Evaluation of a rapid, quantitative real-time PCR method for enumeration of pathogenic Candida cells in water

Abstract

Quantitative PCR (QPCR) technology, incorporating fluorigenic 5' nuclease (TaqMan) chemistry, was utilized for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C. lusitaniae) in water. Known numbers of target cells were added to distilled and tap water samples, filtered, and disrupted directly on the membranes for recovery of DNA for QPCR analysis. The assay's sensitivities were between one and three cells per filter. The accuracy of the cell estimates was between 50 and 200% of their true value (95% confidence level). In similar tests with surface water samples, the presence of PCR inhibitory compounds necessitated further purification and/or dilution of the DNA extracts, with resultant reductions in sensitivity but generally not in quantitative accuracy. Analyses of a series of freshwater samples collected from a recreational beach showed positive correlations between the QPCR results and colony counts of the corresponding target species. Positive correlations were also seen between the cell quantities of the target Candida species detected in these analyses and colony counts of Enterococcus organisms. With a combined sample processing and analysis time of less than 4 h, this method shows great promise as a tool for rapidly assessing potential exposures to waterborne pathogenic Candida species from drinking and recreational waters and may have applications in the detection of fecal pollution.

FIG. 1.

Determination of amplification efficiency and extrapolated cell detection limit for QPCR analysis of Candida species, showing C. albicans as an example. DNA extracts were prepared by a rapid bead-milling method, as described in the text, from cell stocks of known concentration (determined by hemocytometer counts) of the strains ATCC 18804 (◊), ATCC11006 (⧫), ATCC 14053 (▴), ATCC 60193 (▵), ATCC 18804 (•), ATCC 24433 (○), ATCC 66027 (▪) (experiment 1), and ATCC 66027 (□) (experiment 2). In this example, there was no lack of fit for a parallel relationship (P = 0.729), and thus the amplification efficiencies were presumed to be equal. The regression of log cell equivalents on C_T is represented by a single solid line. The dashed lines represent the 95% confidence limits for the individual results.

FIG. 2.

Experimental sensitivity and accuracy of the CFE and QPCR analysis method for quantifying Candida cells. Known numbers of fluorescently labeled C. albicans cells were collected by filtration on polyethylene terephthalate filters and corroborated by solid-phase cytometry and microscopy. The counted cells on the filters were extracted in the presence of G. candidum reference cells by using the CFE method, and C. albicans cells were quantified by QPCR and ΔΔC_T comparative C_T analyses. The regression of log cell numbers per filter determined by direct counts on log cell numbers determined by QPCR analysis is represented by a single solid line. The dashed lines represent the 95% confidence limits for the individual results.

FIG. 3.

Relationship between cellular enumeration of target Candida species in Lake Michigan beach water samples by culture plate counts (CFU) and by QPCR analysis. Water samples were filtered on nitrocellulose filters for plate counts on BiGGY agar medium, and the resulting CFU of target Candida species were enumerated as described in Table 5 and in the text. Equivalent water sample volumes were filtered through polycarbonate filters, the filters were extracted in the presence of G. candidum reference cells, and the extracts were diluted or further purified as indicated in Table 5. Quantities of each of the six Candida populations were determined by using QPCR assays and ΔΔC_T comparative C_T analyses. Results are shown for the six water samples indicated in Table 5 for which both methods of analysis were performed and are based on the total cell numbers of the six pathogenic Candida species, as determined by each method for each sample. The regression value for the line is R = 0.91.

FIG. 4.

Relationship between cellular enumeration of Enterococcus species by culture plate counts (CFU) and target Candida species by QPCR analysis. Water samples were filtered on nitrocellulose filters for plate counts on mEI basal medium, and the resulting colonies with blue halos were enumerated as presumptive enterococcal CFU. Results are presented on the basis of a 100-ml water sample volume. Quantities of each of the six Candida populations were determined as described in the legend for Fig. 3, with results presented on the basis of a 300-ml water sample volume. The regression value for the line is R = 0.93.