Intro -- FOREWORD -- ACKNOWLEDGEMENTS -- ACRONYMS AND ABBREVIATIONS -- CHAPTER 1 Introduction -- 1.1 Aims and objectives -- 1.2 Microbiology and clinical aspects of diphtheria and other related infections -- 1.2.1 C. diphtheriae -- 1.2.2 C. ulcerans -- 1.2.3 C. pseudotuberculosis -- 1.3 Transmission and carriage -- 1.4 Serological testing for population and individual immunity/susceptibility to diphtheria -- 1.5 Role of the laboratory in the diagnosis of diphtheria -- 1.5.1 Roles of reference laboratories -- 1.5.2 Specialized testing -- 1.5.2.1 Molecular epidemiological studies -- 1.5.2.2 Clinical diagnostic microbiology laboratories -- 1.5.3 Notification of potentially toxigenic strains of C. diphtheriae, C. ulcerans or C. pseudotuberculosis -- 1.6 Innovations in diphtheria diagnosis and analysis -- 1.6.1 Tests for detection of diphtheria toxin -- 1.6.2 Molecular typing and gene sequencing -- CHAPTER 2 Procedures for collection, storage transportation of clinical samples and revival of isolates -- 2.1 Criteria for screening suspected specimens of Corynebacterium species -- 2.2 Specimen collection, storage and transport from suspected cases of respiratory or cutaneous diphtheria, and contacts -- 2.2.1 Collection of samples for laboratory examination -- 2.2.2 Transport, preservation, storage and revival of cultures -- CHAPTER 3 Procedures to isolate and biotype C. diphtheriae, C. ulcerans and C. pseudotuberculosis -- 3.1 Laboratory procedures for primary isolation of potentially toxigenic corynebacteria -- 3.2 Primary culture and isolation -- 3.3 Criteria for recognizing suspect colonies that require further evaluation -- 3.4 Presumptive identification and screening of potentially toxigenic and non-toxigenic Corynebacterium species -- 3.4.1 Cystinase test (Tinsdale).

3.5 Microscopic examination and staining procedures for suspect colonies / cultures -- 3.5.1 Gram stain -- 3.6 Biochemical identification -- 3.6.1 API®-Coryne test system -- 3.6.1.1 Control strains specifically for the API® Coryne system -- 3.6.1.2 Quality control for the API®-Coryne system -- 3.7 Minimal laboratory criteria for reporting a specimen as culture positive -- 3.8 Laboratory data reporting -- CHAPTER 4 Phenotypic detection of toxigenicity: Elek test -- 4.1 Recognition and significance of non-toxigenic C. diphtheriae -- 4.2 Methodology to detect the diphtheria toxin: Elek test -- 4.3 Control strains and other quality recommendations -- CHAPTER 5 Automated identification systems -- 5.1 MALDI-TOF MS -- 5.1.1 MALDI-TOF Biotyper® and VITEK® MS -- 5.1.2 Strain relatedness using the MALDI-TOF MS assay -- 5.1.3 Measuring and interpreting MALDI-TOF MS results -- 5.2 VITEK® 2 -- 5.3 BD Phoenix™ system -- 5.4 MicroSEQ® microbial identification system -- CHAPTER 6 Molecular methods confirming the presence of toxigenic C. diphtheriae, C. ulcerans, C. pseudotuberculosis -- 6.1 PCR for detecting diphtheria toxin gene -- 6.2 PCR in the context of an outbreak investigation -- CHAPTER 7 Molecular typing and gene sequencing -- 7.1 Molecular typing of Corynebacterium species -- 7.1.1 MLST -- 7.1.1.1 MLST of C. ulcerans -- 7.1.2 goeBURST analysis -- 7.1.3 Locus variant analysis -- 7.2 Gene sequencing for identifying Corynebacterium species -- 7.3 Novel advances in genomics and proteomics -- 7.3.1 WGS -- 7.3.2 Transcriptomics -- CHAPTER 8 Procedures for serological testing to assess individual and population immunity/susceptibility to diphtheria -- 8.1 Procedures for assaying diphtheria antitoxin -- 8.2 ELISA assays -- 8.2.1 Performance characteristics -- 8.2.2 Bead-based multiplex assay or multiplex immunoassay.

CHAPTER 9 Procedures for antimicrobial susceptibility testing of C. diphtheriae, C. ulcerans and C. pseudotuberculosis -- 9.1 Purpose of antimicrobial susceptibility testing -- 9.2 Review of common treatment choices and in vitro susceptibility of C. diphtheriae -- 9.3 Methods for antimicrobial susceptibility testing of Corynebacterium species -- CHAPTER 10 Quality management -- 10.1 Basis of laboratory quality assurance -- 10.2 Staff and staffing levels -- 10.3 Space allocation -- 10.4 Standard Operating Procedures -- 10.5 Documentation and equipment -- 10.6 Reference materials and reagents -- 10.7 Laboratory safety -- 10.8 Audits and accreditation -- 10.9 Training workshops and internal quality assurance/external quality assurance -- 10.10 Benefits of EQA -- References -- ANNEX 1 Collecting clinical specimens -- A1. Procedures for collecting samples for the laboratory diagnosis of diphtheria -- A1.1 Materials required for clinical sampling: -- A1.2 Oropharyngeal/throat swabs -- A1.3 Nasopharyngeal swabs -- A1.4 Nasal swabs -- 1. -- 2. -- 2.3 -- 2.3.4 -- 2.3.5 -- A1.5 Cutaneous lesions -- 3.3.4 -- 3.3.5 -- A1.6 Pseudomembrane -- ANNEX 2 Temporary storage and transportation of isolates -- A2.1 Silica gel packets for temporary storage and transportation of swabs and bacterial isolates -- A2.1.1 Use of silica packets for temporary storage and transporting bacterial cultures -- A2.1.2 Procedure for storage and transporting isolates or clinical swabs: -- A2.1.3 Procedure for retrieving isolates transported in silica packets: -- ANNEX 3 Long-term storage of strains -- A3.1 Storage of strains -- A3.1.1 Glycerol broth 16% (v/v) for frozen storage of isolates. -- A3.1.2 Skimmed milk tryptone glucose glycerol (STGG) medium -- A3.1.2.1 Preparation of STGG medium -- A3.1.2.2 Cryobeads -- A3.2 Reviving strains from frozen glycerol blood broth.

A3.3 Reviving isolates preserved on cryobeads -- A3.4 Loeffler's serum slopes -- ANNEX 4 Preparation of bacteriological media -- A4.1 Blood agar plates - 5% to 10% horse or sheep blood agar plate -- A4.2 Columbia blood agar -- A4.3 Tellurite-containing blood agar plate (Hoyle's Tellurite) -- A4.4 Tinsdale medium -- A4.5 PIZU medium for detecting cystinase -- A4.5.1 Preparation of medium -- A4.5.2 Control strains -- ANNEX 5 Screening and identification tests -- A5.1 Tinsdale -- A5.1.1 Reading test -- A5.2 Pyrazinamidase test -- ANNEX 6 Staining methods for laboratory identification of C. diphtheriae -- ANNEX 7 Elek toxigenicity test -- A7.1 Elek agar medium materials and preparation -- A7.2 Preparation of Elek agar medium -- A7.3 Preparation of Elek agar medium plates -- A7.4 Preparation of antitoxin strips and discs -- A7.5 Setting up the Elek toxigenicity test -- A7.5.1. Recommended controls: -- A7.6 Reading test -- A7.7 Interpretation of the Elek test -- ANNEX 8 Matrix-assisted laser desorption ionization - time of flight (MALDI-TOF) mass spectrometry (MS) as a tool for rapid identification of Corynebacterium species -- A8.1 Matrix solution composition -- A8.2 Sample preparation (protein extraction protocol) -- 1. -- 2. -- 3. -- 4. -- 5. -- 6. -- 7. -- 8. -- 8.1 -- 8.2 -- A8.3 Measurements and interpretation of MALDI-TOF MS results -- A8.4 QC systems -- A8.5 Maintenance of the MALDI-TOF system -- A8.6 Discrepancies/Troubleshooting -- ANNEX 9 Conventional and qPCR -- A9.1 Sample preparation from bacterial cultures (de Zoysa et al. 2016) - crude extraction/boiling -- A9.1.1 Alterative sample preparation for bacterial cultures and clinical specimens (using extraction kit) -- A9.2 PCR mixture preparation: conventional and modified sample preparation -- A9.2.1 Conventional PCR mix preparation -- A9.2.2 Modified conventional PCR mix preparation.

This is an example of a simplified PCR mix, using the HotStarTaq Mastermix (Qiagen). This Taq DNA polymerase requires a 15-minute denaturation step at the start of the PCR. -- A9.2.3 Analysis of PCR products: conventional gel electrophoresis -- A9.2.3.1 Electrophoresis of the PCR products -- A9.2.4 Using pre-cast gels (Invitrogen E-Gels) for running PCR products -- Wear gloves and plug E-Gel PowerBase to the electricity socket. -- A9.3 qPCR for detection of tox gene and Corynebacterium species (DeZoysa et al. 2016) -- A9.3.1 Purpose and rationale of Corynebacterium species qPCR assay -- A9.3.2 Type of sample -- A9.3.3 Type of RT-PCR assay: dual labelled hybridization probes -- A9.3.4 Materials -- A9.3.5 DNA extraction -- A9.3.6 Preparation of a 20x primer/probe mix -- A9.3.7 Data analysis -- A9.3.8 Interpretation of results -- A9.4 Modifications to the real-time multiplex qPCR -- ANNEX 10 Multilocus sequence typing -- A10.1 MLST for C. diphtheriae -- A10.1.1 PCR reaction preparation (to be done for each allele) -- A10.2 Multilocus Sequence Typing (MLST) of C. ulcerans -- ANNEX 11 ELISA assays -- ANNEX 12 Diphtheria antitoxin assay in Vero cell (in vitro toxin neutralization) -- 12.1 Materials -- 12.1.1 Critical reagents and standards -- 12.1.2 Equipment -- 12.1.3 Buffers and reagents -- 12.1.4 Complete culture medium for Vero cells -- 12.2 Procedures -- 12.2.1 Culture, harvesting and counting of Vero cells -- 12.2.2 Determination of the test dose of diphtheria toxin -- 12.2.3 Staining of Vero cells -- 12.2.4 Determination of potency of the antitoxin -- 12.3 Calculation of results -- 12.4 Validity of the test -- ANNEX 13 Antimicrobial Susceptibility Testing -- A13.1 Materials required for disk and gradient MIC strips (E-test) antimicrobial susceptibility testing -- A13.2 AST methods -- A13.2.1 Disk diffusion by Kirby-Bauer method.

A13.2.2 Preparation of bacterial inoculum for disk susceptibility and MIC.