Abstract
BackgroundQuantitative culture is the most common method to determine the fungal burden and sterility of cerebrospinal fluid (CSF) among persons with cryptococcal meningitis. A major drawback of cultures is a long turnaround-time. Recent evidence demonstrates that live and dead Cryptococcus yeasts can be distinguished using trypan blue staining. We hypothesized that trypan blue staining combined with haemocytometer counting may provide a rapid estimation of quantitative culture count and detection of CSF sterility. To test this, we evaluated 194 CSF specimens from 96 HIV-infected participants with cryptococcal meningitis in Kampala, Uganda. Cryptococcal meningitis was diagnosed by CSF cryptococcal antigen (CRAG). We stained CSF with trypan blue and quantified yeasts using a haemocytometer. We compared the haemocytometer readings versus quantitative Cryptococcus CSF cultures.ResultsHaemocytometer counting with trypan blue staining had a sensitivity of 98% (64/65), while CSF cultures had a sensitivity of 95% (62/65) with reference to CSF CRAG for diagnostic CSF specimens. For samples that were positive in both tests, the haemocytometer had higher readings compared to culture. For diagnostic specimens, the median of log10 transformed counts were 5.59 (n = 64, IQR = 5.09 to 6.05) for haemocytometer and 4.98 (n = 62, IQR = 3.75 to 5.79) for culture; while the overall median counts were 5.35 (n = 189, IQR = 4.78–5.84) for haemocytometer and 3.99 (n = 151, IQR = 2.59–5.14) for cultures. The percentage agreement with culture sterility was 2.4% (1/42). Counts among non-sterile follow-up specimens had a median of 5.38 (n = 86, IQR = 4.74 to 6.03) for haemocytometer and 2.89 (n = 89, IQR = 2.11 to 4.38) for culture. At diagnosis, CSF quantitative cultures correlated with haemocytometer counts (R2 = 0.59, P < 0.001). At 7–14 days, quantitative cultures did not correlate with haemocytometer counts (R2 = 0.43, P = 0.4).ConclusionDespite a positive correlation, the haemocytometer counts with trypan blue staining did not predict the outcome of quantitative cultures in patients receiving antifungal therapy.
Highlights
Quantitative culture is the most common method to determine the fungal burden and sterility of cerebrospinal fluid (CSF) among persons with cryptococcal meningitis
Of the 194 cryptococcal antigen (CRAG) positive CSF samples, 33% (65/194) were obtained at diagnosis, with the remaining CSF samples obtained at day 3 (n = 22), day 7 (n = 9), day 14 (n = 10) and as clinically indicated (n = 88) during amphotericin therapy
Demographics were similar to other cohorts of HIVinfected patients with cryptococcal meningitis and reflected the larger trial (Table 1) [8], with 38% (34/90) of the participants women and an overall median age of 35 years (IQR = 30–40 years)
Summary
Quantitative culture is the most common method to determine the fungal burden and sterility of cerebrospinal fluid (CSF) among persons with cryptococcal meningitis. Recent evidence demonstrates that live and dead Cryptococcus yeasts can be distinguished using trypan blue staining. Culture of cerebrospinal fluid (CSF) is currently the gold standard for diagnosis and the only way of determining both sterility of CSF and viability of Cryptococcus yeasts in CSF [3, 5]. Since Cryptococcus cultures have a long turnaround time of up to 10–14 days, rapid evaluation of treatment success or the diagnosis of relapse is often delayed. Recent studies performed on experimental (spiked) samples indicate that trypan blue stain may be beneficial in distinguishing between viable and dead cryptococcal cells in persons with cryptococcal meningitis [7]
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