title: Rapid microscopy and use of vital dyes: potential to determine viability of Cryptococcus neoformans in the clinical laboratory.
abstract: BACKGROUND
  Cryptococcus neoformans is the commonest cause of fungal meningitis, with a substantial mortality despite appropriate therapy. Quantitative culture of cryptococci in cerebrospinal fluid (CSF) during antifungal therapy is of prognostic value and has therapeutic implications, but is slow and not practicable in many resource-poor countries.
  
  
  METHODS
  We piloted two rapid techniques for quantifying viable cryptococci using mixtures of live and heat-killed cryptococci cultured in vitro: (i) quantitative microscopy with exclusion staining using trypan blue dye, and (ii) flow cytometry, using the fluorescent dye 2'-7'-Bis-(2-carboxyethyl)-5-(6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM). Results were compared with standard quantitative cryptococcal cultures. Quantitative microscopy was also performed on cerebrospinal fluid (CSF) samples.
  
  
  RESULTS
  Both microscopy and flow cytometry distinguished between viable and non-viable cryptococci. Cell counting (on log scale) by microscopy and by quantitative culture were significantly linearly associated (p<0.0001) and Bland-Altman analysis showed a high level of agreement. Proportions of viable cells (on logit scale), as detected by flow cytometry were significantly linearly associated with proportions detected by microscopy (p<0.0001) and Bland-Altman analysis showed a high level of agreement.
  
  
  CONCLUSIONS
  Direct microscopic examination of trypan blue-stained cryptococci and flow-cytometric assessment of BCECF-AM-stained cryptococci were in good agreement with quantitative cultures. These are promising strategies for rapid determination of the viability of cryptococci, and should be investigated in clinical practice.
authors.affiliation: Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute, University of Sydney at Westmead Hospital, Westmead, NSW, Australia; Centre for Infectious Diseases and Microbiology Laboratory Services, Westmead Hospital, Westmead, NSW, Australia.
authors.forename: Brendan J
authors.initials: BJ
authors.lastname: McMullan
authors.affiliation: Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute, University of Sydney at Westmead Hospital, Westmead, NSW, Australia; Marie Bashir Institute for Infectious Diseases and Biosecurity, University of Sydney, Westmead, NSW, Australia.
authors.forename: Desmarini
authors.initials: D
authors.lastname: Desmarini
authors.affiliation: Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute, University of Sydney at Westmead Hospital, Westmead, NSW, Australia; Marie Bashir Institute for Infectious Diseases and Biosecurity, University of Sydney, Westmead, NSW, Australia.
authors.forename: Julianne T
authors.initials: JT
authors.lastname: Djordjevic
authors.affiliation: Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute, University of Sydney at Westmead Hospital, Westmead, NSW, Australia; Centre for Infectious Diseases and Microbiology Laboratory Services, Westmead Hospital, Westmead, NSW, Australia; ICPMR-Pathology West, Westmead Hospital, Westmead, NSW, Australia.
authors.forename: Sharon C-A
authors.initials: SC
authors.lastname: Chen
authors.affiliation: Discipline of Infectious Diseases and Immunology, Westmead Clinical School, Sydney Medical School, The University of Sydney, Westmead, NSW, Australia.
authors.forename: Michael
authors.initials: M
authors.lastname: Roper
authors.affiliation: Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute, University of Sydney at Westmead Hospital, Westmead, NSW, Australia; Marie Bashir Institute for Infectious Diseases and Biosecurity, University of Sydney, Westmead, NSW, Australia.
authors.forename: Tania C
authors.initials: TC
authors.lastname: Sorrell
mesh_terms: D003453:Cryptococcosis / Q000134:cerebrospinal fluid / Q000175:diagnosis / Q000382:microbiology; D003455:Cryptococcus neoformans / Q000502:physiology*; D005434:Flow Cytometry; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D050296:Microbial Viability; D008856:Microscopy, Fluorescence; D013194:Staining and Labeling; D014343:Trypan Blue / Q000737:chemistry
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; C057433:2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester; D014343:Trypan Blue