1887

Chapter 43 : Sequence-Based Fungal Identification and Classification

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.

Preview this chapter:
Zoom in
Zoomout

Sequence-Based Fungal Identification and Classification, Page 1 of 2

| /docserver/preview/fulltext/10.1128/9781555816834/9781555814977_Chap43-1.gif /docserver/preview/fulltext/10.1128/9781555816834/9781555814977_Chap43-2.gif

Abstract:

Identification and classification of fungi from clinical samples are important for antifungal susceptibility testing and epidemiological investigation. Sequence-based molecular techniques are increasingly used in the identification and taxonomy characterization of fungal infections. Phenotypic characteristics used for the recognition and classification of fungi are those that are either easily observed or measured, or a combination of both. An ultimate goal of fungal classification is to draw inferred phylogenetic relationships. In brief, it is sequencing by synthesis, and it is based on the synthesis of cDNA from PCR amplicons. DNA targets that have been used for fungal identification and classification include rDNA, cytochrome b, β-tubulin, calmodulin, enolase, chitin synthase, heat shock protein, and other housekeeping and functional genes. The increased use of gene sequences to recognize different clades within traditional medically important species emphasizes the importance of using sequence data in contrast to species-specific probes to identify particular fungal species. Clinical laboratories are under increasing pressure to provide rapid identification and classification of fungal infections due to the growing number of immunocompromised patients that are susceptible to fungal infection, and the availability of targeted antifungal agents. Sequencing and database comparison of PCR amplicons coupled with phylogenetic methods provide a robust strategy for species recognition, especially for uncommon and emerging pathogenic fungi.

Citation: Dong J, Loeffelholz M, McGinnis M. 2011. Sequence-Based Fungal Identification and Classification, p 669-676. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch43

Key Concept Ranking

Cryptococcus neoformans var. neoformans
0.5113493
Fungal Infections
0.42225477
Fungal Classification
0.4217566
Antifungal Agents
0.4213558
0.5113493
Highlighted Text: Show | Hide
Loading full text...

Full text loading...

Figures

Image of FIGURE 1
FIGURE 1

Organization of fungal rDNA. ETS, external transcribed spacer; SSU, small subunit; LSU, large subunit.

Citation: Dong J, Loeffelholz M, McGinnis M. 2011. Sequence-Based Fungal Identification and Classification, p 669-676. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch43
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 2
FIGURE 2

26S rDNA ESs.

Citation: Dong J, Loeffelholz M, McGinnis M. 2011. Sequence-Based Fungal Identification and Classification, p 669-676. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch43
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 3
FIGURE 3

Phylogenetic tree for isolates using large-subunit (26S) rDNA sequence data. These groupings correlate with six proposed phylogenetic clades. Numbers in the tree are UTMB culture collection numbers.

Citation: Dong J, Loeffelholz M, McGinnis M. 2011. Sequence-Based Fungal Identification and Classification, p 669-676. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch43
Permissions and Reprints Request Permissions
Download as Powerpoint

References

/content/book/10.1128/9781555816834.ch43
1. Alcazar-Fuoli, L.,, E. Mellado,, A. Alastruey-Izquierdo,, M. Cuenca-Estrella, and, J. L. Rodriguez-Tudela. 2008. Aspergillus section Fumigati: antifungal susceptibility patterns and sequence-based identification. Antimicrob. Agents Chemother. 52:12441251.
2. Aroca, A.,, and R. Raposo. 2007. PCR-based strategy to detect and identify species of Phaeoacremonium causing grapevine diseases. Appl. Environ. Microbiol. 73:29112918.
3. Baddley, J. W.,, L. Mostert,, R. C. Summerbell, and, S. A. Moser. 2006. Phaeoacremonium parasiticum infections confirmed by beta-tubulin sequence analysis of case isolates. J. Clin. Microbiol. 44:22072211.
4. Bain, J. M.,, A. Tavanti,, A. D. Davidson,, M. D. Jacobsen,, D. Shaw,, N. A. Gow, and, F. C. Odds. 2007. Multilocus sequence typing of the pathogenic fungus Aspergillus fumigatus. J. Clin. Microbiol. 45:14691477.
5. Balajee, S. A.,, J. Gribskov,, M. Brandt,, J. Ito,, A. Fothergill, and, K. A. Marr. 2005. Mistaken identity: Neosartorya pseudofischeri and its anamorph masquerading as Aspergillus fumigatus. J. Clin. Microbiol. 43:59965999.
6. Balajee, S. A.,, J. Houbraken,, P. E. Verweij,, S. B. Hong,, T. Yaghuchi,, J. Varga, and, R. A. Samson. 2007. Aspergillus species identification in the clinical setting. Stud. Mycol. 59:3946.
7. Balajee, S. A.,, M. D. Lindsley,, N. Iqbal,, J. Ito,, P. G. Pappas, and, M. E. Brandt. 2007. Nonsporulating clinical isolate identified as Petromyces alliaceus (anamorph Aspergillus alliaceus) by morphological and sequence-based methods. J. Clin. Microbiol. 45:27012703.
8. Balajee, S. A.,, D. Nickle,, J. Varga, and, K. A. Marr. 2006. Molecular studies reveal frequent misidentification of Aspergillus fumigatus by morphotyping. Eukaryot. Cell 5:17051712.
9. Balajee, S. A.,, L. Sigler, and, M. E. Brandt. 2007. DNA and the classical way: identification of medically important molds in the 21st century. Med. Mycol. 45:475490.
10. Balajee, S. A.,, S. T. Tay,, B. A. Lasker,, S. F. Hurst, and, A. P. Rooney. 2007. Characterization of a novel gene for strain typing reveals substructuring of Aspergillus fumigatus across North America. Eukaryot. Cell 6:13921399.
11. Bar-Meir, M.,, D. A. Sutton,, B. Wickes,, C. P. Kurtzman,, S. Goldman, and, X. Zheng. 2006. Catheter-related fungemia due to Candida thermophila. J. Clin. Microbiol. 44:30353036.
12. Bishop, J. A.,, N. Chase,, S. S. Magill,, C. P. Kurtzman,, M. J. Fiandaca, and, W. G. Merz. 2008. Candida bracarensis detected among isolates of Candida glabrata by peptide nucleic acid fluorescence in situ hybridization: susceptibility data and documentation of presumed infection. J. Clin. Microbiol. 46:443446.
13. Biswas, S. K.,, L. Wang,, K. Yokoyama, and, K. Nishimura. 2005. Molecular phylogenetics of the genus Trichosporon inferred from mitochondrial cytochrome b gene sequences. J. Clin. Microbiol. 43:51715178.
14. Biswas, S. K.,, K. Yokoyama,, K. Nishimura, and, M. Miyaji. 2001. Molecular phylogenetics of the genus Rhodotorula and related basidiomycetous yeasts inferred from the mitochondrial cytochrome b gene. Int. J. Syst. Evol. Microbiol. 51:11911199.
15. Borman, A. M.,, C. J. Linton,, S. J. Miles, and, E. M. Johnson. 2008. Molecular identification of pathogenic fungi. J. Antimicrob. Chemother. 61(Suppl.1):i7i12.
16. Borman, A. M.,, R. Petch,, C. J. Linton,, M. D. Palmer,, P. D. Bridge, and, E. M. Johnson. 2008. Candida nivariensis, an emerging pathogenic fungus with multidrug resistance to antifungal agents. J. Clin. Microbiol. 46:933938.
17. Boyanton, B. L., Jr.,, R. A. Luna,, L. R. Fasciano,, K. G. Menne, and, J. Versalovic. 2008. DNA pyrosequencing-based identification of pathogenic Candida species by using the internal transcribed spacer 2 region. Arch. Pathol. Lab. Med. 132:667674.
18. Brandt, M. E.,, C. A. Kauffman,, P. G. Pappas,, N. Iqbal,, B. A. Arthington-Skaggs,, W. Lee-Yang, and, M. T. Smith. 2004. Fungemia caused by Zygoascus hellenicus in an allogeneic stem cell transplant recipient. J. Clin. Microbiol. 42:33633365.
19. Carrero, L. L.,, G. Nino-Vega,, M. M. Teixeira,, M. J. Carvalho,, C. M. Soares,, M. Pereira,, R. S. Jesuino,, J. G. McEwen,, L. Mendoza,, J. W. Taylor,, M. S. Felipe, and, G. San-Blas. 2008. New Paracoccidioides brasiliensis isolate reveals unexpected genomic variability in this human pathogen. Fungal Genet. Biol. 45:605612.
20. Carter, D. A.,, J. W. Taylor,, B. Dechairo,, A. Burt,, G. L. Koenig, and, T. J. White. 2001. Amplified single-nucleotide polymorphisms and a (GA)(n) microsatellite marker reveal genetic differentiation between populations of Histoplasma capsulatum from the Americas. Fungal Genet. Biol. 34:3748.
21. Chang, A.,, D. Neofytos, and, D. Horn. 2008. Candidemia in the 21st century. Future Microbiol. 3:463472.
22. Chowdhary, A.,, J. Guarro,, H. S. Randhawa,, J. Gene,, J. Cano,, R. K. Jain,, S. Kumar, and, G. Khanna. 2008. A rare case of chromoblastomycosis in a renal transplant recipient caused by a non-sporulating species of Rhytidhysteron. Med. Mycol. 46:163166.
23. Ciardo, D. E.,, G. Schar,, E. C. Bottger,, M. Altwegg, and, P. P. Bosshard. 2006. Internal transcribed spacer sequencing versus biochemical profiling for identification of medically important yeasts. J. Clin. Microbiol. 44:7784.
24. Clinical and Laboratory Standards Institute. 2008. Interpretive Criteria for Identification of Bacteria and Fungi by DNA Target Sequencing; Approved Guideline. CLSI document MM18-A. Clinical and Laboratory Standards Institute, Wayne, PA.
25. de Valk, H. A.,, C. H. Klaassen, and, J. F. Meis. 2008. Molecular typing of Aspergillus species. Mycoses 51:463476.
26. Diaz, M. R.,, T. Boekhout,, T. Kiesling, and, J. W. Fell. 2005. Comparative analysis of the intergenic spacer regions and population structure of the species complex of the pathogenic yeast Cryptococcus neoformans. FEMS Yeast Res. 5:11291140.
27. Enache-Angoulvant, A.,, J. Chandenier,, F. Symoens,, P. Lacube,, J. Bolognini,, C. Douchet,, J. L. Poirot, and, C. Hennequin. 2007. Molecular identification of Cryptococcus neoformans serotypes. J. Clin. Microbiol. 45:12611265.
28. Fisher, M. C.,, G. L. Koenig,, T. J. White, and, J. W. Taylor. 2002. Molecular and phenotype description of Coccidioides posadasii sp. nov., previously recognized as the non-Californian population of Coccidioides immitis. Mycologia 94:7384.
29. Frealle, E.,, M. Rodrigue,, N. Gantois,, C. M. Aliouat,, E. Delaporte,, D. Camus,, E. Dei-Cas,, C. Kauffmann-Lacroix,, J. Guillot, and, L. Delhaes. 2007. Phylogenetic analysis of Trichophyton mentagrophytes human and animal isolates based on MnSOD and ITS sequence comparison. Microbiology 153:34663477.
30. Gilgado, F.,, J. Cano,, J. Gene, and, J. Guarro. 2005. Molecular phylogeny of the Pseudallescheria boydii species complex: proposal of two new species. J. Clin. Microbiol. 43:49304942.
31. Guarro, J.,, J. Gené, and, A. M. Stchigel. 1999. Developments in fungal taxonomy. Clin. Microbiol. Rev. 12:454500.
32. Hall, L.,, K. A. Doerr,, S. L. Wohlfiel, and, G. D. Roberts. 2003. Evaluation of the MicroSeq system for identification of mycobacteria by 16S ribosomal DNA sequencing and its integration into a routine clinical mycobacteriology laboratory. J. Clin. Microbiol. 41:14471453.
33. Hall, L.,, S. Wohlfiel, and, G. D. Roberts. 2003. Experience with the MicroSeq D2 large-subunit ribosomal DNA sequencing kit for identification of commonly encountered, clinically important yeast species. J. Clin. Microbiol. 41:50995102.
34. Hall, L.,, S. Wohlfiel, and, G. D. Roberts. 2004. Experience with the MicroSeq D2 large-subunit ribosomal DNA sequencing kit for identification of filamentous fungi encountered in the clinical laboratory. J. Clin. Microbiol. 42:622626.
35. Hassouna, N.,, B. Michot, and, J. P. Bachellerie. 1984. The complete nucleotide sequence of mouse 28S rRNA gene. Implications for the process of size increase of the large subunit rRNA in higher eukaryotes. Nucleic Acids Res. 12:35633583.
36. Hennequin, C.,, E. Abachin,, F. Symoens,, V. Lavarde,, G. Reboux,, N. Nolard, and, P. Berche. 1999. Identification of Fusarium species involved in human infections by 28S rRNA gene sequencing. J. Clin. Microbiol. 37:35863589.
37. Herr, R. A.,, E. J. Tarcha,, P. R. Taborda,, J. W. Taylor,, L. Ajello, and, L. Mendoza. 2001. Phylogenetic analysis of Lacazia loboi places this previously uncharacterized pathogen within the dimorphic Onygenales. J. Clin. Microbiol. 39:309314.
38. Hong, S. B.,, S. J. Go,, H. D. Shin,, J. C. Frisvad, and, R. A. Samson. 2005. Polyphasic taxonomy of Aspergillus fumigatus and related species. Mycologia 97:13161329.
39. Kasuga, T.,, J. W. Taylor, and, T. J. White. 1999. Phylogenetic relationships of varieties and geographical groups of the human pathogenic fungus Histoplasma capsulatum Darling. J. Clin. Microbiol. 37:653663.
40. Kasuga, T.,, T. J. White,, G. Koenig,, J. McEwen,, A. Restrepo,, E. Castaneda,, C. Da Silva Lacaz,, E. M. Heins-Vaccari,, R. S. De Freitas,, R. M. Zancope-Oliveira,, Z. Qin,, R. Negroni,, D. A. Carter,, Y. Mikami,, M. Tamura,, M. L. Taylor,, G. F. Miller,, N. Poonwan, and, J. W. Taylor. 2003. Phylogeography of the fungal pathogen Histoplasma capsulatum. Mol. Ecol. 12:33833401.
41. Knutsen, A. K.,, M. Torp, and, A. Holst-Jensen. 2004. Phylogenetic analyses of the Fusarium poae, Fusarium sporotrichioides and Fusarium langsethiae species complex based on partial sequences of the translation elongation factor-1 alpha gene. Int. J. Food Microbiol. 95:287295.
42. Kurtzman, C. P.,, and C. J. Robnett. 1997. Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5′ end of the large-subunit (26S) ribosomal DNA gene. J. Clin. Microbiol. 35:12161223.
43. Kuzoff, R. K.,, J. A. Sweere,, D. E. Soltis,, P. S. Soltis, and, E. A. Zimmer. 1998. The phylogenetic potential of entire 26S rDNA sequences in plants. Mol. Biol. Evol. 15:251263.
44. Leaw, S. N.,, H. C. Chang,, H. F. Sun,, R. Barton,, J. P. Bouchara, and, T. C. Chang. 2006. Identification of medically important yeast species by sequence analysis of the internal transcribed spacer regions. J. Clin. Microbiol. 44:693699.
45. Lee, J. S.,, J. H. Shin,, M. N. Kim,, S. I. Jung,, K. H. Park,, D. Cho,, S. J. Kee,, M. G. Shin,, S. P. Suh, and, D. W. Ryang. 2007. Kodamaea ohmeri isolates from patients in a university hospital: identification, antifungal susceptibility, and pulsed-field gel electrophoresis analysis. J. Clin. Microbiol. 45:10051010.
46. Li, J.,, Y. C. Xu, and, F. Y. Bai. 2006. Candida pseudorugosa sp. nov., a novel yeast species from sputum. J. Clin. Microbiol. 44:44864490.
47. Linton, C. J.,, A. M. Borman,, G. Cheung,, A. D. Holmes,, A. Szekely,, M. D. Palmer,, P. D. Bridge,, C. K. Campbell, and, E. M. Johnson. 2007. Molecular identification of unusual pathogenic yeast isolates by large ribosomal subunit gene sequencing: 2 years of experience at the United Kingdom Mycology Reference Laboratory. J. Clin. Microbiol. 45:11521158.
48. Lutzoni, F.,, and M. Pagel. 1997. Accelerated evolution as a consequence of transitions to mutualism. Proc. Natl. Acad. Sci. USA 94:1142211427.
49. Marimon, R.,, J. Cano,, J. Gene,, D. A. Sutton,, M. Kawasaki, and, J. Guarro. 2007. Sporothrix brasiliensis, S. globosa, and S. mexicana, three new Sporothrix species of clinical interest. J. Clin. Microbiol. 45:31983206.
50. Marimon, R.,, C. Serena,, J. Gene,, J. Cano, and, J. Guarro. 2008. In vitro antifungal susceptibilities of five species of Sporothrix. Antimicrob. Agents Chemother. 52:732734.
51. McGinnis, M. R.,, and L. Pasarell. 1998. In vitro testing of susceptibilities of filamentous ascomycetes to voriconazole, itraconazole, and amphotericin B, with consideration of phylogenetic implications. J. Clin. Microbiol. 36:23532355.
52. McGinnis, M. R.,, M. B. Smith, and, E. Hinson. 2006. Use of the Coccidioides posadasii Δchs5 strain for quality control in the ACCUPROBE culture identification test for Coccidioides immitis. J. Clin. Microbiol. 44:42504251.
53. Nucci, M.,, and J. R. Perfect. 2008. When primary antifungal therapy fails. Clin. Infect. Dis. 46:14261433.
54. O’Donnell, K.,, H. C. Kistler,, E. Cigelnik, and, R. C. Ploetz. 1998. Multiple evolutionary origins of the fungus causing Panama disease of banana: concordant evidence from nuclear and mitochondrial gene genealogies. Proc. Natl. Acad. Sci. USA 95:20442049.
55. Peterson, S. W.,, and L. Sigler. 1998. Molecular genetic variation in Emmonsia crescens and Emmonsia parva, etiologic agents of adiaspiromycosis, and their phylogenetic relationship to Blastomyces dermatitidis (Ajellomyces dermatitidis) and other systemic fungal pathogens. J. Clin. Microbiol. 36:29182925.
56. Petti, C. A. 2007. Detection and identification of microorganisms by gene amplification and sequencing. Clin. Infect. Dis. 44:11081114.
57. Phillips, A. J. 2006. Homology assessment and molecular sequence alignment. J. Biomed. Inform. 39:1833.
58. Pounder, J. I.,, K. E. Simmon,, C. A. Barton,, S. L. Hohmann,, M. E. Brandt, and, C. A. Petti. 2007. Discovering potential pathogens among fungi identified as nonsporulating molds. J. Clin. Microbiol. 45:568571.
59. Poutanen, S. M.,, D. E. Low,, B. Henry,, S. Finkelstein,, D. Rose,, K. Green,, R. Tellier,, R. Draker,, D. Adachi,, M. Ayers,, A. K. Chan,, D. M. Skowronski,, I. Salit,, A. E. Simor,, A. S. Slutsky,, P. W. Doyle,, M. Krajden,, M. Petric,, R. C. Brunham, and, A. J. McGeer. 2003. Identification of severe acute respiratory syndrome in Canada. N. Engl. J. Med. 348:19952005.
60. Pujol, C.,, K. J. Daniels,, S. R. Lockhart,, T. Srikantha,, J. B. Radke,, J. Geiger, and, D. R. Soll. 2004. The closely related species Candida albicans and Candida dubliniensis can mate. Eukaryot. Cell 3:10151027.
61. Putignani, L.,, M. G. Paglia,, E. Bordi,, E. Nebuloso,, L. P. Pucillo, and, P. Visca. 2008. Identification of clinically relevant yeast species by DNA sequence analysis of the D2 variable region of the 25-28S rRNA gene. Mycoses 51:209227.
62. Qian, J.,, M. Dolled-Filhart,, J. Lin,, H. Yu, and, M. Gerstein. 2001. Beyond synexpression relationships: local clustering of time-shifted and inverted gene expression profiles identifies new, biologically relevant interactions. J. Mol. Biol. 314:10531066.
63. Rainer, J.,, and G. S. De Hoog. 2006. Molecular taxonomy and ecology of Pseudallescheria, Petriella and Scedosporium prolificans (Microascaceae) containing opportunistic agents on humans. Mycol. Res. 110:151160.
64. Rozynek, P.,, S. Gilges,, T. Bruning, and, M. Wilhelm. 2004. Quality test of the MicroSeq D2 LSU Fungal Sequencing Kit for the identification of fungi. Int. J. Hyg. Environ. Health 207:297299.
65. Sugita, T.,, A. Nishikawa,, R. Ikeda, and, T. Shinoda. 1999. Identification of medically relevant Trichosporon species based on sequences of internal transcribed spacer regions and construction of a database for Trichosporon identification. J. Clin. Microbiol. 37:19851993.
66. Summerbell, R. C.,, C. A. Levesque,, K. A. Seifert,, M. Bovers,, J. W. Fell,, M. R. Diaz,, T. Boekhout,, G. S. de Hoog,, J. Stalpers, and, P. W. Crous. 2005. Microcoding: the second step in DNA barcoding. Philos. Trans. R. Soc. Lond. B 360:18971903.
67. Tavanti, A.,, A. D. Davidson,, N. A. Gow,, M. C. Maiden, and, F. C. Odds. 2005. Candida orthopsilosis and Candida metapsilosis spp. nov. to replace Candida parapsilosis groups II and III. J. Clin. Microbiol. 43:284292.
68. Valenza, G.,, R. Valenza,, J. Brederlau,, M. Frosch, and, O. Kurzai. 2006. Identification of Candida fabianii as a cause of lethal septicaemia. Mycoses 49:331334.
69. Valerio, A.,, E. Tronconi,, F. Mazza,, G. Fantoni,, C. Atzori,, F. Tartarone,, P. Duca, and, A. Cargnel. 2007. Genotyping of Pneumocystis jiroveci pneumonia in Italian AIDS patients. Clinical outcome is influenced by dihydropteroate synthase and not by internal transcribed spacer genotype. J. Acquir. Immune Defic. Syndr. 45:521528.
70. Wang, L.,, K. Yokoyama,, H. Takahasi,, N. Kase,, Y. Hanya,, K. Yashiro,, M. Miyaji, and, K. Nishimura. 2001. Identification of species in Aspergillus section Flavi based on sequencing of the mitochondrial cytochrome b gene. Int. J. Food Microbiol. 71:7586.
71. Yamada, Y.,, K. Makimura,, K. Uchida,, H. Yamaguchi, and, M. Osumi. 2004. Phylogenetic relationships among medically important yeasts based on sequences of mitochondrial large subunit ribosomal RNA gene. Mycoses 47:2428.
72. Yong, P. V.,, P. P. Chong,, L. Y. Lau,, R. S. Yeoh, and, F. Jamal. 2008. Molecular identification of Candida orthopsilosis isolated from blood culture. Mycopathologia 165:8187.

Tables

Generic image for table
TABLE 1

Target genes used for sequence identification of fungi

Citation: Dong J, Loeffelholz M, McGinnis M. 2011. Sequence-Based Fungal Identification and Classification, p 669-676. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch43
Generic image for table
TABLE 2

Traditional and sequence-based species names

Citation: Dong J, Loeffelholz M, McGinnis M. 2011. Sequence-Based Fungal Identification and Classification, p 669-676. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch43

This is a required field
Please enter a valid email address
Please check the format of the address you have entered.
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error