Chip-Based Molecular Evaluation of a DNA Extraction Protocol for Candida Species from Positive Blood Cultures

Abstract

The diagnosis of Candida bloodstream infection (BSI) may rely on a PCR-based analysis of a positive blood culture (PBC) obtained from the patient at the time of BSI. In this study, a yeast DNA extraction protocol for use on PBCs was developed and evaluated with the molecular mouse (MM) yeast blood (YBL) chip-based PCR assay, which allowed us to detect nine medically relevant Candida species. We studied 125 simulated or clinical PBCs for Candida species. A positive correlation between the DNA concentration and colony-forming unit count was found for simulated (Spearman's ρ = 0.58; p < 0.0001) and clinical (Spearman's ρ = 0.23, p = 0.09) PBCs. The extracted DNA yielded positive results with the MM YBL chip assay that agreed with the Candida species-level identification results for 63 (100%) of 63 isolates from simulated PBCs and 66 (99.5%) of 67 isolates from clinical PBCs. The false-negative result was for one C. tropicalis isolate that grew together with C. albicans in PBC. None of the 30 (Candida)-negative clinical BCs included as negative controls yielded a positive result with the MM YBL chip assay. Our DNA extraction protocol for the Candida species couples efficiency and simplicity together. Nevertheless, further studies are needed before it can be adopted for use with the MM YBL chip assay.

Keywords: Candida species; PCR assay; blood culture; bloodstream infection; molecular detection; yeast blood chip.

Figure 1

In-house protocol steps to extract DNA from PBC samples for Candida species. Once the process is complete, the DNA solution is available for testing within approximately 60 min of sample collection.

Figure 2

Workflow for testing a DNA sample with the MM YBL chip assay. It takes less than one hour from the time the sample-loaded chip is inserted into the MM instrument until the sample result is obtained.

Figure 3

Relationship between DNA concentration and CFU count for simulated or clinical PBC samples for Candida species. According to Spearman’s correlation coefficient (ρ), a positive correlation was observed for both sample groups.

Figure 4

Distribution of PCR Ct values for simulated or clinical PBC samples with DNAs that were tested with the MM YBL chip assay. Values were grouped according to the Candida species detected. In each scatter dot plot (a different color is used to mark each Candida species), the central line indicates the mean Ct value, and the area between the top and bottom lines indicates the standard deviation value. There was statistical significance (p < 0.001) between groups of simulated or clinical PBC samples, as assessed using a one-way analysis of variance (ANOVA) with Tukey’s multiple-comparison test. Ct, cycle threshold.