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Molecular Genetics and Genomics of Fungal Infections

  • Shivaprakash M. Rudramurthy
  • Arunaloke Chakrabarti
Chapter

Abstract

Fungal infections of central nervous system (CNS) are increasingly encountered in various clinical settings. Any pathogenic fungus is able to produce CNS infection, though only certain groups are neurotropic. The outcome of CNS fungal infections depends on early diagnosis and prompt management. But, the early diagnosis faces stiff challenges due to general considerations of rarity of CNS fungal infection, non-specific clinical symptoms and signs and difficulty in collection of samples for conventional diagnosis. Application of genomic-based molecular techniques is usually rapid and sensitive and may detect the causative agent accurately and help appropriate and prompt antifungal therapy. These techniques can be applied either for detection of nucleic acid in clinical samples or for the accurate identification of fungi isolated from the clinical specimen. Detection of nucleic acid in clinical sample faces the challenge of extraction of nucleic acid, inadequate amount nucleic acid in sample and contamination during test procedure. The current chapter describes the existing knowledge on genomic-based molecular tests for diagnosis and monitoring of CNS fungal diseases.

Keywords

Central nervous system Diagnostic procedures DNA extraction Fungal infections Genomics Molecular diagnosis Molecular genetics Molecular test formats 

Abbreviations

AIDS

Acquired immunodeficiency syndrome

CDC

Centers for Disease Control and Prevention

CM

Cryptococcal meningitis

CNS

Central nervous system

CSF

Cerebrospinal fluid

CT

Computed tomography

EF

Elongation factor

HIV

Human immunodeficiency virus

HRCA

Hyperbranched rolling circle amplification

ISHAM

International Society for Human and Animal Mycology

ITS

Internal transcribed spacer

LAMP

Loop-mediated isothermal amplification

LFA

Lateral flow assay

ME

Meningitis/encephalitis

MIC

Minimal inhibitory concentration

MRI

Magnetic resonance imaging

PCR

Polymerase chain reaction

PLB

Phospholipase B

References

  1. de Aguirre L, Hurst SF, Choi JS, Shin JH, Hinrikson HP, Morrison CJ. Rapid differentiation of Aspergillus species from other medically important opportunistic molds and yeasts by PCR-enzyme immunoassay. J Clin Microbiol. 2004;42:3495–504.CrossRefGoogle Scholar
  2. Alonso R, Pisa D, Fernández-Fernández AM, Rábano A, Carrasco L. Fungal infection in neural tissue of patients with amyotrophic lateral sclerosis. Neurobiol Dis. 2017;108:249–60.CrossRefGoogle Scholar
  3. Atkins SD, Clark IM. Fungal molecular diagnostics: a mini review. J Appl Genet. 2004;45:3–15.PubMedGoogle Scholar
  4. Babady NE, Buckwalter SP, Hall L, Le Febre KM, Binnicker MJ, Wengenack NL. Detection of Blastomyces dermatitidis and Histoplasma capsulatum from culture isolates and clinical specimens by use of real-time PCR. J Clin Microbiol. 2011;49:3204–8.CrossRefGoogle Scholar
  5. Badiee P, Alborzi A. Assessment of a real-time PCR method to detect human non-cryptococcal fungal meningitis. Arch Iran Med. 2011;14:381–4.PubMedGoogle Scholar
  6. Balajee SA, Gribskov JL, Hanley E, Nickle D, Marr KA. Aspergillus lentulus sp. nov., a new sibling species of A. fumigatus. Eukaryot Cell. 2005;4:625–32.CrossRefGoogle Scholar
  7. Barnes RA. Early diagnosis of fungal infection in immunocompromised patients. J Antimicrob Chemother. 2008;61(Suppl 1):i3–6.CrossRefGoogle Scholar
  8. Bialek R, Weiss M, Bekure-Nemariam K, Najvar LK, Alberdi MB, Graybill JR, et al. Detection of Cryptococcus neoformans DNA in tissue samples by nested and real-time PCR assays. Clin Diagn Lab Immunol. 2002;9:461–9.PubMedPubMedCentralGoogle Scholar
  9. Bialek R, Konrad F, Kern J, Aepinus C, Cecenas L, Gonzalez GM, et al. PCR based identification and discrimination of agents of mucormycosis and aspergillosis in paraffin wax embedded tissue. J Clin Pathol. 2005;58:1180–4.CrossRefGoogle Scholar
  10. Binnicker MJ, Popa AS, Catania J, Alexov M, Tsaras G, Lloyd F, et al. Meningeal coccidioidomycosis diagnosed by real-time polymerase chain reaction analysis of cerebrospinal fluid. Mycopathologia. 2011;171:285–9.CrossRefGoogle Scholar
  11. Buitrago MJ, Bernal-Martínez L, Castelli MV, Rodríguez-Tudela JL, Cuenca-Estrella M. Histoplasmosis and paracoccidioidomycosis in a non-endemic area: a review of cases and diagnosis. J Travel Med. 2011;18:26–33.CrossRefGoogle Scholar
  12. Centers for Disease Control and Prevention (CDC). Multistate fungal meningitis outbreak – interim guidance for treatment. MMWR Morb Mortal Wkly Rep. 2012;61:842.Google Scholar
  13. Chew KL, Lee CK, Cross GB, Lum LHW, Yan B, Jureen R. Culture-confirmed cryptococcal meningitis not detected by Cryptococcus PCR on the Biofire meningitis/encephalitis panel®. Clin Microbiol Infect. 2018;24(7):791–2.CrossRefGoogle Scholar
  14. Demogines A, Fouch S, Everhart K, Leber A, Barney T, Daly JA, et al. Multi-center clinical evaluation of a multiplex meningitis/encephalitis PCR panel for simultaneous detection of bacteria, yeast, and viruses in cerebrospinal fluid specimens. J Clin Microbiol. 2015;54:2251–61.Google Scholar
  15. Elsayed S, Fitzgerald V, Massey V, Hussain Z. Evaluation of the Candigen enzyme-linked immunosorbent assay for quantitative detection of Candida species antigen. Arch Pathol Lab Med. 2001;125:344–6.PubMedGoogle Scholar
  16. Farina C, Perin S, Andreoni S, Conte M, Fazii P, Lombardi G, et al. Evaluation of the peptide nucleic acid fluorescence in situ hybridisation technology for yeast identification directly from positive blood cultures: an Italian experience. Mycoses. 2012;55:388–92.CrossRefGoogle Scholar
  17. Frías-De-León MG, Ramírez-Bárcenas JA, Rodríguez-Arellanes G, Velasco-Castrejón O, Taylor ML, Reyes-Montes M d R. Usefulness of molecular markers in the diagnosis of occupational and recreational histoplasmosis outbreaks. Folia Microbiol (Praha). 2017;62:111–6.CrossRefGoogle Scholar
  18. Gade L, Scheel CM, Pham CD, Lindsley MD, Iqbal N, Cleveland AA, et al. Detection of fungal DNA in human body fluids and tissues during a multistate outbreak of fungal meningitis and other infections. Eukaryot Cell. 2013;12:677–83.CrossRefGoogle Scholar
  19. Gade L, Grgurich DE, Kerkering TM, Brandt ME, Litvintseva AP. Utility of real-time PCR for detection of exserohilum rostratum in body and tissue fluids during the multistate outbreak of fungal meningitis and other infections. J Clin Microbiol. 2015;53:618–25.CrossRefGoogle Scholar
  20. Gunaratne PS, Wijeyaratne CN, Seneviratne HR. Aspergillus meningitis in Sri Lanka – a post-tsunami effect? N Engl J Med. 2007;356:754–6.CrossRefGoogle Scholar
  21. Hanson KE. The first fully automated molecular diagnostic panel for meningitis and encephalitis: how well does it perform, and when should it be used? J Clin Microbiol. 2016;54:2222–4.CrossRefGoogle Scholar
  22. de Hoog GS, Nishikaku AS, Fernandez-Zeppenfeldt G, Padín-González C, Burger E, Badali H, et al. Molecular analysis and pathogenicity of the Cladophialophora carrionii complex, with the description of a novel species. Stud Mycol. 2007;58:219–34.CrossRefGoogle Scholar
  23. Hummel M, Spiess B, Kentouche K, Niggemann S, Böhm C, Reuter S, et al. Detection of Aspergillus DNA in cerebrospinal fluid from patients with cerebral aspergillosis by a nested PCR assay. J Clin Microbiol. 2006;44:3989–93.CrossRefGoogle Scholar
  24. Kami M, Ogawa S, Kanda Y, Tanaka Y, Machida U, Matsumura T, et al. Early diagnosis of central nervous system aspergillosis using polymerase chain reaction, latex agglutination test, and enzyme-linked immunosorbent assay. Br J Haematol. 1999a;106:536–7.CrossRefGoogle Scholar
  25. Kami M, Shirouzu I, Mitani K, Ogawa S, Matsumura T, Kanda Y, et al. Early diagnosis of central nervous system aspergillosis with combination use of cerebral diffusion-weighted echo-planar magnetic resonance image and polymerase chain reaction of cerebrospinal fluid. Intern Med. 1999b;38:45–8.CrossRefGoogle Scholar
  26. Kaocharoen S, Wang B, Tsui KM, Trilles L, Kong F, Meyer W. Hyperbranched rolling circle amplification as a rapid and sensitive method for species identification within the Cryptococcus species complex. Electrophoresis. 2008;29:3183–91.PubMedGoogle Scholar
  27. Khot PD, Fredricks DN. PCR-based diagnosis of human fungal infections. Expert Rev Anti Infect Ther. 2009;7:1201–21.CrossRefGoogle Scholar
  28. Klingspor L, Jalal S. Molecular detection and identification of Candida and Aspergillus spp. from clinical samples using real-time PCR. Clin Microbiol Infect. 2006;12:745–53.CrossRefGoogle Scholar
  29. Komatsu H, Fujisawa T, Inui A, Horiuchi K, Hashizume H, Sogo T, et al. Molecular diagnosis of cerebral aspergillosis by sequence analysis with panfungal polymerase chain reaction. J Pediatr Hematol Oncol. 2004;26:40–4.CrossRefGoogle Scholar
  30. Landlinger C, Preuner S, Willinger B, Haberpursch B, Racil Z, Mayer J, et al. Species-specific identification of a wide range of clinically relevant fungal pathogens by use of Luminex xMAP technology. J Clin Microbiol. 2009;47:1063–73.CrossRefGoogle Scholar
  31. Lass-Flörl C, Mutschlechner W, Aigner M, Grif K, Marth C, Girschikofsky M, et al. Utility of PCR in diagnosis of invasive fungal infections: real-life data from a multicenter study. J Clin Microbiol. 2013;51:863–8.CrossRefGoogle Scholar
  32. Leeflang MM, Debets-Ossenkopp YJ, Visser CE, Scholten RJ, Hooft L, Bijlmer HA, et al. Galactomannan detection for invasive aspergillosis in immunocompromised patients. Cochrane Database Syst Rev. 2008;(4):CD007394.Google Scholar
  33. Liesman RM, Strasburg AP, Heitman AK, Theel ES, Patel R, Binnicker MJ. Evaluation of a commercial multiplex molecular panel for diagnosis of infectious meningitis and encephalitis. J Clin Microbiol. 2018;56:1–27.CrossRefGoogle Scholar
  34. López LF, Muñoz CO, Cáceres DH, Tobón ÁM, Loparev V, Clay O, et al. Standardization and validation of real time PCR assays for the diagnosis of histoplasmosis using three molecular targets in an animal model. PLoS One. 2017;12:e0190311.CrossRefGoogle Scholar
  35. Mandviwala T, Shinde R, Kalra A, Sobel JD, Akins RA. High-throughput identification and quantification of Candida species using high resolution derivative melt analysis of panfungal amplicons. J Mol Diagn. 2010;12:91–101.CrossRefGoogle Scholar
  36. Martins M d A, Brighente KBS, de Matos TA, Vidal JE, de Hipólito DDC, Pereira-Chioccola VL. Molecular diagnosis of cryptococcal meningitis in cerebrospinal fluid: comparison of primer sets for Cryptococcus neoformans and Cryptococcus gattii species complex. Braz J Infect Dis. 2015;19:62–7.CrossRefGoogle Scholar
  37. Mitchell M, Dizon D, Libke R, Peterson M, Slater D, Dhillon A. Development of a real-time PCR assay for identification of Coccidioides immitis by use of the BD Max system. J Clin Microbiol. 2015;53:926–9.  https://doi.org/10.1128/JCM.02731-14.CrossRefPubMedPubMedCentralGoogle Scholar
  38. McCarthy MW, Walsh TJ. Molecular diagnosis of invasive mycoses of the central nervous system. Expert Rev Mol Diagn. 2017;17:129–39.CrossRefGoogle Scholar
  39. Moling O, Lass-Floerl C, Verweij PE, Porte M, Boiron P, Prugger M, et al. Case reports. Chronic and acute Aspergillus meningitis – Falldarstellungen. Chronische und akute Aspergillus-Meningitis. Mycoses. 2002;45:504–11.PubMedGoogle Scholar
  40. Chen M, Zhou J, Li J, Li M, Sun J, Fang WJ, Al-Hatmi AMS, Xu J, Boekhout T, Liao WQ, Pan WH. Evaluation of five conventional and molecular approaches for diagnosis of cryptococcal meningitis in non-HIV-infected patients. Mycoses. 2016;59(8):494–502.  https://doi.org/10.1111/myc.12497.CrossRefPubMedGoogle Scholar
  41. O’Donnell K, Sutton DA, Rinaldi MG, Sarver BAJ, Balajee SA, Schroers H-J, et al. Internet-accessible DNA sequence database for identifying fusaria from human and animal infections. J Clin Microbiol. 2010;48:3708–18.CrossRefGoogle Scholar
  42. Pappas PG, Rex JH, Sobel JD, Filler SG, Dismukes WE, Walsh TJ, et al. Guidelines for treatment of candidiasis. Clin Infect Dis. 2004;38:161–89.CrossRefGoogle Scholar
  43. Paschoal RC, Hirata MH, Hirata RC, Melhem MDSC, Dias ALT, Paula CR. Neurocryptococcosis: diagnosis by PCR method. Rev Inst Med Trop Sao Paulo. 2004;46:203–7.CrossRefGoogle Scholar
  44. Preuner S, Lion T. Towards molecular diagnostics of invasive fungal infections. Expert Rev Mol Diagn. 2009;9:397–401.CrossRefGoogle Scholar
  45. Qishui O, Ling J, Ni L, Bin Y, Wen L. Comparison of real-time florescence quantitative PCR measurements of VAD1 mRNA with three conventional methods in diagnosis and follow-up treatment of Cryptococcus neoformans infection. Mycoses. 2012;55:326–32.CrossRefGoogle Scholar
  46. Ralph ED, Hussain Z. Chronic meningitis caused by Candida albicans in a liver transplant recipient: usefulness of the polymerase chain reaction for diagnosis and for monitoring treatment. Clin Infect Dis. 1996;23:191–2.CrossRefGoogle Scholar
  47. Rappelli P, Are R, Casu G, Fiori PL, Cappuccinelli P, Aceti A. Development of a nested PCR for detection of Cryptococcus neoformans cerebrospinal fluid. J Clin Microbiol. 1998;36:3438–40.PubMedPubMedCentralGoogle Scholar
  48. Reinwald M, Buchheidt D, Hummel M, Duerken M, Bertz H, Schwerdtfeger R, et al. Diagnostic performance of an Aspergillus-specific nested PCR assay in cerebrospinal fluid samples of immunocompromised patients for detection of central nervous system Aspergillosis. PLoS One. 2013;8:1–6.CrossRefGoogle Scholar
  49. Rhein J, Bahr NC, Hemmert AC, Cloud JL, Bellamkonda S, Oswald C, et al. Diagnostic performance of a multiplex PCR assay for meningitis in an HIV-infected population in Uganda. Diagn Microbiol Infect Dis. 2016;84:268–73.CrossRefGoogle Scholar
  50. Saha DC, Xess I, Biswas A, Bhowmik DM, Padma MV. Detection of Cryptococcus by conventional, serological and molecular methods. J Med Microbiol. 2009;58:1098–105.  https://doi.org/10.1099/jmm.0.007328-0.CrossRefPubMedGoogle Scholar
  51. Shamim MS, Enam SA, Ali R, Anwar S. Craniocerebral aspergillosis: a review of advances in diagnosis and management. J Pak Med Assoc. 2010;60:573–9.PubMedGoogle Scholar
  52. Simoneau E, Kelly M, Labbe AC, Roy J, Laverdière M. What is the clinical significance of positive blood cultures with Aspergillus sp in hematopoietic stem cell transplant recipients? A 23 year experience. Bone Marrow Transplant. 2005;35:303–6.CrossRefGoogle Scholar
  53. Tavares ER, Azevedo CS, Panagio LA, Pelisson M, Pinge-Filho P, Venancio EJ, et al. Accurate and sensitive real-time PCR assays using intergenic spacer 1 region to differentiate Cryptococcus gattii sensu lato and Cryptococcus neoformans sensu lato. Med Mycol. 2016;54:89–96.PubMedGoogle Scholar
  54. Trilles L, Meyer W, Wanke B, Guarro J, Lazéra M. Correlation of antifungal susceptibility and molecular type within the Cryptococcus neoformans/C. gattii species complex. Med Mycol. 2012;50:328–32.CrossRefGoogle Scholar
  55. Trilles L, Wang B, Firacative C, Lazéra MDS, Wanke B, Meyer W. Identification of the major molecular types of Cryptococcus neoformans and C. gattii by Hyperbranched rolling circle amplification. PLoS One. 2014;9:e94648.CrossRefGoogle Scholar
  56. Valero C, De L, Cruz-Villar L, Zaragoza Ó, Buitrago MJ. New panfungal real-time PCR assay for diagnosis of invasive fungal infections. J Clin Microbiol. 2016;54:2910–8.CrossRefGoogle Scholar
  57. Veron V, Simon S, Blanchet D, Aznar C. Real-time polymerase chain reaction detection of Cryptococcus neoformans and Cryptococcus gattii in human samples. Diagn Microbiol Infect Dis. 2009;65:69–72.CrossRefGoogle Scholar
  58. Verweij PE, Brinkman K, Kremer HPH, Kullberg B, Meis JFGM. Aspergillus meningitis: diagnosis by non-culture-based microbiological methods and management Aspergillus meningitis: diagnosis by non-culture-based microbiological methods and management. J Clin Microbiol. 1999;37:1186–9.PubMedPubMedCentralGoogle Scholar
  59. Vucicevic D, Blair JE, Binnicker MJ, McCullough AE, Kusne S, Vikram HR, et al. The utility of Coccidioides polymerase chain reaction testing in the clinical setting. Mycopathologia. 2010;170:345–51.CrossRefGoogle Scholar
  60. White TJ, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Shinsky JJ, White TJ, editors. PCR protocols: a guide to methods and applications. New York: Academic Press Inc.; 1990.Google Scholar
  61. Zaman K, Rudramurthy SM, Das A, Panda N, Honnavar P, Kaur H, et al. Molecular diagnosis of rhino-orbito-cerebral mucormycosis from fresh tissue samples. J Med Microbiol. 2017;66:1124–9.CrossRefGoogle Scholar
  62. Zhao Y, Petraitiene R, Walsh TJ, Perlin DS. A real-time PCR assay for rapid detection and quantification of Exserohilum rostratum, a causative pathogen of fungal meningitis associated with injection of contaminated methylprednisolone. J Clin Microbiol. 2013;51:1034–6.CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Shivaprakash M. Rudramurthy
    • 1
  • Arunaloke Chakrabarti
    • 1
    • 2
  1. 1.Department of Medical MicrobiologyPostgraduate Institute of Medical Education and ResearchChandigarhIndia
  2. 2.Center of Advance Research in Medical Mycology, WHO Collaborating Center for Reference and Research of Fungi of Medical ImportanceNational Culture Collection of Pathogenic FungiChandigarhIndia

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