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Table 1 Advantages and limitations of approaches for clinical diagnosis of microbes

From: The dynamic wound microbiome

Technique Definition Pros Cons Used in clinics
Conventional culture Growth in culture to isolate a pure sample followed by phenotypic analysis. Identification of features such as the ability to grow on specific culture mediums, antibiotic resistance, and biochemical attributes such as the ability to alter particular substrates. Possible only for a small fraction of organisms, and highly variable growth rate may lead to biases in detection.
Additionally, the method is slow and takes 48–72 h to generate results.
Microscopy Microscopy-based approach that utilizes a short culture period followed by fluorescence-based tagging of microbes with antibodies and DNA probes. The sample then flows across the microscope field with automated computer-based identification of both microbial shape and automated fluorescence detection. A large number of samples can be processed rapidly. Biases in culture approaches to enrich the microbes, and in detection. Difficult to adapt to non-bacterial pathogens. Yes
Mass spectrometry Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry for the identification of bacteria. A pure culture is ionized and the mass spectra of the resulting protein fragments compared to a reference database to match patterns of known organisms. Recent advances to the method allow cruder preparations to be analyzed and even negate the need for culture. Rapid and low-cost once the initial equipment has been installed. Difficulty in detecting drug resistance and virulence, and in detecting viruses, fungi, and parasites. Additionally, complex mixtures of organisms can present an unsolvable mass spectrum making its utility best suited to scenarios in which only a single organism is expected. Yes
Polymerase chain reaction Sequencing of specific unique sequences in the genomes or transcriptomes of organisms, leading to the creation of an amplicon used to detect the presence of that organism. Additionally, primers can be designed to indicate the presence of drug resistance and virulence gene sequences. Rapid and low-cost. Limited range of organisms that can be identified. Inability to identify organisms that are not present in the panel design, and the assumption that target sequences is unique to a particular organism. Yes
Microarray Multiple DNA probes are fixed to a solid surface and hybridized to sample DNA fragments that are modified (typically with fluorescent tags or a means to generate fluorescence following hybridization) that allow detection of probes with hybridized sample DNA. Ability to detect and identify a broad range of organisms and their drug resistance and virulence sequences in a single assay. Can be used to analyze mixtures of organisms. Difficulties in designing appropriate probes, difficulties in distinguishing organisms at the strain level. Issues of specificity and time required to perform the assay. No
DGGE/TGGE on the SSU rRNA gene Molecular fingerprinting targeting the SSU rRNA gene. Rapid visualization of prokaryotes community changes. Do not allow the identification of the specific prokaryotic species involved. No
16S rRNA sequencing Amplicon sequencing on the SSU rRNA gene. Allow the identification and characterization of microbial diversity. Do not allow strain-level description of the community, do not take into account the viral and eukaryotic fraction of the population. No
Metagenomic next-generation sequencing (mNGS) DNA is sheared randomly into small segments and sequenced. Taxonomic identification of bacteria to the species or strain level, detection of virulence factors and antibiotic-resistance genes. High amounts of host DNA contamination, high cost of sequencing. No