Candida spp. DNA Extraction in the Age of Molecular Diagnosis

Abstract

The standard procedure for the detection of candidemia is blood culture, a method that might require 3–5 days for a positive result. Compared with culturing, molecular diagnosis techniques can provide faster diagnosis. The current paper aimed to present the main strengths and constraints of current molecular techniques for Candida spp. DNA extraction, analyzing their efficiency from a time, price, and ease of usage point of view. A comprehensive search was conducted using the PubMed NIH database for peer-reviewed full-text articles published before October 2022. The studies provided adequate data on the diagnosis of the infection with the Candida spp. DNA extraction is a relevant step in yielding pure qualitative DNA to be amplified in molecular diagnostic techniques. The most used fungal DNA extraction strategies are: mechanical (bead beating, ultrasonication, steel-bullet beating), enzymatic (proteinase K, lysozyme, lyticase), and chemical extraction (formic acid, liquid nitrogen, ammonium chloride). More clinical studies are needed to formulate adequate guidelines for fungal DNA extraction as the current paper highlighted discrepancies in the reported outcome.

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

“…Over the past few decades, accumulated experience with manual (including in-house) or automated DNA extraction protocols for Candida (or other yeast) species has shown that the yield of DNA extracted from whole blood samples can be variable (limit of detection ranges from 10 0 to 10 6 CFU/mL for manual protocols and from 10 1 to 10 6 CFU/mL for automated protocols) [ 11 ]. This variability is imputable as to whether pretreatment (mechanical, enzymatic, chemical, physical, or thermal) or no treatment was applied to the sample before proceeding with DNA isolation from Candida cells [ 11 ].…”

“…Over the past few decades, accumulated experience with manual (including in-house) or automated DNA extraction protocols for Candida (or other yeast) species has shown that the yield of DNA extracted from whole blood samples can be variable (limit of detection ranges from 10 0 to 10 6 CFU/mL for manual protocols and from 10 1 to 10 6 CFU/mL for automated protocols) [ 11 ]. This variability is imputable as to whether pretreatment (mechanical, enzymatic, chemical, physical, or thermal) or no treatment was applied to the sample before proceeding with DNA isolation from Candida cells [ 11 ]. Very recently, Menu et al [ 10 ] used human blood samples artificially spiked with the Candida species ( C. albicans , C. glabrata , C. parapsilosis , C. tropicalis , and C. krusei , for which inoculum sizes ranged from 0 to 10 8 CFU/mL) to evaluate the efficiency of 11 automated DNA extraction protocols.…”

“…In both assays, multiplexed nucleic acid extraction, amplification, and amplicon detection are performed automatically using all reagents contained in one pouch (FilmArray BCID2 panel) or cartridge (BCID-FP panel). The performance of these assays may, however, be suboptimal [ 9 ], perhaps due to the inefficient recovery of DNA from fungal (yeast) cells [ 10 , 11 ]. It should be recalled that the rigid cell walls of Candida (or other fungi) make sample preparation very complex, possibly requiring chemical, physical, or enzymatic steps before PCR amplification is performed [ 12 ].…”