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Neurotropic arboviruses are increasingly recognised as causative agents of neurological disease in Europe but underdiagnosis is still suspected. Capability for accurate diagnosis is a prerequisite for adequate clinical and public health response.


To improve diagnostic capability in EVD-LabNet laboratories, we organised an external quality assessment (EQA) focusing on molecular detection of Toscana (TOSV), Usutu (USUV), West Nile (WNV) and tick-borne encephalitis viruses (TBEV).


Sixty-nine laboratories were invited. The EQA panel included two WNV RNA-positive samples (lineages 1 and 2), two TOSV RNA-positive samples (lineages A and B), one TBEV RNA-positive sample (Western subtype), one USUV RNA-positive sample and four negative samples. The EQA focused on overall capability rather than sensitivity of the used techniques. Only detection of one, clinically relevant, concentration per virus species and lineage was assessed.


The final EQA analysis included 51 laboratories from 35 countries; 44 of these laboratories were from 28 of 31 countries in the European Union/European Economic Area (EU/EEA). USUV diagnostic capability was lowest (28 laboratories in 18 countries), WNV detection capacity was highest (48 laboratories in 32 countries). Twenty-five laboratories were able to test the whole EQA panel, of which only 11 provided completely correct results. The highest scores were observed for WNV and TOSV (92%), followed by TBEV (86%) and USUV (75%).


We observed wide variety in extraction methods and RT-PCR tests, showing a profound absence of standardisation across European laboratories. Overall, the results were not satisfactory; capacity and capability need to be improved in 40 laboratories.


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  1. Glaser C, Bloch K. Encephalitis: why we need to keep pushing the envelope. Clin Infect Dis. 2009;49(12):1848-50.  https://doi.org/10.1086/648420  PMID: 19929385 
  2. Papa A, Kotrotsiou T, Papadopoulou E, Reusken C, GeurtsvanKessel C, Koopmans M. Challenges in laboratory diagnosis of acute viral central nervous system infections in the era of emerging infectious diseases: the syndromic approach. Expert Rev Anti Infect Ther. 2016;14(9):829-36.  https://doi.org/10.1080/14787210.2016.1215914  PMID: 27458693 
  3. Reusken CBEM, Ieven M, Sigfrid L, Eckerle I, Koopmans M. Laboratory preparedness and response with a focus on arboviruses in Europe. Clin Microbiol Infect. 2018;24(3):221-8.  https://doi.org/10.1016/j.cmi.2017.12.010  PMID: 29274465 
  4. Alkan C, Bichaud L, de Lamballerie X, Alten B, Gould EA, Charrel RN. Sandfly-borne phleboviruses of Eurasia and Africa: epidemiology, genetic diversity, geographic range, control measures. Antiviral Res. 2013;100(1):54-74.  https://doi.org/10.1016/j.antiviral.2013.07.005  PMID: 23872312 
  5. Marchi S, Trombetta CM, Kistner O, Montomoli E. Seroprevalence study of Toscana virus and viruses belonging to the Sandfly fever Naples antigenic complex in central and southern Italy. J Infect Public Health. 2017;10(6):866-9.  https://doi.org/10.1016/j.jiph.2017.02.001  PMID: 28237695 
  6. Alkan C, Allal-Ikhlef AB, Alwassouf S, Baklouti A, Piorkowski G, de Lamballerie X, et al. Virus isolation, genetic characterization and seroprevalence of Toscana virus in Algeria. Clin Microbiol Infect. 2015;21(11):1040.e1-9.  https://doi.org/10.1016/j.cmi.2015.07.012  PMID: 26235198 
  7. Sakhria S, Bichaud L, Mensi M, Salez N, Dachraoui K, Thirion L, et al. Co-circulation of Toscana virus and Punique virus in northern Tunisia: a microneutralisation-based seroprevalence study. PLoS Negl Trop Dis. 2013;7(9):e2429.  https://doi.org/10.1371/journal.pntd.0002429  PMID: 24069484 
  8. Charrel RN, Gallian P, Navarro-Mari JM, Nicoletti L, Papa A, Sánchez-Seco MP, et al. Emergence of Toscana virus in Europe. Emerg Infect Dis. 2005;11(11):1657-63.  https://doi.org/10.3201/eid1111.050869  PMID: 16318715 
  9. Gossner CM, Marrama L, Carson M, Allerberger F, Calistri P, Dilaveris D, et al. West Nile virus surveillance in Europe: moving towards an integrated animal-human-vector approach. Euro Surveill. 2017;22(18):30526.  https://doi.org/10.2807/1560-7917.ES.2017.22.18.30526  PMID: 28494844 
  10. Barrett ADT. West Nile in Europe: an increasing public health problem. J Travel Med. 2018;25(1).  https://doi.org/10.1093/jtm/tay096  PMID: 30289526 
  11. Eybpoosh S, Fazlalipour M, Baniasadi V, Pouriayevali MH, Sadeghi F, Ahmadi Vasmehjani A, et al. Epidemiology of West Nile Virus in the Eastern Mediterranean region: A systematic review. PLoS Negl Trop Dis. 2019;13(1):e0007081.  https://doi.org/10.1371/journal.pntd.0007081  PMID: 30695031 
  12. Sambri V, Capobianchi MR, Cavrini F, Charrel R, Donoso-Mantke O, Escadafal C, et al. Diagnosis of West Nile virus human infections: overview and proposal of diagnostic protocols considering the results of external quality assessment studies. Viruses. 2013;5(10):2329-48.  https://doi.org/10.3390/v5102329  PMID: 24072061 
  13. Zannoli S, Sambri V. West Nile virus and Usutu virus co-circulation in Europe: epidemiology and implications. Microorganisms. 2019;7(7):E184.  https://doi.org/10.3390/microorganisms7070184  PMID: 31248051 
  14. European Centre for Disease Prevention and Control (ECDC). Epidemiological situation of tick-borne encephalitis in the European Union and European Free Trade Association countries. Stockholm: ECDC; 2012. Available from: https://ecdc.europa.eu/sites/portal/files/media/en/publications/Publications/TBE-in-EU-EFTA.pdf
  15. European Centre for Disease Prevention and Control (ECDC). Surveillance report: Annual Epidemiological Report for 2015, West Nile fever. Stockholm: ECDC; 2017. Available from: https://ecdc.europa.eu/sites/portal/files/documents/AER_for_2015-West-Nile-fever.pdf
  16. West Nile virus, lineage 1 strain UVE/WNV/2001/FR/DON2001. Marseille: European Virus Archive. [Accessed: 29 Nov 2019]. Available from: https://www.european-virus-archive.com/virus/west-nile-virus-strain-uvewnv2001frdon2001
  17. Toscana virus lineage A, strain UVE/TOSV/2010/TN/T152. Marseille: European Virus Archive. [Accessed: 29 Nov 2019]. Available from: https://www.european-virus-archive.com/virus/toscana-virus-strain-uvetosv2010tn-t152
  18. Toscana virus lineage B, strain UVE/TOSV/2010/FR/4319: EVAg. Marseille: European Virus Archive. [Accessed: 29 Nov 2019]. Available from: https://www.european-virus-archive.com/virus/toscana-virus-strain-uvetosv2010fr4319
  19. Tick-borne encephalitis virus Western subtype, strain UVE/TBEV/1953/CZ/Hypr. Marseille: European Virus Archive. [Accessed: 29 Nov 2019]. Available from: https://www.european-virus-archive.com/virus/tick-borne-encephalitis-virus-western-subtype-strain-uvetbev1953czhypr
  20. Usutu virus, strain Turdus merula Netherlands 2016. Marseille: European Virus Archive. [Accessed: 29 Nov 2019]. Available from: https://www.european-virus-archive.com/virus/usuv-turdus-merula-netherlands-2016
  21. Donoso Mantke O, Aberle SW, Avsic-Zupanc T, Labuda M, Niedrig M. Quality control assessment for the PCR diagnosis of tick-borne encephalitis virus infections. J Clin Virol. 2007;38(1):73-7.  https://doi.org/10.1016/j.jcv.2006.09.001  PMID: 17070730 
  22. Linke S, Mackay WG, Scott C, Wallace P, Niedrig M. Second external quality assessment of the molecular diagnostic of West Nile virus: are there improvements towards the detection of WNV? J Clin Virol. 2011;52(3):257-60.  https://doi.org/10.1016/j.jcv.2011.08.010  PMID: 21893429 
  23. Niedrig M, Linke S, Zeller H, Drosten C. First international proficiency study on West Nile virus molecular detection. Clin Chem. 2006;52(10):1851-4.  https://doi.org/10.1373/clinchem.2005.064451  PMID: 16887901 
  24. Epelboin L, Hausfater P, Schuffenecker I, Riou B, Zeller H, Bricaire F, et al. Meningoencephalitis due to Toscana virus in a French traveler returning from central Italy. J Travel Med. 2008;15(5):361-3.  https://doi.org/10.1111/j.1708-8305.2008.00221.x  PMID: 19006512 
  25. Kay MK, Gibney KB, Riedo FX, Kosoy OL, Lanciotti RS, Lambert AJ. Toscana virus infection in American traveler returning from Sicily, 2009. Emerg Infect Dis. 2010;16(9):1498-500.  https://doi.org/10.3201/eid1609.100505  PMID: 20735948 
  26. Howell BA, Azar MM, Landry ML, Shaw AC. Toscana virus encephalitis in a traveler returning to the United States. J Clin Microbiol. 2015;53(4):1445-7.  https://doi.org/10.1128/JCM.03498-14  PMID: 25673791 
  27. Arden KE, Heney C, Shaban B, Nimmo GR, Nissen MD, Sloots TP, et al. Detection of Toscana virus from an adult traveler returning to Australia with encephalitis. J Med Virol. 2017;89(10):1861-4.  https://doi.org/10.1002/jmv.24839  PMID: 28464308 
  28. Dominati A, Sap L, Vora S. [Fever in a returning traveler from Tuscany]. Rev Med Suisse. 2018;14(592):294-6. PMID: 29384278 
  29. Pérez-Ruiz M, Collao X, Navarro-Marí JM, Tenorio A. Reverse transcription, real-time PCR assay for detection of Toscana virus. J Clin Virol. 2007;39(4):276-81.  https://doi.org/10.1016/j.jcv.2007.05.003  PMID: 17584525 
  30. Weidmann M, Sanchez-Seco MP, Sall AA, Ly PO, Thiongane Y, MM, et al. Rapid detection of important human pathogenic Phleboviruses. J Clin Virol. 2008;41(2):138-42.  https://doi.org/10.1016/j.jcv.2007.10.001  PMID: 18006376 
  31. Brisbarre N, Plumet S, Cotteaux-Lautard C, Emonet SF, Pages F, Leparc-Goffart I. A rapid and specific real time RT-PCR assay for diagnosis of Toscana virus infection. J Clin Virol. 2015;66:107-11.  https://doi.org/10.1016/j.jcv.2015.03.007  PMID: 25866349 
  32. Marlinge M, Crespy L, Zandotti C, Piorkowski G, Kaphan E, Charrel RN, et al. A febrile meningoencephalitis with transient central facial paralysis due to Toscana virus infection, southeastern France, 2014. Euro Surveill. 2014;19(48):20974.  https://doi.org/10.2807/1560-7917.ES2014.19.48.20974  PMID: 25496570 
  33. Nougairede A, Bichaud L, Thiberville SD, Ninove L, Zandotti C, de Lamballerie X, et al. Isolation of Toscana virus from the cerebrospinal fluid of a man with meningitis in Marseille, France, 2010. Vector Borne Zoonotic Dis. 2013;13(9):685-8.  https://doi.org/10.1089/vbz.2013.1316  PMID: 23808972 
  34. Pfleiderer C, Blümel J, Schmidt M, Roth WK, Houfar MK, Eckert J, et al. West Nile virus and blood product safety in Germany. J Med Virol. 2008;80(3):557-63.  https://doi.org/10.1002/jmv.21110  PMID: 18205233 
  35. Lai L, Lee TH, Tobler L, Wen L, Shi P, Alexander J, et al. Relative distribution of West Nile virus RNA in blood compartments: implications for blood donor nucleic acid amplification technology screening. Transfusion. 2012;52(2):447-54.  https://doi.org/10.1111/j.1537-2995.2011.03289.x  PMID: 21827506 
  36. Lanteri MC, Lee TH, Wen L, Kaidarova Z, Bravo MD, Kiely NE, et al. West Nile virus nucleic acid persistence in whole blood months after clearance in plasma: implication for transfusion and transplantation safety. Transfusion. 2014;54(12):3232-41.  https://doi.org/10.1111/trf.12764  PMID: 24965017 
  37. Fischer C, Pedroso C, Mendrone A Jr, Bispo de Filippis AM, Vallinoto ACR, Ribeiro BM, et al. External Quality Assessment for Zika Virus Molecular Diagnostic Testing, Brazil. Emerg Infect Dis. 2018;24(5):888-92.  https://doi.org/10.3201/eid2405.171747  PMID: 29470164 
  38. Dundas N, Leos NK, Mitui M, Revell P, Rogers BB. Comparison of automated nucleic acid extraction methods with manual extraction. J Mol Diagn. 2008;10(4):311-6.  https://doi.org/10.2353/jmoldx.2008.070149  PMID: 18556770 
  39. Knepp JH, Geahr MA, Forman MS, Valsamakis A. Comparison of automated and manual nucleic acid extraction methods for detection of enterovirus RNA. J Clin Microbiol. 2003;41(8):3532-6.  https://doi.org/10.1128/JCM.41.8.3532-3536.2003  PMID: 12904351 
  40. Witlox KJ, Nguyen TN, Bruggink LD, Catton MG, Marshall JA. A comparative evaluation of the sensitivity of two automated and two manual nucleic acid extraction methods for the detection of norovirus by RT-PCR. J Virol Methods. 2008;150(1-2):70-2.  https://doi.org/10.1016/j.jviromet.2008.02.010  PMID: 18400313 
  41. Wozniak A, Geoffroy E, Miranda C, Castillo C, Sanhueza F, García P. Comparison of manual and automated nucleic acid extraction methods from clinical specimens for microbial diagnosis purposes. Diagn Microbiol Infect Dis. 2016;86(3):268-9.  https://doi.org/10.1016/j.diagmicrobio.2016.07.008  PMID: 27543377 
  42. Yang JL, Wang MS, Cheng AC, Pan KC, Li CF, Deng SX. A simple and rapid method for extracting bacterial DNA from intestinal microflora for ERIC-PCR detection. World J Gastroenterol. 2008;14(18):2872-6.  https://doi.org/10.3748/wjg.14.2872  PMID: 18473413 
  43. Sánchez-Seco MP, Echevarría JM, Hernández L, Estévez D, Navarro-Marí JM, Tenorio A. Detection and identification of Toscana and other phleboviruses by RT-nested-PCR assays with degenerated primers. J Med Virol. 2003;71(1):140-9.  https://doi.org/10.1002/jmv.10465  PMID: 12858420 
  44. Lambert AJ, Lanciotti RS. Consensus amplification and novel multiplex sequencing method for S segment species identification of 47 viruses of the Orthobunyavirus, Phlebovirus, and Nairovirus genera of the family Bunyaviridae. J Clin Microbiol. 2009;47(8):2398-404.  https://doi.org/10.1128/JCM.00182-09  PMID: 19535518 
  45. Linke S, Ellerbrok H, Niedrig M, Nitsche A, Pauli G. Detection of West Nile virus lineages 1 and 2 by real-time PCR. J Virol Methods. 2007;146(1-2):355-8.  https://doi.org/10.1016/j.jviromet.2007.05.021  PMID: 17604132 
  46. Eiden M, Vina-Rodriguez A, Hoffmann B, Ziegler U, Groschup MH. Two new real-time quantitative reverse transcription polymerase chain reaction assays with unique target sites for the specific and sensitive detection of lineages 1 and 2 West Nile virus strains. J Vet Diagn Invest. 2010;22(5):748-53.  https://doi.org/10.1177/104063871002200515  PMID: 20807934 
  47. Tang Y, Anne Hapip C, Liu B, Fang CT. Highly sensitive TaqMan RT-PCR assay for detection and quantification of both lineages of West Nile virus RNA. J Clin Virol. 2006;36(3):177-82.  https://doi.org/10.1016/j.jcv.2006.02.008  PMID: 16675298 
  48. Lanciotti RS, Kerst AJ, Nasci RS, Godsey MS, Mitchell CJ, Savage HM, et al. Rapid detection of west nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a TaqMan reverse transcriptase-PCR assay. J Clin Microbiol. 2000;38(11):4066-71. PMID: 11060069 
  49. Shi P, Kauffman E, Ren P, Felton A, Tai J, Dupuis A, et al. High-throughput detection of West Nile virus RNA. J Clin Microbiol. 2001;39(4):1264-71.
  50. Chaskopoulou A, Dovas C, Chaintoutis S, Bouzalas I, Ara G, Papanastassopoulou M. Evidence of enzootic circulation of West Nile virus (Nea Santa-Greece-2010, lineage 2), Greece, May to July 2011. Euro Surveill. 2011;16(31):19933. PMID: 21871217 
  51. Scaramozzino N, Crance JM, Jouan A, DeBriel DA, Stoll F, Garin D. Comparison of flavivirus universal primer pairs and development of a rapid, highly sensitive heminested reverse transcription-PCR assay for detection of flaviviruses targeted to a conserved region of the NS5 gene sequences. J Clin Microbiol. 2001;39(5):1922-7.  https://doi.org/10.1128/JCM.39.5.1922-1927.2001  PMID: 11326014 
  52. Sánchez-Seco MP, Rosario D, Domingo C, Hernández L, Valdés K, Guzmán MG, et al. Generic RT-nested-PCR for detection of flaviviruses using degenerated primers and internal control followed by sequencing for specific identification. J Virol Methods. 2005;126(1-2):101-9.  https://doi.org/10.1016/j.jviromet.2005.01.025  PMID: 15847925 
  53. Moureau G, Temmam S, Gonzalez JP, Charrel RN, Grard G, de Lamballerie X. A real-time RT-PCR method for the universal detection and identification of flaviviruses. Vector Borne Zoonotic Dis. 2007;7(4):467-77.  https://doi.org/10.1089/vbz.2007.0206  PMID: 18020965 
  54. Patel P, Landt O, Kaiser M, Faye O, Koppe T, Lass U, et al. Development of one-step quantitative reverse transcription PCR for the rapid detection of flaviviruses. Virol J. 2013;10(1):58.  https://doi.org/10.1186/1743-422X-10-58  PMID: 23410000 
  55. Briese T, Jia XY, Huang C, Grady LJ, Lipkin WI. Identification of a Kunjin/West Nile-like flavivirus in brains of patients with New York encephalitis. Lancet. 1999;354(9186):1261-2.  https://doi.org/10.1016/S0140-6736(99)04576-6  PMID: 10520637 
  56. Vina-Rodriguez A, Sachse K, Ziegler U, Chaintoutis SC, Keller M, Groschup MH, et al. A Novel Pan-Flavivirus Detection and Identification Assay Based on RT-qPCR and Microarray. BioMed Res Int. 2017;2017:4248756.  https://doi.org/10.1155/2017/4248756  PMID: 28626758 
  57. Vázquez A, Sánchez-Seco MP, Palacios G, Molero F, Reyes N, Ruiz S, et al. Novel flaviviruses detected in different species of mosquitoes in Spain. Vector Borne Zoonotic Dis. 2012;12(3):223-9.  https://doi.org/10.1089/vbz.2011.0687  PMID: 22022811 
  58. Nikolay B, Weidmann M, Dupressoir A, Faye O, Boye CS, Diallo M, et al. Development of a Usutu virus specific real-time reverse transcription PCR assay based on sequenced strains from Africa and Europe. J Virol Methods. 2014;197:51-4.  https://doi.org/10.1016/j.jviromet.2013.08.039  PMID: 24036076 
  59. Cavrini F, Della Pepa ME, Gaibani P, Pierro AM, Rossini G, Landini MP, et al. A rapid and specific real-time RT-PCR assay to identify Usutu virus in human plasma, serum, and cerebrospinal fluid. J Clin Virol. 2011;50(3):221-3.  https://doi.org/10.1016/j.jcv.2010.11.008  PMID: 21156352 
  60. Jöst H, Bialonski A, Maus D, Sambri V, Eiden M, Groschup MH, et al. Isolation of Usutu virus in Germany. Am J Trop Med Hyg. 2011;85(3):551-3.  https://doi.org/10.4269/ajtmh.2011.11-0248  PMID: 21896821 
  61. Weissenböck H, Bakonyi T, Rossi G, Mani P, Nowotny N. Usutu virus, Italy, 1996. Emerg Infect Dis. 2013;19(2):274-7.  https://doi.org/10.3201/eid1902.121191  PMID: 23347844 
  62. Del Amo J, Sotelo E, Fernández-Pinero J, Gallardo C, Llorente F, Agüero M, et al. A novel quantitative multiplex real-time RT-PCR for the simultaneous detection and differentiation of West Nile virus lineages 1 and 2, and of Usutu virus. J Virol Methods. 2013;189(2):321-7.  https://doi.org/10.1016/j.jviromet.2013.02.019  PMID: 23499258 
  63. Schwaiger M, Cassinotti P. Development of a quantitative real-time RT-PCR assay with internal control for the laboratory detection of tick borne encephalitis virus (TBEV) RNA. J Clin Virol. 2003;27(2):136-45.  https://doi.org/10.1016/S1386-6532(02)00168-3  PMID: 12829035 
  64. Achazi K, Růžek D, Donoso-Mantke O, Schlegel M, Ali HS, Wenk M, et al. Rodents as sentinels for the prevalence of tick-borne encephalitis virus. Vector Borne Zoonotic Dis. 2011;11(6):641-7.  https://doi.org/10.1089/vbz.2010.0236  PMID: 21548766 
  65. Puchhammer-Stöckl E, Kunz C, Mandl CW, Heinz FX. Identification of tick-borne encephalitis virus ribonucleic acid in tick suspensions and in clinical specimens by a reverse transcription-nested polymerase chain reaction assay. Clin Diagn Virol. 1995;4(4):321-6.  https://doi.org/10.1016/0928-0197(95)00022-4  PMID: 15566853 
  66. Bagó Z, Bauder B, Kolodziejek J, Nowotny N, Weissenböck H. Tickborne encephalitis in a mouflon (Ovis ammon musimon). Vet Rec. 2002;150(7):218-20.  https://doi.org/10.1136/vr.150.7.218  PMID: 11878442 
  67. Schrader C, Süss J. A nested RT-PCR for the detection of tick-borne encephalitis virus (TBEV) in ticks in natural foci. Zentralbl Bakteriol. 1999;289(3):319-28.  https://doi.org/10.1016/S0934-8840(99)80069-3  PMID: 10467662 
  68. Briggs BJ, Atkinson B, Czechowski DM, Larsen PA, Meeks HN, Carrera JP, et al. Tick-borne encephalitis virus, Kyrgyzstan. Emerg Infect Dis. 2011;17(5):876-9.  https://doi.org/10.3201/eid1705.101183  PMID: 21529400 
  69. Gäumann R, Mühlemann K, Strasser M, Beuret C. High-throughput procedure for tick surveys of tick-borne encephalitis virus and its application in a national surveillance study in Switzerland. Appl Environ Microbiol. 2010;76(13):4241-9.
  70. Klaus C, Hoffmann B, Hering U, Mielke B, Sachse K, Beer M, et al. Tick-borne encephalitis (TBE) virus prevalence and virus genome characterization in field-collected ticks (Ixodes ricinus) from risk, non-risk and former risk areas of TBE, and in ticks removed from humans in Germany. Clin Microbiol Infect. 2010;16(3):238-44.  https://doi.org/10.1111/j.1469-0691.2009.02764.x  PMID: 19906276 
  71. Brinkley C, Nolskog P, Golovljova I, Lundkvist Å, Bergström T. (Tick-borne encephalitis virus natural foci emerge in western Sweden. Int J Med Microbiol. 2008;298(Suppl. 1):73-80.  https://doi.org/10.1016/j.ijmm.2007.12.005 
  72. Andreassen A, Jore S, Cuber P, Dudman S, Tengs T, Isaksen K, et al. Prevalence of tick borne encephalitis virus in tick nymphs in relation to climatic factors on the southern coast of Norway. Parasit Vectors. 2012;5(1):177.  https://doi.org/10.1186/1756-3305-5-177  PMID: 22913287 
  73. Skarpaas T, Golovljova I, Vene S, Ljøstad U, Sjursen H, Plyusnin A, et al. Tickborne encephalitis virus, Norway and Denmark. Emerg Infect Dis. 2006;12(7):1136-8.  https://doi.org/10.3201/eid1207.051567  PMID: 16836835 

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