Clinical and Laboratory Standards Institute guideline MM18—Interpretive Criteria for Identification of Bacteria and Fungi by Targeted DNA Sequencing includes information intended for use with molecular diagnostic testing procedures published in CLSI documents MM031 and MM09.2 These guidelines contain information about developing, evaluating, and applying nucleic acid–based testing for infectious diseases in medical laboratories. Historically, microorganism identification has relied on phenotypic methods and, more recently, matrix-assisted laser desorption/ionization time-of-flight technology. Patient isolates for bacterial and fungal identification may have ambiguous biochemical profiles or mass spectra and cannot be reliably characterized. Laboratories can apply broad-range DNA sequencing for microorganism identification and as a standardized, portable method for data sharing. This guideline includes the most current information for microbial classification by targeted DNA sequencing, with particular emphasis on interpreting and reporting results.

This guideline replaces the previous edition of the approved guideline, MM18-A, published in 2008. Several changes were made in this edition, including: 

• Reorganized to fit the CLSI quality management system and path of workflow format 

• Revised all bacteriology tables (Tables 6 to 14) to reflect current taxonomy and to outline where sequencing of the 16S rRNA gene’s V1-V3 region (ie, first ˜ 500 base pairs) provides genus- and/or species-level identification and where diversity occurs within the entire gene to distinguish each genus and/or species 

• Deleted table on bacterial agents of bioterrorism and its introductory text and added discussion of each agent to the group-specific tables (Tables 6 to 14) 

• Revised all organism tables to include information on how MALDI-TOF MS may be used to complement sequencing for identification 

• Deleted all dendrograms 

• Revised organism tables to include emerging, clinically relevant microorganisms 

• Updated organism nomenclature

This guideline specifies recommendations for interpreting and reporting results of Sanger-based (dideoxynucleotide chain termination) sequencing of broad-range DNA targets for identifying pure isolates of bacteria, mycobacteria, and fungi from cultured patient isolates. Partial- and full-gene sequencing with 16S ribosomal RNA (rRNA) genes for bacterial and mycobacterial identification, as well as internal transcribed spacer (ITS) regions (ie, ITS-1 and ITS-2) for fungal identification are covered, including alternative DNA targets when appropriate. Although massively parallel (next-generation) sequencing technologies are rapidly emerging, this guideline’s scope is limited to Sanger-based targeted DNA sequencing. 

To assist the medical laboratory, guidance is provided for: 

• Selecting DNA targets and sizes for amplification and sequencing 

• Establishing QC parameters for amplification and sequencing 

• Measuring sequence quality 

• Assessing reference sequences and databases 

• Comparing sequences for identification 

• Establishing interpretive criteria for identity scores generated by targeted DNA sequencing 

• Developing clinically relevant reporting strategies for specific microorganism groups 

• Identifying the limitations of targeted DNA sequencing for microbial identification The intended users of this guideline are medical laboratories and laboratories performing amplification and Sanger-based (dideoxynucleotide chain termination) sequencing of broad-range DNA targets for identifying bacteria, mycobacteria, and fungi from cultured patient isolates. 

This guideline does not: 

• Include procedures for performing microbial sequencing. 

• Include RNA targets for sequencing. 

• Provide guidance on definitive taxonomical criteria for microorganism classification or identification methods for novel microorganisms. 

• Cover alternative sequencing systems or specific molecular assays designed with these broad-range DNA targets. 

• Discuss typing strains for epidemiological purposes. 

• Discuss virus or parasite identification. 

• Discuss amplification and sequencing directly from patient specimens.