Clinical Microbiology Procedures Handbook, Multi-Volume

Gold Standard consensus-based procedures from the experts.

The Clinical Microbiology Procedures Handbook, 5^th edition, provides those engaged in microbial analysis of clinical specimens with procedures for the detection, identification, and characterization of microorganisms involved in human infections. This unique and valuable collection of step-by-step descriptions of the numerous testing modalities used in the clinical microbiology laboratory was written and edited by highly knowledgeable laboratorians. The 5^th edition features two new sections, one on blood cultures and one on MALDI-TOF MS, and the sections on molecular diagnostics, virology, and serology were extensively revised and updated. Presented over multiple volumes, this handbook enables laboratory staff to perform all analyses, including appropriate quality control recommendations, from the receipt of the specimen through processing, testing, interpretation, presentation of the final report, and subsequent consultation.

If you are looking for online access to the latest from this reference or site access for your lab, please visit www.wiley.com/learn/clinmicronow.

SECTION 1           Procedure Coding, Reimbursement, and Billing Compliance

SECTION EDITORS: Vickie Baselski and Alice Weissfeld

1.1.        Introduction

Vickie Baselski and Alice Weissfeld………………………………………………………………………………………..1.1.1

1.2.        Procedure Coding, Reimbursement, and Billing Compliance

Vickie Baselski and Alice Weissfeld…………………………………………………………………………………….1.2.1.1

1.2.1 Procedure Coding……………………………………………………………………………………………………..1.2.1.1

1.2.2 Procedure BillingB………………………………………………………………………………………………...1.2.2.1

1.2.3 Coverage of Laboratory Services………………………………………………………………………………1.2.3.1

1.2.4 Billing Compliance Programs…………………………………………………………………………………….1.2.4.1

SECTION 2          

Specimen Collection, Transport, and Acceptability

SECTION EDITORS: Andrea Linscott and Huanyu Wang

2.1.        Collection, Transport, and Manipulation of Clinical Specimens and Initial Laboratory Concerns

Andrea Linscott………..2.1.1

Table 2.1-1.  “Rule-Out” Clinical Impressions and Potential Etiological Agents………..2.1.6

Table 2.1-2.  General Principles for Specimen Collection………..2.1.17

Table 2.1-3.  Common Transport Media………..2.1.17

Table 2.1-4.  Collection of Specimens for Bacteriological Analysis………..2.1.18

Table 2.1-5.  Rejection Criteria for Microbiological Specimens………..2.1.25

Table 2.1-6.  Collection of Specimens to Detect Infrequently Encountered Organisms………..2.1.28

Table 2.1-7.   Collection of Specimens for Virological Analysis…2.1.30

Table 2.1-8.  Laboratory Approaches to Suspected Fungal Infections………..2.1.34

Table 2.1-9.  Collection of Specimen to Detect Parasites………..2.1.35

Table 2.1-10.  Specimen Processing Triage………..2.1.37

Table 2.1-11.  Procedure for Processing Clinical Specimens in Microbiology………..2.1.37

Table 2.1-12.  Critical in Microbiology………..2.1.39

Table 2.1-13.  Alert Request………..2.1.39

SECTION 3           Aerobic Bacteriology

SECTION EDITOR: Amanda T. Harrington and Melanie L. Yarbrough

3.1.        Introduction

Amanda T. Harrington and Melanie L. Yarbrough…...……..……..……..……..……..……..……..……..3.1.1

3.2.        Staining Procedures

                Steven Dallas and Amanda T. Harrington………………………………………………………………………..3.2.1.1

3.2.1.  Gram Stain………..……..……..……..……..……..……..……..……..……..……..……..……..…………....3.2.1.1

Appendix 3.2.1–1.  Preparation of Gram Stain Reagents…………..……..……..……..3.2.1.X

Appendix 3.2.1–2.  Rejection Criteria for Sputum

Culture……………..……..……..……..……..……..……..……..……..……..……..……....3.2.1.X

Appendix 3.2.1–3.  Reporting Gram-Stained Vaginal Smears To Diagnose Bacterial

Vaginosis and Vaginitis…………..……..……..……..……..……..……..……..……..…..3.2.1.X

3.2.2.  Acridine Orange Stain …………..……..……..……..……..……..……..……..……..……..……..…..3.2.2.1     

Appendix 3.2.2–1.  Preparation of Acridine Orange Stain…………..……..……..……..…..3.2.2.X

3.2.3. Vaginal Wet Mount…………………………………………………………………………………………..….3.2.3.1

3.2.4. Wet Mount for Detection of Leukocytes and Microorganisms ………..……..……..…....3.2.3.1

3.3.         Processing, Isolation, Detection, and Interpretation of Aerobic Bacteriology Cultures

Amanda T. Harrington and Melanie L. Yarbrough ..……..……..……..……..……..……..……..……..3.3.1.1

3.3.1.  Processing of Specimens for Aerobic Bacteriology …………..……..……….3.3.1.1

3.3.2.  Interpretation and Rapid Identification of Bacterial Growth on Primary Culture Media...3.3.2.1

3.4.         Body Fluid Cultures (Excluding Blood, Cerebrospinal Fluid, and Urine)

                Nicholas Moore……………..……..……..……..……..……..……..……..……..……..……..……..……..……....3.4.1

3.5.        Cerebrospinal Fluid Cultures

Rangaraj Selvarangan ..……....……..……..……..……..……..……..……..……..……..……..……..……..…..3.7.1

3.6          Medical Devices: Pre- and Postimplant testing……………………………………………………………………3.6.1

3.7.         Fecal and Other Gastrointestinal Cultures and Toxin Assays

Dylan R. Pillai and Thomas Griener.……..……..……..……..……..……..……..……..……..……..……..…..3.7.1.1

                3.8.1.  Fecal Culture for Aerobic Pathogens of Gastroenteritis …………..……..……..……..…..3.8.1.1

                                Appendix 3.7.1–1.  Detection of Somatic O Antigen Serogroups of Bacteria………..3.8.1.X

                                Appendix 3.7.1–2.  Detection of Escherichia coli O157 by Latex

Agglutination………………………………………………………………………………………….3.8.1.X

                                Appendix 3.7.1–3. Detection of Shiga Toxin by Immunochromatographic Assay…3.7.1.X

                3.7.2.  Fecal Culture for Campylobacter and Related Organisms ……..……..……..……..………..3.7.2.1  

                3.7.3.  Helicobacter pylori Cultures ………..……..……..……..……..……..……..……..……..……..……..3.7.3.1

                                Appendix 3.8.4–1.  Helicobacter pylori Antigen Assay (HpSA)…… ……..……..……..…..3.7.3.X

                3.7.4.  Quantitative Culture of Small-Bowel Contents……………………………..……..……..……..3.7.4.1

3.8.         Genital Cultures

                Laura Filkins and Omai Garner……………………………………………………………………………………….…3.8.1.1

3.8.1.  Guidelines for Performance of Genital Cultures

3.8.2.  Group B Streptococcus Cultures ……………………………..………..……..……..……..……..……..3.8.2.1

3.8.3.  Neisseria gonorrhoeae Cultures …………………………..……………..……..…….…..……..……..3.8.3.1

3.8.4.  Haemophilus ducreyi Cultures ………………………….……..……..……..……..……..……..……...3.8.4.1

3.9.         Ocular Cultures

                Eileen Burd………….……………..……..……..……..……..……..……..……..……..……..……..…………..……....3.9.1

3.10.      Respiratory Tract Cultures………..………..……..……..……..……..……..……..……..……..…….…..3.10.1.1

3.10.1.  Guidelines for Performance of Respiratory Tract Cultures • Kevin Alby and Jason Smedberg………..……..……..……..……..……..……..……..……..…….…..3.10.1.1

3.10.2.  Lower Respiratory Tract Cultures • Kevin Alby and Jason Smedberg.…..…….3.10.2.1

Appendix 3.11.2–1.  Quantitative Culture of Protected Specimen Brush and

Bronchoalveolar Lavage Fluid Specimens……..……..……..……..……..………..3.10.2.X

3.10.3.  Respiratory Cultures from Cystic Fibrosis Patients • Kevin Alby and Jason Smedberg ……..……..……..……..……..…………………………………………………………...……..3.10.3.1

3.10.4.  Legionella Cultures and Urinary Antigen Testing• Diane SJ Lindsay and Maria L.  Ricci………………………………………………………………………………………………………………..3.10.4.1

                Appendix 3.10.4–1. Urinary Antigen Testing…………………………………………………..3.10.4.X

                3.10.5.  Ear Cultures • Melanie L. Yarbrough and Carey-Ann D. Burnham…………………..3.10.5.1

                3.10.6.  Bordetella Cultures • Amy L. Leber …………………………………..……..……..……..…..3.10.6.1

3.10.7.  Culture of Corynebacterium diphtheriae and Other Diphtheria Toxin-Producing Bacterial Species • Allen C. Bateman…………………………………………………….………..3.10.7.1

                3.10.8.  Throat Culture and Nonculture Tests for Pharyngitis • Mark D. Gonzalez………..3.10.8.1

                                Appendix 3.11.8–1. Beta-Hemolytic Streptococcal Latex Agglutination Method..3.10.8.X

                3.10.9.  Nasal Sinus Cultures • Morgan A. Pence………..……..……..……..……..……..………..…..3.10.9.1

3.11.      Urine Cultures

Melanie L. Yarbrough.……..……..……..……..……..……..……..……..……………………………………………..3.11.1

3.11.1.  Urine Cultures…………………………………………………………………………………………….……..3.11.1.1

                Appendix 3.11.1–1.  Validation of Urine Inoculation Methods………………………..…..3.11.1.X

3.11.2.  Stone Cultures……………………………………………………………………………………………...…3.11.2.1

3.12.      Wound Cultures

Alexandra Bryson and Christopher Doern…..……..……..……..……..…….……..……..……..………..3.12.1.1

                3.12.1.  Wound/Abscess and Soft Tissue Cultures …………..……..…………………………………..3.12.1.1

                3.12.2.  Quantitative Cultures of Wound Tissues …………..……..………..……..……..……..……..3.12.2.1

3.13.      Leptospira Culture

                Renee L. Galloway…………..……..……..………..……..……..………..……..……..………..……..……..……..3.13.1

                                Appendix 3.13–1.  Summary of Diagnostic Tests for Leptospirosis…………………….3.13.X

3.14.      Detection of Human Mycoplasmas and Ureaplasmas from Clinical Specimens by Culture and PCR

3.14.1.  Mycoplasma pneumoniae, Mycoplasma hominis, and Ureaplasma Cultures. • Cécile Bébéar and Ken B. Waites ……………………………………………………………………….3.14.1.1

                Appendix 3.14.1–1.  Medium Formulations for Cultivation of Mycoplasmas and Ureaplasmas from Humans………………………………………………………………………3.14.1.X

3.14.2.  Antimicrobial Susceptibility Testing of Mycoplasmas and Ureaplasmas. • Cécile Bébéar and Ken B. Waites ……………..……..……..………..……..……..……………………………..3.14.2.1

3.14.3.  Detection of Mycoplasma pneumoniae by nucleic acid amplification tests. • Cécile Bébéar ………………………………………………………………………………………………………..3.14.3.1

3.14.4.  Detection of Mycoplasma genitalium, Mycoplasma hominis and Ureaplasma species by nucleic acid amplification tests. • Cécile Bébéar ………..3.14.4.1

3.15.      Bartonella Cultures

                Jill Clarridge and Amanda T. Harrington……………………………………………………………….3.15.1

3.16.      Guidelines for Biochemical Identification of Aerobic Bacteria

                James J. Dunn…………..……..……..………..……..……..………..……..……..………..……..……..……..3.16.1

3.17.      Biochemical Tests for the Identification of Aerobic Bacteria

Richard Davis and Marie Pezzlo…..……..………..……..……..…………..……..……..…..3.17.1.1

                3.17.1.  Acetamide Utilization Test (Acetamide Agar) …………..……..……..………..……..……17.1.1

                3.17.2.  Acetate Utilization Test – (Acetate Differential Agar) …………..……..……..………3.17.2.1

3.17.3.  ALA (δ-Aminolevulinic Acid) Test for Porphyrin Synthesis – Tube or Disk Test ..3.17.3.1

3.17.4.  Antimicrobial Disk Tests for Identification ……..3.17.4.1

3.17.5.  Bile-Esculin and Esculin Tests ………..……..……..………..……..……..………..3.17.5.1

3.17.6.  Bile Solubility Test ……………..……..……..………..……..……..………..……..……..…..3.17.6.1

3.17.7.  Butyrate Esterase Test…….……..……..……..………..……..……..………..……..……..….3.17.7.1

3.17.8.  CAMP Factor Tests (Standard/ Rapid, Reverse and Inhibition) ………3.17.8.1

3.17.9.  Carbohydrate Utilization Tests ………..……..……..………..……..……..………..3.17.9.1

3.17.10.  Catalase Test ……..……..……..………..……..……..………..……..……..………..……..……..…3.17.10.1

3.17.11.  Cetrimide Test…………..……..……..………..……..……..………..……..……..…..3.17.11.1

3.17.12.  Citrate Utilization Test (Simmons) ………..……..……..………..……..……..………..3.17.12.1

3.17.13.  Coagulase Test—Protein A/Clumping Factor Agglutination Method…..3.17.13.1

3.17.14.  Coagulase Test—Rabbit Plasma Method ………..……..……..………..3.17.14.1

3.17.15.  Decarboxylase-Dihydrolase Tests…………..……..……..………..……..……..…..3.17.15.1

3.17.16.  DNase Test-Rapid Thermonuclease Test ………..……..……..………..……..……..……..3.17.16.1

3.17.17.  Fluorescent-Pigment Agars for Pseudomonas Identification….……..……..……...3.17.17.1

                3.17.18.  Gelatin Liquefaction……………..……..……..………..……..……..…..3.17.18.1

                3.17.19.  Gram Reaction Enzymatic Test…………..……..……..………..……..……..…..3.17.19.1

                3.17.20.  Hippurate Hydrolysis Rapid Test …………..……..……..………..……..……..……..3.17.20.1

                3.17.21.  Hydrogen Sulfide Production ……………..……..……..………..……..……..…..3.17.21.1

                3.17.22.  Indole Test ……………..……..……..………..……..……..………..……..……..…..3.17.22.1

                                Appendix 3.17.22–1.  Reagent Preparation…………..……..……..……..3.17.22.X

                3.17.23.  Indoxyl Acetate Disk Test…………..……..……..………..……..……..……..3.17.23.1

                3.17.24.  Kligler's Iron Agar Test and Triple Sugar Iron Agar Test.. ……..……..……..…..3.17.24.1

                3.17.25.  LAP (Leucine Aminopeptidase) Test …………..……..……..………..……..……..……..3.17.25.1

                3.17.26.  Lecithinase and Lipase Detection ……………..……..……..………..……..……..…..3.17.26.1

                                Appendix 3.17.26–1.  Preparation of Egg Yolk Agar Medium………..3.17.26.X

                3.17.27.  Lipophilism Test for Corynebacterium …………..……..……..………..……..……..…..3.17.27.1

                3.17.28.  Malonate Test ……………..……..……..………..……..……..………..……..……..…..3.17.28.1

                3.17.29.  MGP (Methyl Glucopyranoside) Test …………..……..……..………..……..……..……..3.17.29.1

                3.17.30.  Motility Tests ……….……..……..……..………..……..……..………..……..……..….3.17.30.1

                3.17.31.  MRS Broth ……………..……..……..………..……..……..………..……..……..…..3.17.31.1

                3.17.32.  MR-VP (Methyl Red–Voges-Proskauer) Tests ………..……..……..………..3.17.32.1

                3.17.33.  MUG (4-Methylumbelliferyl-β-D-Glucuronide) Test ……………..……..……..…..3.17.33.1

                3.17.34.  Nitrate/Nitrite Reduction Test ……………..……..……..………..……..……..…..3.17.34.1

                3.17.35.  O/129 Disk Susceptibility Testing for Vibrio and Aeromonas spp. …..3.17.35.1

                3.17.36.  ONPG (o-Nitrophenyl-β-D-Galactopyranoside) Test ……………..……..……..…..3.17.36.1

                3.17.37.  Optochin Susceptibility Test …………..……..……..………..……..……..……..3.17.37.1

                3.17.38.  Oxidase Test ……………..……..……..………..……..……..………..……..……..…..3.17.38.1

                3.17.39.  Phenylalanine Deaminase Test …………..……..……..………..……..……..……..3.17.39.1

                3.17.40.  PYR (L-Pyrrolidonyl-β-Naphthylamide) Test …………..……..……..……..3.17.40.1

                3.17.41.  Quellung Reaction for Streptococcus pneumoniae (Neufeld test) ………….…..3.17.41.1

                3.17.42.  6.5% Salt and Temperature Tolerance Test …………..……..……..……..3.17.42.1

                3.17.43.  Satellite Test ……………..……..……..………..……..……..………..……..……..…..3.17.43.1

                3.17.44.  SPS (Sodium Polyanethol Sulfonate) Disk Test ………..……..……..………..3.17.44.1

                3.17.45.  Starch Hydrolysis Test ……………..……..……..………..……..……..………..……..……..…..3.17.45.1

                3.17.46.  Urease Test …………..……..……..………..……..……..………..……..……..……..3.17.46.1

3.18.      Identification of Gram-Positive Bacteria

Kathryn Bernard…………..……..……..………..……..…………………………………………….…..……..3.18.1

3.19.  Identification of Gram-Negative Bacteria • Erik Munson and Raymon Podzoeaki………..3.19.1

SECTION 4           Anaerobic Bacteriology

SECTION EDITOR: Audrey N. Schuetz

4.1.        Introduction

Audrey N. Schuetz..........................................................4.1.1

4.2          Taxonomy Updates for Anaerobes

Samantha K. Shannon and Morgan A. Pence….4.2.1

4.3.        Specimen Selection, Collection, and Transport for Anaerobic Culture

Melphine M. Harriott and Kristen L. Brown…..4.3.1

4.4.        Culture Media for Anaerobes

Jennifer Dien Bard, Sarah Copsey-Mawer, and Mimi Precit...................................................4.4.1

4.4.1.  Primary Culture Media for Anaerobes…………………………………4.4.1.1

                Appendix 4.3.1–1.  Formulas of Media for Anaerobes

4.4.2.  Secondary Culture Media for Anaerobes………………………………..4.4.2.1

4.5.        Processing Specimens for Anaerobic Culture

                Trefor E. Morris………………………………………………..4.5.1

4.6.        Incubation Techniques for Anaerobic Bacteriology Specimens

Trefor E. Morris.......................................................4.6.1

4.7.        Examination of Primary Culture Plates for Anaerobic Bacteria

Trefor E. Morris.....................................................4.7.1

4.8.        A Practical Guide to the  Workup of Anaerobic Cultures

Samantha K. Shannon and Morgan A. Pence....... 4.8.1

4.9.        Rapid Disk, Spot Tests, and Other Rapid or Primary Methods for the Identification of Anaerobes

Andrew Clark and Margaret Ordonez Smith de Daines.........4.9.1.1

4.9.1. Introduction ................................................................. 4.9.1.1

4.9.2. Indole Test ........................................................... 4.9.2.1

4.9.3. Nitrate Disk Reduction Test ............................................ 4.9.3.1

4.9.4. Catalase Test ................................................................ 4.9.4.1

4.9.5. Identification by Using Special-Potency Disks .................... 4.9.5.1

4.9.6. Sodium Polyanethol Sulfonate Disk for Differentiation of Anaerobic Cocci... 4.9.6.1

4.9.7. Bile Test/Bacteroides Bile Esculin Agar for Differentiation of Anaerobic Gram Negative

Rods …………….4.9.7.1

4.9.8. Fluorescence ................................................................ 4.9.8.1

4.9.9. Lipase Test .................................................................. 4.9.9.1

4.9.10. Lecithinase Test .........................................................4.9.10.1

4.9.11. Pigment Production ....................................................4.9.11.1

4.9.12. Urease Test ...............................................................4.9.12.1

4.9.13. Gelatinase Production …………………………………………4.9.13.1

4.9.14 Alkaline Phosphatase ..................................................... 4.9.14.1

4.9.15. Glutamic Acid Decarboxylase ......................................... 4.9.15.1

4.9.16. L-Alanyl-Alanylaminopeptidase ........................................ 4.9.16.1

4.9.17. L-Proline-Aminopeptidase ............................................... 4.9.17.1

4.9.18. 4-Methylumbelliferone Derivative Substrates ..................... 4.9.18.1

4.9.19. Combination Enzymatic Tablets for Nitrophenol, Aminopeptidase, and

Methylumbelliferyl Substrates ................ 4.9.19.1

4.10.      Commercial Kit and Rapid Enzymatic Systems for the Identification of Anaerobes

Frances Valencia-Shelton and Jennifer Dien Bard ……4.10.1

4.11.      Storage and Stocking of Anaerobes

                Frances Valencia-Shelton and Kristen L. Brown…………………4.11.1

4.12.      Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight for the Identification of

Anaerobic Bacteria

Ashley L. Paulick and Sarah Copsey-Mawer.................... 4.12.1

4.13.      Anaerobic Gram-Negative Bacilli

Rosemary C. She and Dierdre L. Church.............................................. 4.13.1

4.14.      Anaerobic Gram-Positive Bacilli

Kathryn Bernard and Camille Hamula............................................... 4.14.1

4.15.      Anaerobic Cocci

Rosemary C. She and Deirdre L. Church.................................................................. 4.15.1

4.16.      Clostridioides (Clostridium) difficile as a Pathogen Involved in Antimicrobial Agent-Associated Diarrhea, Colitis, and Pseudomembranous Colitis

Karen C. Carroll and Ashley L. Paulick................................................ 4.16.1

Section 5: Blood Culture                                               

5.1          Introduction       Amanda Harrington        

5.2          Preanalytic Considerations and Laboratory Processing of Blood Culture   Amanda Harrington and Robert Tibbetts              

5.3          Continuous Monitoring Blood Culture Systemsf or Detection of Aerobic and Anaerobic Bacteria   Evann Hilt Evann Hilt and Sophonie Jean    

5.4          Lysis Centrifugation        Erin McElvania  

5.5          Continuous Monitoring Blood Culture Systems for Detection of Acid-Fast Bacilli and Fungi              Bill Lainhart and Paul Leuthy

5.6          Quality Control and Quality Assurance    Raquel Martinez              

5.7          Culture Methods for Detection of Aerobic and Anaerobic Bacteria from Positive Blood Cultures   Evann Hilt and Sophonie Jean         

5.8          Culture Methods for Detection of Acid-fast Bacilli and Fungi from Positive Blood Culture Bill Lainhart and Paul Leuthy 

5.9          Post Analytical Considerations for Blood Culture Joan-Miquel Balada-Llasat and Kamal Kamboj    

5.10.      Supplementation of Positive Blood Culutres with Chromogenic Media      Matthew Faron

5.11        Specialized Processing of Blood  Robin Chamberland       

5.11.1    Transfusion Reactions    Robin Chamberland       

5.11.2    "Autopsy Blood Cultures               "             Robin Chamberland       

5.11.3    Special Media and Stains for Fastidious and Infrequently Encountered Organism Robin Chamberland       

5.12        Catheter Tip Cultures      Steve Miller       

5.13        Multi-Plex Molecular Panels for Positive Blood Cultures  Rosemary She and Tam Van       

5.14        Broad Range PCR and Emerging Technologies for the Detection of Microorganisms from Blood Specimens                Dan Greene      

5.15        Phenotypic Antimicrobial Susceptibility Testing for Positive Blood Culture              April Abbott and Shelley Campeau             may not be included in print

Section 6: MALDI-TOF                                   

6.1          Introduction       Erin McElvania  

6.2          MALDI-TOF) for Bacterial and Yeast Identification              Margaret V. Powers-Fletcher and Mark Fisher    

6.3          High-Consequence Bacterial Pathogens (BT Agents)         Anthony Tran    

6.4          Nocardia and Mycobacteria         Adrian Zelazny and Joan Miquel Balada-Llasat    

6.5          Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) for Filamentous Fungi Identification                Kimberly Hanson*, Juliana Sobczyk, and Emily Snavely   

6.6          MALDI-TOF MS for Identification of Bacteria and Yeast from Positive Blood Culture Broth                Erin McElvania           

SECTION 7           Antimicrobial Susceptibility Testing

SECTION EDITORS: Romney M. Humphries and Patricia J. Simner

7.1.         Introduction

                Romney M. Humphries and Patricia J. Simner

                7.1.1.     Types of Antimicrobial Susceptibility Tests ……………….7.1.1.1

                                Appendix 7.1.1–1. Surrogate and equivalent agent tests for commonly isolated bacteria

                                                that grow aerobically ………….7.1.1.X

                7.1.2.     Selecting and Applying Clinical Breakpoints ………………7.1.2.1

ROUTINE TESTS PERFORMED FOR AST

7.2.         Disk Diffusion Testing

                Eric M. Ransom, Carey-Ann D. Burnham, and Erika Matuschek………………………………………………….………7.2.1

                                Appendix 7.2–1. Quality Control: CLSI …………………….7.2.1.X

                                Appendix 7.2–2. Quality Control: EUCAST ……………….7.2.1.X

                Appendix 7.2–3. Where to find CLSI and EUCAST disk diffusion criteria for various organisms……………………….7.2.1.X

                Appendix 5.1–4. Zone reading ……………….7.2.1.X

7.3.         Disk Diffusion from Positive Blood Culture

                Gunnar Kahlmeter and Shelley Campeau …….7.3.1

                                Appendix 7.3–1. Procedures for CLIS and EUCAST disk diffusion from positive blood

                                                cultures ……7.3.X

7.4.         Broth Microdilution MIC Test

                7.4.1.     Broth Microdilution MIC Test• Dulini Gamage and Sukantha Chandrasekaran………………..7.4.1.1

Appendix 7.4.1–1. Reference list for performing broth microdilution MIC tests……………………………….5.2.1.X

                                Appendix 7.4.1–2. Broth microdilution QC log sheet……………………………….5.2.1.X

                Appendix 5.2.1–3. Haemophilus influenzae and Haemophilus parainfluenzae

……………………………….5.2.1.X

Appendix 7.4.1–4. Streptococcus pneumoniae and Streptococcus spp. (beta hemolytic group viridans group…….5.2.1.X

Appendix 7.4.1–5. Neisseria meningitidis …………5.2.1.X.

Appendix 7.4.1–6. Breakpoint MIC panels ………..5.2.1.X

Appendix 7.4.1–7. Example of a portion of a MIC breakpoint panel….5.2.1.X

                7.4.2.     Broth Microdilution MIC Test for Anaerobic Bacteria• Darcie Carpenter............................................7.4.2.1

                                Appendix 7.4.2–1. Preparation of Media and Reagents ……7.4.2.X

                                Appendix 7.4.2–2. Anaerobe broth microdilution QC ………7.4.2.X

7.5.         Gradient Diffusion Tests

                Hari P. Dwivedi, Fabio Brocco, Erika Matuschek …………………………………………...…….7.5.1

                                Appendix 7.5–1. Gradient test QC table ……………7.5.X

                                Appendix 7.5–2. Reference list for performing gradient diffusion tests ……7.5.X

                                Appendix 7.5–3. Photographic reading guide for bacteriostatic vs. bactericidal agents

…7.5.X

7.6.         Agar Dilution MIC Test

                Samir N. Patel…………………….7.5.1

                7.6.1.     Agar Dilution MIC Test for Aerobic Bacteria…………………………………………...7.6.1.1

                                Appendix 7.6.1–1. Neisseria meningitidis ……………….7.6.1.X

                                Appendix 7.6.1–2. Neisseria gonorrhoeae ………………7.6.1.X

                                Appendix 7.6.1–3. Helicobacter pylori…………………….7.6.1.X

                                Appendix 7.6.1–4. Reference guide for performing agar dilution MIC tests….7.6.1.X

                                Appendix 7.6.1–5. Preparation of MHA deeps……………………………………..7.6.1.X

Appendix 7.6.1–6. Volumes of components required for preparation of agar dilution

plates when using round or square petri plates……………..7.6.1.X

Appendix 7.6.1–7. Agar dilution QC. ………………….7.6.1.X

Appendix 7.6.1–8. Timetable for agar dilution susceptibility testing of aerobic bacteria

……7.6.1.X

                                Appendix 7.6.1–9. Preparation of antimicrobial dilutions from stock solutions….7.6.1.X

                                Appendix 7.6.1–10. Agar dilution MIC worksheet…………………………….7.6.1.X

                7.6.2.     Agar Dilution MIC Test for Anaerobic Bacteria……………………………………...7.6.1.1

                                Appendix 7.6.2–1. Preparation of media and reagents….7.6.2.X

                                Appendix 7.6.2–2. Agar dilution QC………………….7.6.2.X

Appendix 7.6.2–3. Timetable for agar dilution susceptibility testing of anaerobic

bacteria…………7.6.2.X

                                Appendix 7.6.2–4. Preparation of antimicrobial dilutions from stock solutions…..7.6.2.X

                                Appendix 7.6.2–5. Volumes of components required for preparation of agar dilution

                                                Plates when using round of square petri plates……7.6.2.X

                                Appendix 7.6.2–6. Agar dilution MIC worksheet…………….7.6.2.X

PHENOTYPIC DETECTION OF RESISTANCE MECHANISMS --GENERAL

7.7.         Beta-lactamase Tests

                Shelley Campeau and Ezster Deak……………………………….7.7.1

                                Appendix 7.7–1. Summary of colorimetric beta-lactamase testing methods and

                                                Method(s) generally satisfactory for various organisms…..7.7.X

                                Appendix 7.7–2. Beta-lactamase test QC……7.7.X

PHENOTYPIC DETECTION OF RESISTANCE MECHANISMS –GRAM POSITIVE BACTERIA

7.8.         Oxacillin Salt-Agar Screen Test to Detect Oxacillin (Methicillin)-Resistant Staphylococcus

aureus

                Patricia J. Simner……………………………………………………….7.8.1

                                Appendix 7.8–1. Staphylococcus aureus oxacillin salt agar test QC….7.8.X

7.9.         Detection of VRSA, VISA, and Vancomycin-Heteroresistant Staphylococcus aureus (hVISA)

                Taylor Morrisette and Michael J. Rybak…………………………………………………….7.9.1

                                Appendix 7.9–1. Preparation of vancomycin (VAN) dilutions …..7.9.X

                                Appendix 7.9–2. Volumes of components required for preparation of vancomycin agar

plates when using 100-mm round petri plates…………7.9.X

                                Appendix 7.9–3. Dilutions for each agar plate…..7.9.X

                                Appendix 7.9–4. Example calculations for AUC……..7.9.X.

                                Appendix 7.9–5. PAP-AUC worksheet…………..7.9.X

7.10.      Detection of high level mupirocin resistance in Staphylococcus aureus

                Peera Hemarajata ………………………….7.10.1

7.11.      Detection of Inducible Clindamycin Resistance in Staphylococcus spp., Streptococcus

pneumoniae, and Streptococcus spp. Beta-Hemolytic Group

                Jennifer Dien Bard ………………………………..7.11.1

                                Appendix 7.11–1. Quick reference list to screen for inducible clindamycin resistance

…………….7.11.X

                                Appendix 7.11–2. D-zone test QC…………..7.11.X

                                Appendix 7.11–3. Inducible clindamycin resistance test—broth microdilution QC…7.11.X

7.12.      Screen Tests to Detect High-level Aminoglycoside resistance in Enterococcus spp.

                Alexander S. Maris and Lili Tao…………………………………………………..…7.12.1

Appendix 7.12–1. Aminoglycoside-modifying enzymes that confer HLAR in Enterococcus

spp……………7.12.X

                                Appendix 7.12–2. Enterococcus HLAR agar screen test QC…………..7.12.X

7.13.      Agar Screen Test to Detect Vancomycin Resistance in Enterococcus spp.

                Alexander S. Maris and Lili Tao……………………………………………………..7.13.1

                                Appendix 7.13–1…………………Enterococcus vancomycin agar screen test QC….7.13.X

PHENOTYPIC DETECTION OF RESISTANCE MECHANISMS –ENTEROBACTERIACEAE

7.14.      Extended-Spectrum Beta-Lactamase Testing for Escherichia coli, Klebsiella pneumoniae,

Klebsiella oxytoca, and Proteus mirabilis

                Johan J. Pitout …………………………………………………….7.14.1

                                Appendix 7.14–1. ESBL disk diffusion test QC……………7.14.X

                                Appendix 7.14–2. ESBL MIC test QC……………..7.14.X

7.15.      AmpC Beta-Lactamase Testing

                Rebecca Yee and Patricia J. Simner……………….7.15.1

                                Appendix 7.15–1. Disk diffusion test QC …………7.15.X

                                Appendix 7.15–2. Disk diffusion test results interpretation examples …7.15.X

7.16. Phenotypic Carbapenemase Detection Methods

                7.16.1. Introduction• Lars F. Westblade and Nicholas Moore ……………7.16.1.1

                7.16.2. Modified Hodge Test and Modified Carbapenem Inactivation Method for Phenotypic

Detection of Carbapenemases• Lars Westblade, Nicholas Moore, Laurent Poirel, and

Patrice Nordman …….7.16.2.1

                7.16.3. Carbapenemase Nordmann-Poirel (Carba NP) Test for the Phenotypic Detection of

Carbapenemases• Lars F. Westblade and Nicholas Moore ……………..7.16.3.1

                7.16.4. Phenotypic Tests for the Detection of Metallo-Beta-Lactamases• Lars F. Westblade and

Nicholas Moore ………..7.16.4.1

SPECIALIZED SUSCEPTIBILITY TESTS

7.17.      Tests to Assess Bactericidal Activity

                Laura M. Koeth………………………………………………………….5.14.1

                7.17.1.   Minimum Bactericidal Concentration Testing……………………………………………7.17.1.1

                                Appendix 7.17.1–1. MBC testing conditions for various bacteria for microdilution and

macrodilution procedures …………………7.17.1.X

                                Appendix 7.17.1–2. Worksheet for MIC and MBC …………..7.17.1.X

                                Appendix 7.17.1–3. Technique for using bent glass rod “hockey sticks” for colony counts

                                                ………..7.17.1.X

                                Appendix 7.17.1–4. Worksheet for MBCs………….7.17.1.X

                                Appendix 7.17.1–5. Rejection value and calculated sensitivity and specificity for each

initial concentration based on duplicate 0.01-ml samples ………..7.17.1.X

                7.17.2.   Time-Kill Assay…………………………………..…7.17.2.1

                                Appendix 7.17.2–1. MBC testing conditions for various bacteria for microdilution and

macrodilution procedures …………..7.17.2.X

Appendix 7.17.2–2. Preparation of inoculum for reaction tubes in time-kill assays

                                                …………….7.17.2.X

Appendix 7.17.2–3. Determining colony counts from control and antimicrobial tubes for

time-kill assays……………7.17.2.X

Appendix 7.17.2–4. Time-kill assay worksheet………….7.17.2.X

Appendix 7.17.2–5. Sample graph for time kill assay of S. aureus with vancomycin

                                                …..7.17.2.X

                7.17.3.   Time-Kill Assay for Determining Synergy…………………………………………………………..7.17.3.1

Appendix 7.17.3–1. Time-kill assay worksheet ………………….7.17.3.X

Appendix 7.17.3–2. Graphic representation of time-kill assay showing synergism and

Antagonism…………………….7.17.3.X

Appendix 7.17.3–3. Sample graph of time-kill assay of P. aeruginosa with piperacillin and

Amikacin ……………………..7.17.3.X

7.18.      Serum Inhibitory and Bactericidal Titers

                Kevin Alby……………………………………………………....7.18.1

                                Appendix 7.18–1. Alternative protocols for variations in sample or diluent….7.18.X

                                Appendix 7.18–2. SIT and SBT test conditions and media used for various bacteria

                                                ……………7.18.X

                                Appendix 7.18–3. Configuration of microdilution plate for SIT and SBT ………….7.18.X

                                Appendix 7.18–4. Worksheet for SIT and SBT tests ……………….7.18.X

                                Appendix 7.18–5. Rejection value and calculated sensitivity and specificity for each

initial concentration based on duplicate 0.01-ml samples………7.18.X

7.19.      Synergism Testing: Broth Microdilution Checkerboard and Broth Macrodilution Methods

                Christopher D. Doern and Alex Van Belkum……………………………………………...7.19.1

                                Appendix 7.19–1. Examples of reported combination interactions ……………7.19.X

                                Appendix 7.19–2. Example of format of broth microdilution checkerboard panel…7.19.X

                                Appendix 7.19–3. Dilution schematics for two-agent broth microdilution checkerboard

…………………7.19.X

                                Appendix 7.19–4. Example of broth microdilution showing synergism, partial synergism,

and indifference…………………..7.19.X

                                Appendix 7.19–5. Example of broth microdilution showing indifference and antagonism

                                                ……………..7.19.X

                                Appendix 7.19–6. Example of limited-series checkerboard format for broth

Microdilution……………….7.19.X

                                Appendix 7.19–7. Dilution schematics for two-agent broth macrodilution limited

checkerboard………………..7.19.X

                                Appendix 7.19–8. Example of broth macrodilution showing synergism ………….7.19.X

                                Appendix 7.19–9. Example of broth macrodilution showing antagonism ……….7.19.X

                                Appendix 7.19–10. Representing checkerboards as isobolograms …………….7.19.X

                                Appendix 7.19–11. Synergy testing by disk agar diffusion methods ……….7.19.X

                                Appendix 7.19–12. Planning studies ……………..7.19.X

MISCELLANEOUS

7.20.      Quality Assurance Measures for Antimicrobial Susceptibility Testing

                Sharon K. Cullen………………………………………………………….7.20.1

                                Appendix 7.20–1. Suggested QC strains for antimicrobial susceptibility tests ……7.20.X

                                Appendix 7.20–2. Example for use of QC strains with “on scale” endpoints ….7.20.X

                                Appendix 7.20–3. Primary variables that must be controlled when performing routine

disk diffusion and broth microdilution MIC tests for nonfastidious bacteria

………………7.20.X

                                Appendix 7.20–4. Competency assessment checklist for antimicrobial susceptibility

                                                Testing ………….7.20.X

7.21.      Creation of an Antibiogram and Verification of Data

                J. Kristie Johnson and Sharon K. Cullen ………………………………………..7.21.1

                                Appendix 7.21–1. Stepwise instructions for generating antibiogram reports based on

                                                antimicrobial susceptibility test system …………7.21.X

7.22.      Evaluation and Verification of Antimicrobial Susceptibility Test Systems

                April M. Bobenchik…………………………………………………….7.22.1

                                Appendix 7.22–1. Suggested recommendations for number of isolates and extent of

verification/validation………………………7.22.X

                                Appendix 7.22–2. Suggested resistant organisms to include in evaluation of AST systems

                                                ………….7.22.X

                                Appendix 7.22–3. Susceptibility category errors ………….7.22.X

                                Appendix 7.22–4. Example of a correlation table for one antimicrobial agent …7.22.X       

                                Appendix 7.22–5. Example of an overall correlation table ………..7.22.X

7.23.      Selecting Antimicrobial Agents for Testing and Reporting

                April M. Bobenchik and Patricia J. Simner………………………………………………..7.23.1

                                Appendix 7.23–1. Reference chart for commonly used antimicrobial agents …..7.23.X

7.24.      Preparation of Routine Media and Reagents Used in Antimicrobial Susceptibility Tests

                David R. Lonsway ……………………………5.24.1

                7.24.1. McFarland Standards ………………………………..7.24.1.1

                                Appendix 7.24.1–1. Preparation of McFarland standards ……….7.21.1.X

                                Appendix 7.24.1–2. Wickerham Card ……….7.24.1.X

                7.24.2. Antimicrobial Stock Solutions …………………..7.24.2.1

                                Appendix 7.24.2–1. Preparation of solvents and diluents ………..7.24.2.X

                                Appendix 7.24.2–2. Worksheet for antimicrobial stock solution QC ………7.24.2.X

                                Appendix 7.24.2–3. Formulas for preparation of antimicrobial stock solutions    

7.24.2.X

                7.24.3. Preparation of Agar and Broth Media Used in Routine Antimicrobial Susceptibility Tests ……………………………………………………………………7.24.3.1

                                Appendix 7.24.3–1. Preparation of additives and supplements …………7.24.3.X

                                Appendix 7.24.3–2. Notes and QC parameters for media …………………7.24.3.X

7.25.      Preparation of Broth Microdilution MIC Trays

                David R. Lonsway ……………………………5.25.1

                                Appendix 7.25–1. Ordering media for broth microdilution MIC tray preparation …7.25.X

                                Appendix 7.25–2. QC log for broth microdilution MIC tray preparation ….7.25.X

                                Appendix 7.25–3. QC of stock solution: form and example …………..7.25.X

                                Appendix 7.25–4. Sample listing of antimicrobial agents, abbreviations, and

Concentrations that might be included in microdilution MIC trays ….7.25.X

                                Appendix 7.25–5. ATCC QC organisms that have on-scale endpoints for the agents and

                                                Concentrations depicted in Appendix 7.25–4 ……..7.25.X

                                Appendix 7.25–6. Preparation of antimicrobial dilutions for broth microdilution MIC

Trays ……..7.25.X

                                Appendix 7.25–7. Preparation of individual broth microdilution MIC trays ………..7.25.X

                                Appendix 7.25–8. Worksheet for microdilution MIC trays and sample sheet …7.25.X

                                Appendix 7.25–9. Broth microdilution MIC tray preparation checklist ……..7.25.X

SECTION 8: AEROBIC ACTINOMYCETES                                  

8.1          Introduction       Michael A. Saubolle        

8.2          Specimen Examination and Primary Isolation       Melissa Bell, Zdenek Sussland   

8.3          Media and Methods Used for Phenotypic Characterization of Aerobic Actinomycetes       Melissa Bell, Zdenek Sussland, and Brian Mochon      

8.4          Definitive Identification of Aerobic Actinomycetes            Melissa Bell, Brian Mochon, Sarah Highlander    

8.5          Susceptibility Testing      Zdenek Sussland and Mike Saubolle        

8.6          Appendixes        Melissa Bell*, Zdenek Sussland, and Mile Saubolle           

Section 9: MYCOBACTERIOLOGY AND ANTIMYCOBACTERIAL SUSCEPTIBILITY TESTING                                      

9.1          Introduction       Nancy Wengenack          

9.2          General Mycobacterial Procedures                          

9.2.1      Safety and Levels of Laboratory Service  Nancy Wengenack          

9.2.2      Digestion Decontamination         Nancy Wengenack          

9.2.3      Acid-Fast Stains Nancy Wengenack          

9.2.4      Reporting            Nancy Wengenack          

9.3          Solid Media Used for Isolation    Nancy Wengenack          

9.4          Liquid Media Used for Isolation                 

9.4.1      BACTEC MGIT  Automated Mycobacterial Culture System              Richard Pfeltz   

9.4.2      VersaTREK Myco Culture System               Nancy Wengenack          

9.5          Identification of Mycobacteria                  

9.5.1      Conventional Biochemicals          Nancy Wengenack          

9.5.2      AccuProbe Mycobacterial Culture Identification Tests      Vivian Jonas      

9.5.3 (or 9.6 TBD)             INNO-LiPA MYCOBACTERIA v2 Line Probe Assay Paula Snippes   

9.5.4      Sequencing (Sanger and WGS)   Kim Musser and Tanya Halse      

9.6          Nucleic Acid Amplification Procedures for Identification from Specimen                 

9.6.1      Cepheid Xpert MTB/RIF Test       Ellen Jo Baron and Devasena Gnashanmugam    

9.6.2      Cepheid Xpert® MTB Test: Xpert® MTB/XDR1      Ellen Jo Baron and Devasena Gnashanmugam    

9.6.3      Cepheid Xpert® MTB/RIF Tests: Xpert®MTB/RIF Ultra1    Ellen Jo Baron and Devasena Gnashanmugam    

9.7          Susceptibility Testing by Liquid Media Methods                

9.7.1      BACTEC MGIT SIRE—Nonradiometric Susceptibility Testing for Mycobacterium tuberculosis complex                Richard Pfeltz   

9.7.2      BACTEC MGIT PZA—Nonradiometric Susceptibility Testing for Mycobacterium tuberculosis            Richard Pfeltz               

9.7.3      VersaTREK—Indirect Susceptibility Testing for Mycobacterium tuberculosis           Nancy Wengenack          

9.8          Susceptibility Testing by Agar Proportion Method for Mycobacterium tuberculosis complex           Vincent Escuyer* and Max Salfinger        

9.9          Susceptibility Testing by TREK Sensititre Microdilution Plates                       

9.9.1      Antimicrobial Susceptibility Testing for Rapidly Growing Mycobacteria     Vincent Escuyer and Max Salfinger               

9.9.2      Slowly Growing Nontuberculous Mycobacterium Antimicrobial Susceptibility Testing        Max Salfinger *and Barbara Brown Elliott     

9.9.3      Antimicrobial Susceptibility Testing for the Mycobacterium tuberculosis complex Using Broth Microdilution Plates    Nikki Parrish and Derek Armstrong          

9.10.      Molecular Susceptibility Testing for Mycobacterium tuberculosis                               

9.10.1    Pyrosequencing prediction of drug resistance in Mycobacterium tuberculosis complex-positive clinical specimens          Kimberlee Musser and Tanya Halse         

SECTION 10: MYCOLOGY AND ANTIFUNGAL SUSCEPTIBILITY TESTING                                      

10.1        Introduction and General Considerations              Shawn Lockhart and Nathan Wiederhold              

10.2        Fungal Taxonomy and Nomenclatures    Shawn Lockhart and Nathan Wiederhold              

10.3        Specimen Selection, Collection, and Transport    Susan Howell and Alireza Abdolrasouli   

10.4        Specimen Examination                 

10.4.1    Microscopic Clinical Specimen Examination          Susan Howell and Alireza Abdolrasouli*

10.4.2    Induced Sputum: Stained Preparations for Detection of Pneumocystis jirovecii     Susan Howell and Alireza Abdolrasouli*   

10.5        Processing Specimens for Fungal Culture               Susan Howell and Alireza Abdolrasouli*

10.6        Examination and Evaluation of Primary Cultures Sean Zhang        

10.7        Presumptive Identification Tests for Yeasts Isolated on Primary Culture   Sixto Leal and Marwan Azar        

10.8        Identification of Moulds on Primary Culture         Sixto Leal and Marwan Azar        

10.9        Yeast Identification - Phenotypic               Sean Zhang        

10.10.    Mould Identification - Phenotypic             Connie Gibas and Nathan Wiederhold   

10.11     Molecular Identification of Yeasts and Molds       Connie Gibas and Nathan Wiederhold   

10.12     Antifungal Susceptibility Testing               

10.12.1  Broth Dilution Antifungal Susceptibility Testing   Shawn Lockhart and Nathan Wiederhold              

10.12.2  Disk Diffusion and Gradient Diffusion Antifungal Susceptibility Testing                    

10.13     Clinical Breakpoints and Epidemiologic Cut-Off Values     Philippe Dufresne and Jeff Fuller              

Section 11: PARASITOLOGY                                        

11.1        Introduction                      

                Part 1. Equipment           Lynne S. Garcia 

                Part 2. Safety                    

                Part 3. Quality Control                  

                Part 4. Quality Assurance                            

                Part 5. STAT Testing in Parasitology                         

11.2        Collection and Preservation of Fecal Specimens                 

11.2.1    Collection of Fresh Specimens    Marc Couturier

11.2.2    Fixation and Preservation of Specimens                

11.2.3    Shipment of Specimens                

11.3        Macroscopic and Microscopic Examination of Fecal Specimens                   

11.3.1    Macroscopic Examination of Fecal Specimens: Age and Physical Description          Wilson Chan      

11.3.2    Microscopic Examination of Fecal Specimens: Direct Smears                        

11.3.3    Microscopic Examination of Fecal Specimens: Concentration by Formalin-Ethyl Acetate Sedimentation                   

11.3.4    Microscopic Examination of Fecal Specimens: Concentration by Zinc Sulfate Flotation                      

11.3.5    Microscopic Examination of Fecal Specimens: Permanent Stained Smear (Trichrome)                      

11.3.6    Microscopic Examination of Fecal Specimens: Iron Hematoxylin Stain (Modified Spencer-Monroe Method)                               

11.4        Special Stains for Coccidia and Microsporidia                      

11.4.1    Special Stains for Coccidia: Modified Kinyoun’s Acid-Fast Stain (Cold)        Blaine A. Mathison         

11.4.2    Special Stains for Coccidia: Modified Ziehl-Neelsen Acid-Fast Stain (Hot)                 

11.4.3    Special Stains for Microsporidia: Modified Trichrome-Weber Green          Lynne S. Garcia 

11.4.4    Special Stains for Microsporidia: Modified Trichrome-Ryan Blue                 

11.4.5    Special Stains for Microsporidia: Acid-Fast Modified Trichrome Stain for Coccidia and the Microsporidia                  

11.4.6    Special Stain for Cyclospora Oocysts: Modified Safranin Technique with Microwave Heating           Blaine A. Mathison            

11.4.7    Autofluorescence for Cyclospora, Cystoisospora, and Sarcocystis               

11.4.8    Calcofluor White for Detection of Microsporidial Spores and Acanthamoeba Cysts             Sixto Leal Jr.      

11.5        Additional Techniques for Stool Examination                      

11.5.1    “Culture” of Larval-Stage Nematodes: Baermann Technique         Ribhi Shawar     

11.5.2    “Culture” of Larval-Stage Nematodes: Harada-Mori Technique                   

11.5.3    “Culture” of Larval-Stage Nematodes: Petri Dish-Filter Paper Slant                            

11.5.4    “Culture” of Larval-Stage Nematodes: Agar Plate Culture for Strongyloides stercoralis       Lynne S. Garcia 

11.5.5    Determination of Egg Viability: Schistosomal Egg Hatching             Ribhi Shawar     

11.5.6    Recovery of Scolices and Proglottids of Cestodes               Lynne S. Garcia 

11.5.7    Qualitative Fecal Fat       Lisa Johnson     

11.5.8    Reducing Substances (Clinitest)                

11.6        Other Specimens from the Intestinal Tract and the Urogenital System                     

11.6.1    Examination for Pinworm: Cellulose Tape Preparation     Heather Morris and Bobbi Pritt 

11.6.2    Sigmoidoscopy Specimen: Direct Wet Smear        Lynne S. Garcia 

11.6.3    Sigmoidoscopy Specimen: Permanent Stained Smear                     

11.6.4    Duodenal Contents: Duodenal Aspirate  Heather Morris and Bobbi Pritt 

11.6.5    Urogenital Specimens: Direct Saline Mount                         

11.6.6    Urogenital Specimens: Permanent Stained Smear (Giemsa)                         

11.6.7    Urine Concentration: Centrifugation                       

11.6.8    Urine Concentration: Membrane Filter (Nuclepore)                         

11.7        Sputum, Aspirates, and Biopsy Material                 

11.7.1    Expectorated Sputum: Direct-Mount and Stained Preparations   Sixto Leal            

11.7.2    Aspirates and Bronchoscopy Specimens               

11.7.3    Biopsy Specimens                           

11.8        Detection of Blood Parasites                      

11.8.1    Detection of Blood Parasites       Richard Davis    

11.8.2    Preparation of Thin Blood Films                

11.8.3    Preparation of Thick Blood Film                 

11.8.4    Combination Thick and Thin Blood Films (Can Be Stained as Either)                           

11.8.5    Giemsa Stain                     

11.8.6    Wright’s Stain                   

11.8.7    Determination of Parasitemia     Lynne S. Garcia and Richard Davis            

11.8.8    Delafield’s Hematoxylin Stain      Richard Davis    

11.8.9    Concentration Procedures: Buffy Coat Concentration                     

11.8.10.                Concentration Procedures: Membrane Filtration Concentration                 

11.8.11  Concentration Procedures: Knott Concentration               

11.8.12  Concentration Procedures: Triple Centrifugation Concentration                 

11.8.13  Algorithmic approach to malaria diagnosis                           

11.9        Culture                

11.9.1    Parasite Culture: Acanthamoeba and Naegleria spp.         Ibne Karim         

11.9.2    Parasite Culture: Trichomonas vaginalis  Evan Secor         

11.9.3    Parasite Culture: Leishmania spp. and Trypanosoma cruzi               Sarah Sapp         

11.10.    Gross Examination of Helminths and Arthropods               Blaine A. Mathison         

11.11     Appendixes                       

11.11.1  Appendix 9.11.1–1—Identification Aids: Artifacts               Bobbi Pritt          

11.11.2  Appendix 9.11.2 -1—Information Tables Lynne S. Garcia 

11.11.3  Appendix 9.11.3–1—Common Problems in Organism Identification                          

11.11.4  Appendix 9.11.4–1—Quality Control Recording Sheets                   

11.11.5  Appendix 9.10.5–1—Flowcharts for stool examinatio                      

11.11.6  Appendix 9.11.6–1—Current OSHA Regulations on the Use of Formaldehyde                       

11.11.7  Appendix 9.11.8–1— Test Report Comments                      

Section 12: Viruses and Chlamydiae                                        

12.1        Introduction: Traditional Methods for Laboratory Diagnosis of Viral Infections      Richard L. Hodinka          

12.2        Selection, Collection, Transport, and Processing of Specimens for Viral Diagnosis Erin Graf             

12.3        Quality Control and Quality Assurance in Clinical Virology              Heba Mostafa  

12.4        Biosafety in the Clinical Virology Laboratory         Anna Marie Cardenas    

12.5        Conventional Tube Culture for Primary Virus Isolation      Richard L. Hodinka and Ryan Relich         

12.6        Shell Vial/Multiwell Plate Culture for Rapid Virus Isolation             Christine Ginocchio and and Ninad Mehta*               

12.7        Serial Propagation and Maintenance of Monolayer Cell Cultures Rick Hodinka     

12.8        Preparation of Cell Culture Media and Supplemental Components            Richard L. Hodinka          

12.9        Tests for Direct Detection of Viruses in Clinical Specimens                            

12.9.1    Immunofluorescence Test for Direct Detection of Viruses in Clinical Specimens    Richard L. Hodinka          

12.9.2    Rapid Solid-Phase Immunoassays for Direct Detection of Viruses in Clinical Specimens - Influenza, RSV and SARS-CoV-2        Rangaraj Selvarangan    

12.9.3    Histologic and Cytologic Procedures for Viral Detection   Laurel J. Glaser and Lyn Thomas

12.10.    Isolation of Chlamydia spp. in Cell Culture             Ellena M. Peterson         

12.11     Neutralization Assay       Benjamin A. Pinsky and Stephen A. Young            

12.12     Hemagglutination Inhibition Test              Stephen A. Young and Benjamin A. Pinsky            

12.13     Health Care-Associated Viral Respiratory Tract Infections Due to Influenza Virus, Respiratory Syncytial Virus, and Other Respiratory Viruses (add SARS-CoV-2) Go to virology section   Christian Renaud and Jesse Papenburg 

Section 13: SEROLOGY                                  

13.1        Immunology Introduction                           

13.1.1    Introduction       Elli Theel             

13.1.2    Immunologic Assays Used in the Serologic Diagnosis of Infectious Diseases            Elli Theel             

13.2        Serologic Diagnosis of Group A Streptococcal Infections                 

13.2.1    Introduction       David Murray    

13.2.2    Anti-Streptolysin O and Anti-Deoxyribonuclease B Test   David Murray    

13.3        Detection of Brucella spp. Antibodies      Robyn Stoddard

13.4        Detection of Francisella tularensis Antibodies      Jeanine Petersen            

13.5        Laboratory Diagnosis of Syphilis                

13.5.1    Introduction       Yetunde Fakile 

13.5.2    VDRL Tests                         

13.5.3    Rapid Plasma Reagin Test                            

13.5.4    Treponema pallidum Particle Agglutination Test                

13.6        Detection of Borrelia burgdorferi Antibodies       Maria E. Aguero-Rosenfield        

13.7        Epstein Barr Virus                           

13.7.1    Introduction       Linda E. Miller   

13.7.2    Detection of Heterophile Antibodies       Linda E. Miller   

13.7.3    Detection of Epstein-Barr Virus-Specific Antibodies          Linda E. Miller   

13.8        Cytomegalovirus Serology                           

13.8.1    Introduction       Harry Prince      

13.8.2    ELISA and Chemiluminescence Assay (CIA) for Cytomegalovirus antibodies            Harry Prince      

13.8.3    Cytomegalovirus IgG avidity testing          Harry Prince      

13.9        HIV Serology                     

13.9.1    Introduction       Erin Kaleta          

13.9.2    HIV Screening Assays      Erin Kaleta          

13.9.3    HIV Supplemental Assays             Erin Kaleta          

13.10.    Hepatitis Viruses                             

13.10.1  Heptatits A Virus Serology            Neil Anderson  

13.10.2  Hepatitis B Virus Serology            Neil Anderson  

13.10.3  Hepatitis C Virus Serology            Neil Anderson  

13.11     Arboviruses        Matthew Collins              

13.12     Laboratory Diagnosis of Histoplasma capsulatum               Patricia Slev       

13.13     Coccidioides spp.              Mark Lindsley   

13.14     Trypanosoma cruzi (Chagas Disease) Serology     Caryn Bern and Jeff Whitman    

13.15     Toxoplasma gondii           Jose Montoya, Christelle Pomares, and Kjerstie Olson     

13.16     Interferon-Gamma Release Assays           Niaz Banaei        

Section 14: Molecular Techinuqes                                           

14.1        Introduction       Alex Greninger & Samia Naccache           

14.2        General Aspects of Molecular Diagnostics                             

14.2.1    Pre-analytical Considerations for Molecular Testing          Rob Cybulski      

14.2.2    "Molecular Workflow and Contamination

Control in a Clinical Microbiology

Laboratory"        Mark Lee            

14.2.3    Biosafety in the molecular laboratory      Joshua Lieberman           

14.2.4    Verification and Validation of Molecular Tests     Matthew J. Binnicker and Jill N. Hill          

14.2.5    Use of Controls and Calibrators  Jeffery B. Stevenson      

14.2.6    Post-analytical Considerations for Molecular testing         Jennifer Dien-Bard; Thao Throng, Tam Van          

14.3        Molecular Methods for Identification of Cultured Microorganisms                            

14.3.1    HIV-1     Alex Greninger & Samia Naccache           

14.3.2    HIV Genotypic Resistance Testing             Larry Reimer and David R. Hillyard           

14.3.3    Molecular Methods for Identification of High-Risk Human Papillomaviruses           Robert Schlaberg             

14.3.4    Molecular Detection of Herpes Simplex Viruses 1 and 2 and Varicella Zoster Virus               Nang L. Nguyen(* (primary corresponding author) & Yi-Wei Tang   

14.3.5    Quantitative Detection of Human Cytomegalovirus DNA by Real-Time PCR             Benjamin A. Pinsky and fellows

14.3.6    Human Cytomegalovirus Drug Resistance Testing              Melanie A. Mallory         

14.3.7    Molecular Diagnostics of Epstein-Barr Virus Infections     Ted E. Schutzbank          

14.3.8    Molecular Diagnostics of BK Virus Infections        Ted E. Schutzbank          

14.3.9    Chlamydia trachomatis, Neisseria gonorrhoeae  Erin H. Graf and Robert Schlaberg            

14.4        Syndromic Panels                            

14.4.1    Syndromic Panels: Gastroenteritis Panels              Marc R. Couturier           

14.4.2    Syndromic Panels:  Respiratory Panels    Esther Babady  

14.4.3    Sydnromic Panels:  Meningitis/Encephalitis panels            Jennifer Dien-Bard and Tam Van               

14.5        Sequence-Based Identification and Typing                           

14.5.1    Sequencing-Based Identification of Microorganisms         Rosemary She  

14.5.2    Deep Amplicon sequencing for prokaryotes and eukaryotes         Shaun Yang        

14.5.3    whole genome sequencing          Varvara Kozyreva            

14.5.4    metagenomic NGS          Shaun Yang        

Section 15: Epidemiologic and Infection Control Microbiology                                     

15.1        Introduction       J. Michael Miller              

15.2        Laboratory Support for Infection Prevention: Collaboration with Benefits for All   Jennifer Kristie Johnson                

15.3        Microbiological Sampling for Pharmacies that Compound Sterile Preparations                     

15.3.1    Environmental Monitoring (EM) by Viable Air Sampling   Alice Weissfeld

15.3.2    Surface Sampling              Mary Frances Schaffer and Paula Heinz Vance    

15.3.3    Gloved Fingertip Testing              Mary Frances Schaffer and Paula Heinz Vance    

15.3.4    Media Fill Testing             Mary Frances Schaffer and Paula Heinz Vance    

15.3.5    USP 71> Sterility Testing               Mary Frances Schaffer and Paula Heinz Vance    

15.3.6    Endotoxin Testing            Mary Frances Schaffer and Paula Heinz Vance    

15.4        Environmental Sampling and Cultures                    

15.4.1    Culture of Hospital Water for Legionellaceae       Janet Stout        

15.4.2    Heterotrophic Bacterium Culture and Endotoxin Assay of Hemodialysis Fluids       Angela D. Coulliette and Matthew J. Arduino       

15.4.3    Air Cultures for Fungi      Paul Luethy       

15.4.4    Environmental Testing in a clinical or environmental laboratory  Gwen Robinson and Kristie Johnson       

15.4.5    Sterity Testing of Blood and Cellular Therapy Products in Blood Banking  (including Stem cells)      Bradley Ford and Annette Schlueter  and Anna Lau     

15.4.6    Culture of Human Cadaveric Tissues for Transplantation Culture of Human Cadaveric Tissues for Transplantation               

15.4.7    Duodenoscope surveillance         Puman Verma  

15.5        Outbreak Investigations: Laboratory and Epidemiologic Concepts                              

15.5.1    Collection and Tracking of Surveillance and Clinical Specimens for Outbreak Investigation               Kristie Johnson              

15.5.2    Collection and Storage of Outbreak Isolates         Kristie Johnson

15.5.3    Systematic Analysis of Nosocomial Outbreaks     Ralf-Peter Vonberg, Doris Weitzel-Kage and Michael Behnke                 

15.5.4    Public Health Reporting of Infectious Diseases: a Collaborative Approach by the Microbiology Laboratory and Clinicians              Priya Nori and Belinda Ostrowsky            

15.6        Microbial Strain Typing for Epidemiology and Infection Control   Mark A. Fisher and Bert K. Lopansri         

15.7        Surveillance Cultures For Hsopital Associated Infections                

15.7.1    Center of Disease Control and Prevention Antimicrbial Resistance Laboratory Network    Kristie Johnson

15.7.2    Screening for Methicillin-Resistant Staphylococcus aureus             William Lainhart              

15.7.3    Screening for Vancomycin-Resistant Enterococci Willian Lainhart

15.7.4    Surveillance Cultures for Multidrug-Resistant Gram-negatives bacteria    Johann Pitout, Sam Bourassa-Blancette and Helen Bibby          

15.7.5    Surveillance Cultures for Candida auris   Shawn Lockhart

15.7.6    Surveillance Cultures from Immunocompromised Hosts Daniel Green    

15.7.7    Infection Surveillance Prior to Transplantation    Kimberly E. Hanson and Hannah Imlay* 

15.8        Infection Control in the Laboratory                          

15.8.1    Immunization of Microbiology Laboratory Personnel       Michael A. Pentella        

15.8.2    Laboratory Support of Blood-Borne Pathogen Exposures Paul Luethy       

15.8.3    Investigation of Laboratory Exposures    Michael A. Pentella        

15.8.4    Laboratory Isolation Codes and Notification Fan-Out for Suspected Biosafety Risk Group 3 and 4 Microorganisms (add how cliancians should notify laboratory)     Deirdre L. Church            

Section 16: Quality Assurance, Quality Control, Laboratory Records, and Water Quality                                   

16.1        Quality Assessment and Improvement (Quality Assurance)           "Sue Kehl

Melphine Harriott"         

16.1.1    Process Improvement    Linoj Samuel     

16.1.2    Using LIS for QC Ron Jackups       

16.1.3    QA/QC and Laboratory Automation         Karissia Culbreath and Cecilia Thompson              

16.2        Quality Control  Mark Cervinski and Nicole Hubbard         

16.3        Validation and Verification           Mike Loeffelholz and Rebecca Martin*  

16.4        CLIA Certification and Accreditation, ISO 15189 Accreditation, and Proficiency Testing      Heather Stang  

16.5        Overview of Individualized Quality Control Plan (IQCP)    Susan Sharp and Kendall Bryant

16.6        Laboratory Records         Rhonda Hensley              

16.7        Preparation and Quality Control of Laboratory Water       Rhonda Hensley              

16.8        Pipette and Loop Calibration                      

16.8.1    Use and Calibration of Microbiological Loops       Rhonda Hensley              

16.8.2    Use and Calibration of Pipettors Rhonda Hensley              

Section 17: Biohazards and Safety                                           

17.1        Introduction       Ryan F. Relich    

17.2        Biological Safety and Biohazard Prevention          Ryan F. Relich and Ryan Demkowicz        

17.1.1    Routes of Infection and Laboratory Activities                      

17.1.2    Safe Work Practices                       

17.1.3    Decontamination                            

17.1.4    Biohazardous Spills                         

17.1.5    Hand Hygiene for Laboratory Personnel Michael A. Pentella        

17.3        Biohazard Containment Ryan F. Relich and Drew Bell       

17.3.1    Introduction                      

17.3.2    Biosafety Levels               

17.3.3    Biological Safety Cabinet                             

17.3.4    PPE and Engineering Controls                    

17.4        Laboratory Instrumentation and Equipment        Blake Buchan and Gerald Capraro            

17.4.1    Introduction                      

17.4.2    Autoclave                           

17.4.3    Centrifuge                         

17.4.4    Compressed Gas Cylinders                          

17.4.5    Pneumatic Tube System               

17.4.6    Specimen/Microorganism Storage and Retention                             

17.4.7    Other Equipment and Devices                   

17.5        Special Pathogens and Employee Safety Gerald Capraro and Corrie Simons           

17.5.1    Introduction                      

17.5.2    Early (Bench Level) Recognition of Agents of Bioterrorism and Laboratory-Acquired Infections                     

17.5.3    Select Agents                    

17.5.4    Environmental, Nonanimal, and Suspicious Specimens and Substances                   

17.5.5    Prions, Prion Diseases (Including Creutzfeldt-Jakob), and Safety Issues Specific to Specimens Suspected of Containing Prions                            

17.5.6    Resources for Information on Specific Pathogens                              

17.6        Packaging and Shipping Infectious Substances    Ryan F. Relich and Laurice Flowers          

17.7        Management of Laboratory Accidents    Blake Buchan and Ryan Relich    

17.8        Management of Infectious Waste             Ryan F. Relich    

17.9        Risk Assessment              Blake Buchan    

Section 18: Bioterrorism                                              

18.1        General Introduction to Bioterrorism and Emerging Infectious Diseases  Marie-Claire Rowlinson

18.2        Levels of Laboratory Safety          Michael Pentella             

18.3        Bacillus anthracis              Philip Lee and Marie-Claire Rowlinson   

18.4        Brucella spp.      Philip Lee and Marie-Claire Rowlinson   

18.5        Yersinia pestis   Susan Sharp       

18.6        Francisella tularensis      Kurt Schaecher

18.7        Melioidosis (Burkholderia pseudomallei) and Glanders (Burkholderia mallei)         Christina Egan and Michael Perry    

18.8        Poxviruses          Miguel Arrroyocazurro 

18.9        Novel Influenza Viruses and Highly Pathogenic Coronaviruses     Michael Loffelholz and Ryan Relich          

18.10.    Coxiella burnetii               Kurt Schaecher

18.11     Botulinum Toxin—Clostridium botulinum and Staphylococcal Enterotoxin B – Staphylococcus aureus                Christina Egan and Michael Perry             

18.12     High-Consequence Viral Pathogens          Ryan Relich        

18.13     Eastern Equine Encephalitis Virus              Sara Vetter        

18.14     Clinical Laboratory Bioterrorism Readiness Plan  Priyanka Uprety

18.15     Biochemical Procedures               

18.15.1  Antimicrobial Disk Tests for Identification             Philip Lee           

18.15.2  Catalase Test                    

18.15.3  Beta-lactamase Test                      

18.15.4  Spot Indole Test                               

18.15.5  Motility Tests                    

18.15.6  Oxidase Test                     

18.15.7  Satellite Test                     

18.15.8  Urease Test                       

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