Evaluation of trypan blue stain in the TC20 automated cell counter as a point-of-care for the enumeration of viable cryptococcal cells in cerebrospinal fluid

ASTRO Team

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

Abstract

Cerebrospinal fluid (CSF) culture can determine a quantitative viability of Cryptococcus yeasts; however, culture has a long turnaround-time. The TC20 automated cell counter (Bio-Rad) is a benchtop instrument used to count cells in 30 seconds. In vitro studies suggest trypan blue staining can distinguish between viable and dead cryptococcal yeasts. We hypothesized that trypan blue staining with automated cell counting may provide rapid quantification of viable CSF Cryptococcus yeasts. In sum, 96 HIV-infected participants with cryptococcal meningitis were enrolled and provided 194 CSF specimens in Kampala, Uganda. Cryptococcosis was diagnosed by CSF cryptococcal antigen (CRAG). CSF was stained with trypan blue and quantified yeasts with the TC20 cell counter. We compared the log10 transformed cell counter readings with gating of 4–10 µm versus log10 quantitative Cryptococcus cultures/ml. TC20 showed more positive results (95.4%) overall than culture (78.4%) with reference to CSF CRAG. TC20 had higher readings compared to culture in most cases with only a 25% level of agreement between the two methods. TC20 had a poor correlation to culture throughout the 14 days of antifungal therapy. The median of log10 transformed counts were 5.22 (IQR = 4.79–5.44) for the TC20 and 3.99 (IQR = 2.59–5.14) for culture. Overall, a linear regression showed no significant relationship between the TC20 and culture (r = −0.0025; P = .92). TC20 automated cell counting with trypan blue staining was poorly predictive of the quantitative CSF culture and could not be used as a substitute for quantitative culture.

Original languageEnglish (US)
Pages (from-to)559-564
Number of pages6
JournalMedical mycology
Volume56
Issue number5
DOIs
StatePublished - Jul 1 2018

Bibliographical note

Funding Information:
We thank institutional support from Drs. Richard Brough, Rosalind Parkes Ratanshi and Mohammed Lamorde. This work was supported by the National Institutes of Health (R01NS086312, T32AI055433 R25TW009345, K01TW010268), Grand Challenges Canada (S4 0296-01), and United Kingdom Medical Research Council (MR/M007413/1).

Publisher Copyright:
© The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved.

Keywords

  • Cryptococcal infection
  • Diagnostic techniques
  • HIV
  • Point-of-care systems
  • Quantitation
  • TC20

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