Quantifying Cryptococcal Meningitis Fungal Burden

We prospectively screened 91 HIV-infected participants with suspected cryptococcal meningitis at Mulago Hospital in Kampala, Uganda from August 13 through November 30, 2013. CSF samples (n=305) were collected at diagnosis (n=86), and at days 3 (n=58), 7 (n=60), 10 (n=44) 14 (n=45), and after day 19 (n=12) of follow-up during the pilot phase of the ASTRO-CM trial (ClinicalTrials.gov: NCT01802385). The ASTRO-CM pilot was a dose finding phase II clinical study of adjunctive sertraline 100–400mg/day added to amphotericin 0.7–1.0 mg/kg/day with fluconazole 800mg/day. Participants provided written informed consent, and applicable institutional review boards approved the study.
Fresh CSF specimens were simultaneously cultured by three different quantitative culture techniques of varying complexity. The “St. George’s Method,” as developed by Robert Larsen, Thomas Harrison, and colleagues, was the reference standard used for comparison.^{5 (link)–11 (link)} This method uses 100 µL input volume of undiluted CSF with five additional 1:10 serial dilutions in sterile H₂O. Each Sabouraud dextrose agar plate received a 100 µL volume of CSF and was distributed using five 4.5mm sterile glass marbles (Figure 1A). The second method, as developed by Robert Larsen and used by the AIDS Clinical Trials Group (ACTG), employs multiple input volumes (1000, 100, 10 µL) of undiluted CSF and two sequential 1:100 dilutions each with two 100 µL and 10 µL input volume per dilution, distributed using an L-shaped spreader (Figure 1B). This ACTG method uses seven culture plates in total. Finally, a third, and deliberately simple “loop” method of a calibrated plastic 10 µL loop was used to plate undiluted and 1:100 diluted CSF onto two culture plates (Figure 1C). Due to delays in receiving supplies, 10 µL loop cultures began in October 2013, one month after the first CSF sample was collected. For each method, CSF vortexing was performed prior to dilutions and prior to plating. For quantification of fungal CSF burden, distinct colonies on agar plates were counted and colony-forming units (CFU) per mL CSF were enumerated on the 10^th day of culturing.
We performed additional quantitative analyses of CSF fungal burden at diagnosis. In order to quantify cryptococcal antigen titers in CSF, we performed Cryptococcal antigen lateral flow assays (CrAg LFA) using a strategy that minimized the number of LFAs needed to determine titer values (Supplemental Appendix). In addition, yeast cells were quantified using automated cell counts performed on 10 µL of undiluted CSF using the TC20 Automated Cell Counter (Bio-Rad, Hercules, CA), and subtracting the CSF white cell count (determined by manual counting).