neisseria meningitidis in Europe: a review of studies undertaken in the region. Expert Rev. Anti Infect. Ther. 9:761-774. http://dx.doi.org/10.1586/eri.11.89
51. Trotter CL, Chandra M, Cano R, Larrauri A, Ramsay ME, Brehony C, Jolley KA, Maiden MC, Heuberger S, Frosch M. 2007. A surveillance network for meningococcal disease in Europe. FEMS Microbiol. Rev. 31: 27-36. http://dx.doi.org/10.1111/j.1574-6976.2006.00060.x. (Pubitemid 46058817)
52. Jacobsson S, Hedberg ST, Mölling P, Unemo M, Comanducci M, Rappuoli R, Olcén P. 2009. Prevalence and sequence variations of the genes encoding the five antigens included in the novel 5CVMB vaccine covering group B meningococcal disease. Vaccine 27:1579-1584. http://dx.doi.org/10.1016/j. vaccine.2008.12.052
53. Marsh JW, Shutt KA, Pajon R, Tulenko MM, Liu S, Hollick RA, Kiehlbauch JA, Clark TA, Stephens DS, Arnold KE, Myers RA, Mayer LW, Harrison LH. 2011. Diversity of factor H-binding protein in Neisseria meningitidis carriage isolates. Vaccine 29:6049-6058. http://dx.doi.org/10.1016/j.vaccine.2011.06.025
54. Anderson AS, Hao L, Jiang Q, Harris SL, Jones TR, Perez JL, York L, Eiden J, Jansen KU. 2012. Potential impact of the bivalent rLP2806 vaccine on Neisseria meningitidis carriage and invasive serogroup B disease. Hum. Vaccin. Immunother. 9:471-479. http://dx.doi.org/10.4161/hv.23222
55. Wang X, Cohn A, Comanducci M, Andrew L, Zhao X, MacNeil JR, Schmink S, Muzzi A, Bambini S, Rappuoli R, Pizza M, Murphy E, Hoiseth SK, Jansen KU, Anderson AS, Harrison LH, Clark TA, Messonnier NE, Mayer LW. 2011. Prevalence and genetic diversity of candidate vaccine antigens among invasive Neisseria meningitidis isolates in the United States. Vaccine 29:4739-4744. http://dx.doi.org/10
56. Lucidarme J, Comanducci M, Findlow J, Gray SJ, Kaczmarski EB, Guiver M, Vallely PJ, Oster P, Pizza M, Bambini S, Muzzi A, Borrow R. 2010. Characterization of fHbp, nhba (gna2132), nadA, porA, and sequence type in group B meningococcal case isolates collected in England and Wales during January 2008 and potential coverage of an investigational group B meningococcal vaccine. Clin. Vaccine Immunol. 17:919-929. http://dx.doi.org/10.1128/CVI.00027-10
57. Plikaytis BD, Stella M, Boccadifuoco G, DeTora LM, Agnusdei M, Santini L, Brunelli B, Orlandi L, Simmini I, Giuliani M, Ledroit M, Hong E, Taha MK, Ellie K, Rajam G, Carlone GM, Claus H, Vogel U, Borrow R, Findlow J, Gilchrist S, Stefanelli P, Fazio C, Carannante A, Oksnes J, Fritzs?nn E, Klem AM, Caugant DA, Abad R, Vázquez JA, Rappuoli R, Pizza M, Donnelly JJ, Medini D. 2012. Interlaboratory standardization of the sandwich enzyme-linked immunosorbent assay designed for MATS, a rapid, reproducible method for estimating the strain coverage of investigational vaccines. Clin. Vaccine Immunol. 19:1609-1617. http://dx.doi.org/10.1128/CVI.00202-12
58. Vogel U, Taha MK, Vazquez JA, Findlow J, Claus H, Stefanelli P, Caugant DA, Kriz P, Abad R, Bambini S, Carannante A, Deghmane AE, Fazio C, Frosch M, Frosi G, Gilchrist S, Giuliani MM, Hong E, Ledroit M, Lovaglio PG, Lucidarme J, Musilek M, Muzzi A, Oksnes J, Rigat F, Orlandi L, Stella M, Thompson D, Pizza M, Rappuoli R, Serruto D, Comanducci M, Boccadifuoco G, Donnelly JJ, Medini D, Borrow R. 2013. Predicted strain coverage of a meningococcal multicomponent vaccine (4CMenB) in Europe: a qualitative and quantitative assessment. Lancet Infect. Dis. 13:416-425. http://dx.doi.org/10.1016/S1473-3099(13)70006-9
59. Comanducci M, Bambini S, Caugant DA, Mora M, Brunelli B, Capecchi B, Ciucchi L, Rappuoli R, Pizza M. 2004. NadA diversity and carriage in Neisseria meningitidis. Infect. Immun. 72:4217-4223. http://dx.doi.org/10.1128/IAI.72.7. 4217-4223.2004 (Pubitemid 38821936)
60. Claus H, Jördens MS, Kriz P, Musilek M, Jarva H, Pawlik MC, Meri S, Vogel U. 2012. Capsule null locus meningococci: typing of antigens used in an investigational multicomponent meningococcus serogroup B vaccine. Vaccine 30:155-160. http://dx.doi.org/10.1016/j.vaccine.2011.11.050.