TABLE 13.

Comparison of MALDI-TOF and nucleic acid sequencing methods for pathogen identification

Comparison	MALDI-TOF	Sanger 16S sequencing	Next-generation sequencing
Availability	Widespread adoption	Limited in clinical labs	Limited in clinical labs
	Application specific	Universal use possible for bacteria/fungi	Sent to a core facility with shared instrumentation
		Suitable for typing/molecular resistance	Universal use for other microorganisms
			Expansion to typing/molecular resistance
Procedure complexity	Low	High; little or no automation	High-limited (application-specific) automation
		Lower when using a commercial kit	Kits available for library preparation
		CLSI MM18-A2 guides analyses
Accuracy	High depending on microorganism/group being interrogated	High if several regions covered (long reads with excellent coverage of 16S feasible	Whole-genome sequencing provides full coverage of 16S operons
	Applicable to pure cultures	Sequences must be edited and trimmed	Application to pure cultures
		Application to pure cultures or normally sterile clinical samples with a single pathogen	Usually less sensitive depending on assembly processes
			Intraoperon diversity problematic
			All pipelines need to be thoroughly developed for various clinical application
			Applicable to pure cultures
Databases	Ongoing development limited for various microorganisms/groups	Covers all microorganisms/groups	Rapidly expanding deposit of WGS data in public databases without curation
	Requires ongoing validation against phenotypic and sequencing results	Several public databases are outlined in Table 2, but coverage for bacterial species of interest and reliability of annotation and sequences must be assessed	Some freely accessible target-specific 16S databases outlined in Table 2 can be used (i.e., in packages such as Qiime, www.qiime.org), but coverage for bacterial species of interest and reliability of annotation and sequences must be assessed
		Commercial databases also available	Whole genomes are available under the genome section of NCBI/GenBank and can be searched using BLAST
			Commercial databases are also available as part of an analysis package
Cost/test	Low, but MS instrument cost, maintenance, and database use need to be considered	High when using a commercial kit linked to specific instruments and reagents	High, but costs decrease when pooling samples
			Cost/sample is rapidly decreasing with increased throughput and read length and depends on read length
Instrumentation	MALDI-TOF MS instruments expensive but supplied by Becton-Dickinson (Bruker) or bioMérieux (Shimatzu)	Requires purchase of an automated genetic analyzer	Requires purchase of one or more NGS instruments, maintenance contracts, and reagent-rental agreements
		Capillary electrophoresis columns must be regularly maintained and replaced	Shared core facility to minimize costs
		Separate installation to avoid contamination	Separate installation to avoid contamination
Quality assurance	Laser must be regularly calibrated	Appropriate controls must be included with each run	Appropriate controls much be included with each run
	QA organisms should be regularly run to verify performance	Appropriate controls must be used for each step of the procedure	Appropriate controls must be included with each step of the procedure
		Sequence trimming/editing allows identification of contamination problems	Checks on read generation, read filtering (elimination of nonspecific reads), mean read length, phred scores, concatenation/assembly efficiency must be done to ensure quality results
Testing capacity and throughput	Hundreds of individual isolates per day can be analyzed depending on the no. of instruments used	Suitable for single and few samples	Higher throughput than Sanger depending on the method and instrument being used
		No more than 8–12 isolates can be run in a day	Pooling of samples is customary to reduce the per-sample cost
		Throughput has never been automated
Data analysis	MALDI-TOF immediately provide an answer by analysis of an isolates spectral profile against the onboard database	Complex and general understanding of BLAST and alignments	Complex and a major barrier to clinical implementation
		Requires sequence editing and analysis against a reference sequence	Requires appropriate storage of large amounts of sequence data
		A multialignment against a close reference sequence should be performed	Requires knowledge in bioinformatics and general informatics
		Reduced errors using curated commercial or online reference database	Delayed results often taking days to complete
		Reliable results analysis using CLSI MM18-A2 guideline