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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.

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 (R2 = 0.59, P < 0.001). At 7-14 days, quantitative cultures did not correlate with haemocytometer counts (R2 = 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.

SUBMITTER: Kwizera R

PROVIDER: S-EPMC5567638 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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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.

Kwizera Richard R Akampurira Andrew A Kandole Tadeo K TK Nielsen Kirsten K Kambugu Andrew A Meya David B DB Boulware David R DR Rhein Joshua J

BMC microbiology 20170822 1

<h4>Background</h4>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 an ...[more]

PMID: 28830348

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Project description:Lumbar cerebrospinal fluid (CSF) parameters are widely studied and have wide clinical applications, but ventricular CSF has rarely been studied since it is relatively difficult to obtain. To determine whether there are differences between ventricular and lumbar CSF parameters and whether the differences have clinical significance, we retrospectively reviewed 77 patients with Cryptococcal meningitis who received a ventriculoperitoneal shunt. We analyzed the following parameters: white blood cell count, total protein concentration, CSF/blood glucose ratio, chloride ion concentration, and Cryptococcal count. All parameters between lumbar and ventricular CSF were remarkably different (all p < 0.001). White blood cell count, total protein level and Cryptococcal count were lower in ventricular CSF than in lumbar CSF, while CSF/blood glucose ratio and chloride ion concentration were higher. Compared to patients without ventriculomegaly, patients with ventriculomegaly had a significantly higher total protein concentration in ventricular CSF (p = 0.047). Compared to patients without surgical complications, patients with complications had a significantly lower CSF/blood glucose ratio in ventricular CSF (p = 0.032). The lumbar CSF parameters had no significant differences between these groups. The changes in lumbar CSF indices over time after shunt placement were also analyzed. After shunt placement, total protein concentration was transiently increased, white blood cell count, CSF/blood glucose ratio and chloride ion concentration were continued at the preoperative level until two months after shunting surgery. These findings suggest that the composition of ventricular CSF differs from that of lumbar CSF, and different CSF parameters have disparate rostro-caudal gradients in patients with Cryptococcal meningitis. Furthermore, ventricular and lumbar CSF parameters may have different clinical implications. Transient deterioration of lumbar CSF parameters after ventriculoperitoneal shunt placement may not be due to disease progression, but to change in CSF flow rate by CSF shunts.

Project description:Understanding the host immune response during cryptococcal meningitis (CM) is of critical importance for the development of immunomodulatory therapies. We profiled the cerebrospinal fluid (CSF) immune-response in ninety patients with HIV-associated CM, and examined associations between immune phenotype and clinical outcome. CSF cytokine, chemokine, and macrophage activation marker concentrations were assayed at disease presentation, and associations between these parameters and microbiological and clinical outcomes were examined using principal component analysis (PCA). PCA demonstrated a co-correlated CSF cytokine and chemokine response consisting primarily of Th1, Th2, and Th17-type cytokines. The presence of this CSF cytokine response was associated with evidence of increased macrophage activation, more rapid clearance of Cryptococci from CSF, and survival at 2 weeks. The key components of this protective immune-response were interleukin (IL)-6 and interferon-γ, IL-4, IL-10 and IL-17 levels also made a modest positive contribution to the PC1 score. A second component of co-correlated chemokines was identified by PCA, consisting primarily of monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-1α (MIP-1α). High CSF chemokine concentrations were associated with low peripheral CD4 cell counts and CSF lymphocyte counts and were predictive of immune reconstitution inflammatory syndrome (IRIS). In conclusion CSF cytokine and chemokine profiles predict risk of early mortality and IRIS in HIV-associated CM. We speculate that the presence of even minimal Cryptococcus-specific Th1-type CD4+ T-cell responses lead to increased recruitment of circulating lymphocytes and monocytes into the central nervous system (CNS), more effective activation of CNS macrophages and microglial cells, and faster organism clearance; while high CNS chemokine levels may predispose to over recruitment or inappropriate recruitment of immune cells to the CNS and IRIS following peripheral immune reconstitution with ART. These results provide a rational basis for future studies of immune modulation in CM, and demonstrate the potential of baseline immune profiling to identify CM patients most at risk of mortality and subsequent IRIS.

Project description:IntroductionSurvival among people with HIV-associated cryptococcal meningitis (CM) remains low, particularly among women, despite the currently optimal use of antifungal drugs. Cryptococcus dissemination into the central nervous system [brain, spinal cord, and cerebrospinal fluid (CSF)] elicits the local production of cytokines, chemokines, and other biomarkers. However, no consistent diagnostic or prognostic neuroimmune signature is reported to underpin the risk of death or to identify mechanisms to improve treatment and survival. We hypothesized that distinct neuroimmune signatures in the CSF would distinguish survivors from people who died on antifungal treatment and who may benefit from tailored therapy.MethodsWe considered baseline clinical features, CSF cryptococcal fungal burden, and CSF neuroimmune signatures with survival at 18 weeks among 419 consenting adults by "gender" (168 women and 251 men by biological sex defined at birth).ResultsSurvival at 18 weeks was significantly lower among women than among men {47% vs. 59%, respectively; hazard ratio (HR) = 1.4 [95% confidence interval (CI), 1.0 to 1.9; p = 0.023]}. Unsupervised principal component analysis (PCA) demonstrated divergent neuroimmune signatures by gender, survival, and intragender-specific survival. Overall, women had lower levels of programmed death ligand 1, Interleukin (IL) (IL-11RA/IL-1F30, and IL-15 (IL-15) than men (all p < 0.028). Female survivors compared with those who died expressed significant elevations in levels of CCL11 and CXCL10 chemokines (both p = 0.001), as well as increased T helper 1, regulatory, and T helper 17 cytokines (all p < 0.041). In contrast, male survivors expressed lower levels of IL-15 and IL-8 compared with men who died (p < 0.044).ConclusionsSurvivors of both genders demonstrated a significant increase in the levels of immune regulatory IL-10. In conclusion, the lower survival among women with CM was accompanied by distinct differential gender-specific neuroimmune signatures. These female and male intragender-specific survival-associated neuroimmune signatures provide potential targets for interventions to advance therapy to improve the low survival among people with HIV-associated CM.

Dataset Information

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

Publications

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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