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
Other versions by this Author
