False negatives were registered more among patients who had received more than seven doses of Amphotericin B. When comparing both India ink and acridine orange to culture, false positive results were registered more among samples with a (+1) grade of positivity ( Table 2). 52% (22/42) of the India ink negative samples were positive with culture while 63% (5/8) of the acridine orange negative samples were positive with culture. However, both India ink (positive predictive value = 86%, negative predictive value = 48%) and acridine orange (PPV = 79%, NPV = 38%) had poor predictive values with reference to culture. When compared to quantitative CSF culture, the sensitivity for India ink increased to 86% (130/152) while that of Acridine orange did not change (97%). In addition, since all samples were from known positive cases (using CSF CrAg), this result was also supported by the fact that India ink had more negative results ( 22%) than acridine orange ( 4%). This confirmed the analysis done in the previous paragraphs. The same statistical analysis also revealed that acridine orange was more sensitive (96%) than India ink (79%) with reference to CSF CrAg. However, there was a 25% correlation between the two tests. Using the fishers exact test, there was a statistically significant difference (p<0.001) in the grades of positivity between acridine orange and India ink at a 95% CI. However, using acridine orange (n = 194), 47% of the samples had a positivity grade of +1, followed by a +2 (29%), then +3 (20%) and the least with zero (negative- 4%) ( Table 2).ĭata presented are number of samples (n) for the different grades of positivity and the number of samples that were positive for culture (gold standard) in each category. Using India ink (n = 194), 33% of the samples had a low positivity grade of +1, followed by a +3 (25%), then zero (negative-22%) and the least with a +2 (21%). ![]() ![]() Acridine orange had a sensitivity of 96% while India ink had a sensitivity of 78% with reference to CSF CrAg (n = 194).įor both India ink and acridine orange tests, the level of positivity was graded on a scale of zero (0) to +3 where zero = negative, +1 = 1–9 cells in 100 fields, +2 = 10–99 cells in 100 fields and +3 is ≥100 cells in one field. The overall (n = 194) median for quantitative cultures was 820 CFU/ml (IQR = 10 to 73000). 127 samples were positive for all these three tests. Of the 194 CrAg LFA positive CSF samples tested from different time points during antifungal therapy, 152 were positive for India ink, 186 were positive for acridine orange, and 151 were positive for quantitative cultures. Performance of India ink and acridine orange against culture and CrAg We hypothesized that with the availability of a florescent microscope in a clinical laboratory, acridine orange may be a more sensitive alternative to India ink light microscopy in the detection of Cryptococcus yeasts in CSF. In this study, we used acridine orange to detect cryptococcal yeasts in fresh whole CSF in comparison to India ink stain at serial time points during antifungal treatment in a clinical setting. Earlier work done by Cohen et al, 1984 showed no apparent advantage of acridine orange over india ink for the rapid identification of Cryptococcus neoformans using three laboratory strains. It emits yellow fluorescence when it binds RNA and green fluorescence when it binds DNA. It has been used to stain samples for viability staining, epifluorescence microscopy and is particularly useful in the rapid screening of sterile biological specimens from clinical and non-clinical materials. The poor sensitivity of India ink may lead to misdiagnosis hence increasing the burden and mortality due to cryptococcal meningitis in resource limited settings where it is still utilized.Īcridine orange is a fluorescence dye used to stain acidic vacuoles, DNA, and RNA in viable cells. The new point of care CrAg lateral flow assay is in fact more sensitive and user friendly in different sample types. However, many recent studies done in sub-Saharan Africa in clinical settings demonstrated low and poor sensitivity of India ink in the diagnosis of cryptococcal meningitis. It is still being used in some resource limited settings of sub-Saharan Africa where the burden due to cryptococcal meningitis is highest. India ink has over time been used in the rapid diagnosis of cryptococcal meningitis using cerebrospinal fluid (CSF) among AIDS patients. However, detection of the cryptococcal antigen (CrAg) in CSF, serum and whole blood has recently gained more popularity with better sensitivity. Culture of cerebrospinal fluid (CSF) is the gold standard for diagnosis of CM. Cryptococcal meningitis (CM) is the most common cause of adult meningitis in Africa accounting for 15%–25% of AIDS-related deaths with majority of the cases registered in sub-Saharan Africa.
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