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Diagnostic Molecular Biology

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Book cover Diagnosis and Treatment of Fungal Infections

Part of the book series: Infectious Disease ((ID))

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Abstract

Clinical mycology is being increasingly challenged by two growing problems: (1) loss of experienced medical mycologists who can identify both common and rare fungi, and (2) a rapidly expanding group of clinically significant fungi that clinicians have rarely or never seen before that are appearing with increasing frequency. As increasingly fewer young, experienced, medical mycologists are entering clinical laboratories, the skills to identify current and future new species of fungi are rapidly disappearing. Unfortunately, patient populations are becoming increasingly immunosuppressed for longer periods of time, which is creating a perfect storm of a growing need for diagnostic skills being unmet by decreasing numbers of young mycologists entering the clinical profession. One of the ways around this issue is to develop new, robust, easy-to-use diagnostic strategies. The evidence is overwhelming that the field of molecular biology can be the source of these new strategies. There currently are multiple approaches that employ new equipment and new techniques that offer a way to fill the growing diagnostic vacuum that has been created in the past 10–20 years. These approaches need to make their way into clinical microbiology laboratories as quickly as possible in order to insure that the diagnostic needs for the field of medical mycology are met today and into the future.

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References

  1. Steinbach WJ, Mitchell TG, Schell WA, Espinel-Ingroff A, Coico RF, Walsh TJ, et al. Status of medical mycology education. Med Mycol. 2003;41(6):457–67.

    Article  PubMed  Google Scholar 

  2. Van Thiel DH George M Moore CM. Fungal infections: their diagnosis and treatment in transplant recipients. Int J Hepatol. 2012;2012:106923.

    PubMed Central  PubMed  Google Scholar 

  3. Wilson LS, Reyes CM, Stolpman M, Speckman J, Allen K, Beney J. The direct cost and incidence of systemic fungal infections. Value Health. 2002;5(1):26–34.

    Article  PubMed  Google Scholar 

  4. Barnes RA. Early diagnosis of fungal infection in immunocompromised patients. The Journal of antimicrob chemother. 2008;61 Suppl 1:i3–6.

    Article  CAS  Google Scholar 

  5. Freydiere AM, Guinet R, Boiron P. Yeast identification in the clinical microbiology laboratory: phenotypical methods. Med Mycol. 2001;39(1):9–33.

    Article  CAS  PubMed  Google Scholar 

  6. Koehler AP, Chu KC, Houang ET, Cheng AF. Simple, reliable, and cost-effective yeast identification scheme for the clinical laboratory. J clin microbiol. 1999;37(2):422–6.

    PubMed Central  CAS  PubMed  Google Scholar 

  7. Wolk DM, Dunne WM. New Technologies in Clinical Microbiology. J clin microbiol. 2011;49(9):S62–S7.

    Article  PubMed Central  Google Scholar 

  8. von Eiff M Roos N Schulten R Hesse M Zuhlsdorf M van de Loo J. Pulmonary aspergillosis: early diagnosis improves survival. Respir int rev thorac dis. 1995;62(6):341–7.

    Google Scholar 

  9. Fredricks DN, Smith C, Meier A. Comparison of six DNA extraction methods for recovery of fungal DNA as assessed by quantitative PCR. J clin microbiol. 2005;43(10):5122–8.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  10. Schrader C, Schielke A, Ellerbroek L, Johne R. PCR inhibitors – occurrence, properties and removal. J appl microbiol. 2012;113(5):1014–26.

    Article  CAS  PubMed  Google Scholar 

  11. Buchheidt D, Baust C, Skladny H, Ritter J, Suedhoff T, Baldus M, et al. Detection of Aspergillus species in blood and bronchoalveolar lavage samples from immunocompromised patients by means of 2-step polymerase chain reaction: clinical results. Clin Infect Dis off publ Infect Dis Soc Am. 2001;33(4):428–35.

    Article  CAS  Google Scholar 

  12. Evriviades D, Jeffery S, Cubison T, Lawton G, Gill M, Mortiboy D. Shaping the military wound: issues surrounding the reconstruction of injured servicemen at the Royal Centre for Defence Medicine. Philos trans R Soc Lond B Biol sci. 2011;366(1562):219–30.

    Article  PubMed Central  PubMed  Google Scholar 

  13. Blow N. Tissue preparation: tissue issues. Nature. 2007;448(7156):959–63.

    Article  PubMed  Google Scholar 

  14. Simner PJ, Buckwalter SP, Uhl JR, Wengenack NL, Pritt BS. Detection and identification of yeasts from formalin-fixed, paraffin-embedded tissue by use of PCR-electrospray ionization mass spectrometry. J clin microbiol. 2013;51(11):3731–4.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988;239(4839):487–91.

    Article  CAS  PubMed  Google Scholar 

  16. Chemaly RF, Tomford JW, Hall GS, Sholtis M, Chua JD, Procop GW. Rapid diagnosis of Histoplasma capsulatum endocarditis using the AccuProbe on an excised valve. J clin microbiol. 2001;39(7):2640–1.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Scalarone GM, Legendre AM, Clark KA, Pusater K. Evaluation of a commercial DNA probe assay for the identification of clinical isolates of Blastomyces dermatitidis from dogs. J med vet mycol bi-mon publ Int Soc Hum Anim Mycol. 1992;30(1):43–9.

    CAS  Google Scholar 

  18. Valesco M, Johnston K. Stability of hybridization activity of Coccidioides immitis in live and heat-killed frozen cultures tested by AccuProbe Coccidioides immitis culture identification test. J clin microbiol. 1997;35(3):736–7.

    PubMed Central  CAS  PubMed  Google Scholar 

  19. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nuc Acids Res. 2000;28(12):E63.

    Article  CAS  Google Scholar 

  20. Njiru ZK. Loop-mediated isothermal amplification technology: towards point of care diagnostics. PLoS Negl Trop Dis. 2012;6(6):e1572.

    Article  PubMed Central  PubMed  Google Scholar 

  21. Niessen L. Loop-mediated isothermal amplification-based detection of Fusarium graminearum. Meth Mol Biol. 2013;968:177–93.

    Article  CAS  Google Scholar 

  22. Denschlag C, Vogel RF, Niessen L. Hyd5 gene-based detection of the major gushing-inducing Fusarium spp. in a loop-mediated isothermal amplification (LAMP) assay. Int J Food Microbiol. 2012;156(3):189–96.

    Article  CAS  PubMed  Google Scholar 

  23. Sun J, Najafzadeh MJ, Vicente V, Xi L, de Hoog GS. Rapid detection of pathogenic fungi using loop-mediated isothermal amplification, exemplified by Fonsecaea agents of chromoblastomycosis. J Microbiol Methods. 2010;80(1):19–24.

    Article  CAS  PubMed  Google Scholar 

  24. Lucas S, da Luz Martins M, Flores O, Meyer W, Spencer-Martins I, Inacio J. Differentiation of Cryptococcus neoformans varieties and Cryptococcus gattii using CAP59-based loop-mediated isothermal DNA amplification. Clin Microbiol Infect Off Publ Eur Soc Clin Microbiol Infect Dis. 2010;16(6):711–4.

    CAS  Google Scholar 

  25. Fakruddin M, Mannan KS, Chowdhury A, Mazumdar RM, Hossain MN, Islam S, et al. Nucleic acid amplification: alternative methods of polymerase chain reaction. J pharm Bioallied sci. 2013;5(4):245–52.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  26. Sun J, de Hoog S. Hyperbranching rolling circle amplification, an improved protocol for discriminating between closely related fungal species. Methods Mol Biol. 2013;968:167–75.

    Article  CAS  PubMed  Google Scholar 

  27. Sun J, Najafzadeh MJ, Zhang J, Vicente VA, Xi L, de Hoog GS. Molecular identification of Penicillium marneffei using rolling circle amplification. Mycoses. 2011;54(6):e751–9.

    Article  CAS  PubMed  Google Scholar 

  28. Najafzadeh MJ, Sun J, Vicente VA, de Hoog GS. Rapid identification of fungal pathogens by rolling circle amplification using Fonsecaea as a model. Mycoses. 2011;54(5):e577–82.

    Article  CAS  PubMed  Google Scholar 

  29. Najafzadeh MJ, Dolatabadi S, Saradeghi Keisari M, Naseri A, Feng P, de Hoog GS. Detection and identification of opportunistic Exophiala species using the rolling circle amplification of ribosomal internal transcribed spacers. J Microbiol Meth. 2013;94(3):338–42.

    Article  CAS  Google Scholar 

  30. Meyer W, Marszewska K, Amirmostofian M, Igreja RP, Hardtke C, Methling K, et al. Molecular typing of global isolates of Cryptococcus neoformans var. neoformans by polymerase chain reaction fingerprinting and randomly amplified polymorphic DNA-a pilot study to standardize techniques on which to base a detailed epidemiological survey. Electrophoresis. 1999;20(8):1790–9.

    Article  CAS  PubMed  Google Scholar 

  31. Versalovic J, Koeuth T, Lupski JR. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res. 1991;19(24):6823–31.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Mutschlechner W, Grif K, Blum G, Lass-Florl C. Rep-PCR and RAPD-PCR fingerprinting of Aspergillus terreus. Med Mycol. 2013;51(8):876–9.

    Article  CAS  PubMed  Google Scholar 

  33. Palencia ER, Klich MA, Glenn AE, Bacon CW. Use of a rep-PCR system to predict species in the Aspergillus section Nigri. J Microbiol Meth. 2009;79(1):1–7.

    Article  CAS  Google Scholar 

  34. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215(3):403–10.

    Article  CAS  PubMed  Google Scholar 

  35. Kurtzman CP, Robnett CJ. 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. 1997;35(5):1216–23.

    PubMed Central  CAS  PubMed  Google Scholar 

  36. White TJ, Bruns TD, Lee SB, Taylor JW. Amplification and sequencing of fungal ribosomal RNA genes for phylogenetics. New York: Academic Press; 1990.

    Google Scholar 

  37. Abe A, Asano K, Sone T. A molecular phylogeny-based taxonomy of the genus Rhizopus. Biosci Biotechnol Biochem. 2010;74(7):1325–31.

    Article  CAS  PubMed  Google Scholar 

  38. Berbee ML, Pirseyedi M, Hubbard S. Cochliobolus phylogenetics and the origin of known, highly virulent pathogens, inferred from ITS and Glyceraldehyde-3-Phosphate Dehydrogenase gene sequences. Mycopath. 1999;91(6):964–77.

    CAS  Google Scholar 

  39. Carbone I, Kohn LM. A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia. 1999;91(3):553–6.

    Article  CAS  Google Scholar 

  40. Gao J, Takashima A. Cloning and characterization of Trichophyton rubrum genes encoding actin, Tri r2, and Tri r4. J Clin Microbiol. 2004;42(7):3298–9.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  41. Glass NL, Donaldson GC. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. App Envir Microbiol. 1995;61(4):1323–30.

    CAS  Google Scholar 

  42. Hong SB, Cho HS, Shin HD, Frisvad JC, Samson RA. Novel Neosartorya species isolated from soil in Korea. Int J Syst Evol Microbiol. 2006;56(Pt 2):477–86.

    Article  CAS  PubMed  Google Scholar 

  43. Lackner M, Najafzadeh MJ, Sun J, Lu Q, Hoog GS. Rapid identification of Pseudallescheria and Scedosporium strains by using rolling circle amplification. Appl Environ Microbiol. 2012;78(1):126–33.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  44. Lasker BA. Nucleotide sequence-based analysis for determining the molecular epidemiology of Penicillium marneffei. J Clin Mic. 2006;44(9):3145–53.

    Article  CAS  Google Scholar 

  45. Marimon R, Gene J, Cano J, Trilles L, Dos Santos Lazera M, Guarro J. Molecular phylogeny of Sporothrix schenckii. J Clin Microbiol. 2006;44(9):3251–6.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  46. O’Donnell K, Kistler HC, Cigelnik E, Ploetz RC. Multiple evolutionary origins of the fungus causing Panama disease of banana: concordant evidence from nuclear and mitochondrial gene genealogies. Proc Natl Acad Sci U S A. 1998;95(5):2044–9.

    Article  PubMed Central  PubMed  Google Scholar 

  47. O’Donnell K, Nirenberg HI, Aoki T, Cigelnik E. A Multigene phylogeny of the Gibberella fujikuroi species complex: detection of additional phylogenetically distinct species. Mycosci. 2000;41:61–78.

    Article  Google Scholar 

  48. Petti CA, Bosshard PP, Brandt ME, Clarridge JE, Feldblyum TV, Foxall P, et al. Interpretive criteria for identification of bacteria and fungi by DNA target sequencing; approved guideline. MM18-P. Wayne, PA2008.

    Google Scholar 

  49. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975;98(3):503–17.

    Article  CAS  PubMed  Google Scholar 

  50. Hung WT, Su SL, Shiu LY, Chang TC. Rapid identification of allergenic and pathogenic molds in environmental air by an oligonucleotide array. BMC Infect Dis. 2011;11:91.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  51. Sato T, Takayanagi A, Nagao K, Tomatsu N, Fukui T, Kawaguchi M, et al. Simple PCR-based DNA microarray system to identify human pathogenic fungi in skin. J Clin Microbiol. 2010;48(7):2357–64.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  52. Aittakorpi A, Kuusela P, Koukila-Kahkola P, Vaara M, Petrou M, Gant V, et al. Accurate and rapid identification of Candida spp. frequently associated with fungemia by using PCR and the microarray-based Prove-it Sepsis assay. J Clin Microbiol. 2012;50(11):3635–40.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  53. Tissari P, Zumla A, Tarkka E, Mero S, Savolainen L, Vaara M, et al. Accurate and rapid identification of bacterial species from positive blood cultures with a DNA-based microarray platform: an observational study. Lancet. 2010;375(9710):224–30.

    Article  CAS  PubMed  Google Scholar 

  54. Buelow DR, Gu Z, Walsh TJ, Hayden RT. Evaluation of multiplexed PCR and liquid-phase array for identification of respiratory fungal pathogens. Med Mycol. 2012;50(7):775–80.

    Article  CAS  PubMed  Google Scholar 

  55. Babady NE, Miranda E, Gilhuley KA. Evaluation of Luminex xTAG fungal analyte-specific reagents for rapid identification of clinically relevant fungi. J Clin Microbiol. 2011;49(11):3777–82.

    Article  PubMed Central  PubMed  Google Scholar 

  56. Balada-Llasat JM, LaRue H, Kamboj K, Rigali L, Smith D, Thomas K, et al. Detection of yeasts in blood cultures by the Luminex xTAG fungal assay. J Clin Microbiol. 2012;50(2):492–4.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  57. Pulcrano G, Iula DV, Vollaro A, Tucci A, Cerullo M, Esposito M, et al. Rapid and reliable MALDI-TOF mass spectrometry identification of Candida non-albicans isolates from bloodstream infections. J Microbiol Meth. 2013;94(3):262–6.

    Article  CAS  Google Scholar 

  58. Stone NR, Gorton RL, Barker K, Ramnarain P, Kibbler CC. Evaluation of PNA-FISH yeast traffic light for rapid identification of yeast directly from positive blood cultures and assessment of clinical impact. J Clin Microbiol. 2013;51(4):1301–2.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  59. Rigby S, Procop GW, Haase G, Wilson D, Hall G, Kurtzman C, et al. Fluorescence in situ hybridization with peptide nucleic acid probes for rapid identification of Candida albicans directly from blood culture bottles. J Clin Microbiol. 2002;40(6):2182–6.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  60. Lau A, Chen S, Sleiman S, Sorrell T. Current status and future perspectives on molecular and serological methods in diagnostic mycology. Fut Microbiol. 2009;4(9):1185–222.

    Article  CAS  Google Scholar 

  61. Putignani L, Del Chierico F, Onori M, Mancinelli L, Argentieri M, Bernaschi P, et al. MALDI-TOF mass spectrometry proteomic phenotyping of clinically relevant fungi. Mol Biosyst. 2011;7(3):620–9.

    Article  CAS  PubMed  Google Scholar 

  62. Sendid B, Ducoroy P, Francois N, Lucchi G, Spinali S, Vagner O, et al. Evaluation of MALDI-TOF mass spectrometry for the identification of medically-important yeasts in the clinical laboratories of Dijon and Lille hospitals. Med Mycol. 2013;51(1):25–32.

    Article  CAS  PubMed  Google Scholar 

  63. Claydon MA, Davey SN, Edwards-Jones V, Gordon DB. The rapid identification of intact microorganisms using mass spectrometry. Nature Biotechnol. 1996;14(11):1584–6.

    Article  CAS  Google Scholar 

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Acknowledgments

BLW is supported by the US Army Medical Research and Materiel Command under grant W81XWH-13-C-0103, Office of Congressionally Directed Medical Research Programs, Joint Warfighter Medical Research Program, and the Army Research Office of the Department of Defense under Contract No.W911NF-11–1-0136.

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Correspondence to Brian L. Wickes .

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Romanelli, A., Wickes, B. (2015). Diagnostic Molecular Biology. In: Hospenthal, D., Rinaldi, M. (eds) Diagnosis and Treatment of Fungal Infections. Infectious Disease. Springer, Cham. https://doi.org/10.1007/978-3-319-13090-3_3

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