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Evaluation of trypan blue stain in a haemocytometer for rapid detection of cerebrospinal fluid sterility in HIV patients with cryptococcal meningitis.

Evaluation of trypan blue stain in a haemocytometer for rapid detection of cerebrospinal fluid sterility in HIV patients with cryptococcal meningitis.
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Kwizera R, Akampurira A, Kandole TK, Nielsen K, Kambugu A, Meya DB, Boulware DR, Rhein J, ,


Kwizera R, Akampurira A, Kandole TK, Nielsen K, Kambugu A, Meya DB, Boulware DR, Rhein J, , (click to view)

Kwizera R, Akampurira A, Kandole TK, Nielsen K, Kambugu A, Meya DB, Boulware DR, Rhein J, ,

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BMC microbiology 2017 08 2217(1) 182 doi 10.1186/s12866-017-1093-4

Abstract
BACKGROUND
Quantitative 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.

RESULTS
Haemocytometer 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 (R(2) = 0.59, P < 0.001). At 7-14 days, quantitative cultures did not correlate with haemocytometer counts (R(2) = 0.43, P = 0.4). CONCLUSION
Despite a positive correlation, the haemocytometer counts with trypan blue staining did not predict the outcome of quantitative cultures in patients receiving antifungal therapy.

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