Reproducibility of CSF quantitative culture methods for estimating rate of clearance in cryptococcal meningitis

Abstract

Quantitative cerebrospinal fluid (CSF) cultures provide a measure of disease severity in cryptococcal meningitis. The fungal clearance rate by quantitative cultures has become a primary endpoint for phase II clinical trials. This study determined the inter-assay accuracy of three different quantitative culture methodologies. Among 91 participants with meningitis symptoms in Kampala, Uganda, during August-November 2013, 305 CSF samples were prospectively collected from patients at multiple time points during treatment. Samples were simultaneously cultured by three methods: (1) St. George's 100 mcl input volume of CSF with five 1:10 serial dilutions, (2) AIDS Clinical Trials Group (ACTG) method using 1000, 100, 10 mcl input volumes, and two 1:100 dilutions with 100 and 10 mcl input volume per dilution on seven agar plates; and (3) 10 mcl calibrated loop of undiluted and 1:100 diluted CSF (loop). Quantitative culture values did not statistically differ between St. George-ACTG methods (P= .09) but did for St. George-10 mcl loop (P< .001). Repeated measures pairwise correlation between any of the methods was high (r≥0.88). For detecting sterility, the ACTG-method had the highest negative predictive value of 97% (91% St. George, 60% loop), but the ACTG-method had occasional (∼10%) difficulties in quantification due to colony clumping. For CSF clearance rate, St. George-ACTG methods did not differ overall (mean -0.05 ± 0.07 log10CFU/ml/day;P= .14) on a group level; however, individual-level clearance varied. The St. George and ACTG quantitative CSF culture methods produced comparable but not identical results. Quantitative cultures can inform treatment management strategies.

Keywords: Cryptococcus; HIV/AIDS; accuracy; culture; meningitis; methodology.

Figure 1.

Three Quantitative CSF Culture Methods.

Figure 2.

Correlation between Quantitative Cultures by St. George Method and ACTG Method. Center reference line displays 1:1 correlation with boundaries displaying ±0.5 log ₁₀ CFU/ml. Paired specimens (n = 229) were collected on day 1 (n = 52), day 3 (n = 49), day 7 (n = 50), day 10 (n = 34), day 14 (n = 36), and after day 18 (n = 8).

Figure 3.

Individual CSF Quantitative Culture differences between ACTG and St. George method. The two methods correlated well (β = 0.88, 95% CI, 0.84 to 0.93) with the ACTG method having slightly higher overall average growth (+0.09 log ₁₀ CFU/mLl). This was most apparent at the extremes of high and low culture values whereby the ACTG method tended to count slightly higher than the St. George method.

Figure 4.

Differences in calculated early fungicidal activity of the rate of CSF clearance for ACTG quantative culture method in comparison with St. George method as reference. Among 47 participants with culture positive cryptococcal meninigitis and ≥2 CSF cultures, the mean difference in rate of clearance was −0.05 log ₁₀ CFU/ml/day (95% CI, −0.11 to 0.02, P = .14).