Test bank for Bailey Scotts Diagnostic Microbiology 13th Edition by Patricia Tille
Test bank for Bailey Scotts Diagnostic Microbiology 13th Edition by Patricia Tille
Known as the #1 bench reference for practicing microbiologists and an excellent text for students in clinical laboratory science programs, Bailey & Scott’s Diagnostic Microbiology, 13th Edition helps you develop and refine the skills you need for effective laboratory testing. In-depth information is useful and easily accessible, with step-by-step instructions for all the procedures. This edition features more than 20 NEW chapters plus updated material on the newest advances and the latest trends in clinical microbiology. Written by expert Dr. Patricia Tille, this classic reference addresses the topics and issues most relevant to you and your success on the job.
Table of Contents
Front Matter
Dedication
Reviewers
Contributors
Preface
Acknowledgments
Part I Basic Medical Microbiology
Chapter 1 Microbial Taxonomy
Objectives
Classification
Species
Genus
Family
Nomenclature
Identification
Box 1-1 Role of Taxonomy in Diagnostic Microbiology
Identification Methods
TABLE 1-1 Identification Criteria and Characteristics for Microbial Classification
Bibliography
Chapter Review
Chapter 2 Bacterial Genetics, Metabolism, and Structure
Objectives
Bacterial Genetics
Nucleic Acid Structure and Organization
Nucleotide Structure and Sequence
Figure 2-1 General overview of bacterial cellular processes.
DNA Molecular Structure
Genes and the Genetic Code
Chromosomes
Nonchromosomal Elements of the Genome
Figure 2-2 A, Molecular structure of DNA depicting nucleotide structure, phosphodiester bonds connecting nucleotides, and complementary base pairing (A, adenine; T, thymine; G, guanine; C, cytosine) between antiparallel nucleic acid strands. B, 5′ and 3′ antiparallel polarity and double helix configuration of DNA.
Figure 2-3 Molecular structure of nucleic acid bases. Pyrimidines: cytosine, thymine, and uracil. Purines: adenine and guanine.
Replication and Expression of Genetic Information
Replication
Figure 2-4 Bacterial DNA replication depicting bidirectional movement of two replication forks from origin of replication. Each parent strand serves as a template for production of a complementary daughter strand and, eventually, two identical chromosomes.
Expression of Genetic Information
Transcription.
Figure 2-5 Overview of gene expression components; transcription for production of mRNA and translation for production of polypeptide (protein).
Translation.
TABLE 2-1 The Genetic Code as Expressed by Triplet-Base Sequences of mRNA*
Figure 2-6 Overview of translation in which mRNA serves as the template for the assembly of amino acids into polypeptides. The three steps include initiation (A), elongation (B and C), and termination (not shown).
Regulation and Control of Gene Expression
Figure 2-7 Transcriptional control of gene expression. A and B, Gene repression. C and D, Induction.
Genetic Exchange and Diversity
Mutation
Genetic Recombination
Genetic Exchange
Transformation.
Transduction.
Figure 2-8 A, Genetic recombination. The mechanisms of genetic exchange between bacteria: transformation (B), transduction (C), and conjugational transfer of chromosomal (D) and plasmid (E) DNA.
Conjugation.
Figure 2-9 Photomicrograph of Escherichia coli sex pilus between donor and recipient cell.
Bacterial Metabolism
Fueling
Acquisition of Nutrients
Figure 2-10 Pathways for bacterial dissemination of plasmids and transposons, together and independently.
Production of Precursor Metabolites
Energy Production
Figure 2-11 Overview of bacterial metabolism, which includes the processes of fueling, biosynthesis, polymerization, and assembly.
Figure 2-12 Overview diagram of the central metabolic pathways (Embden-Meyerhof-Parnas [EMP], the tricarboxylic acid [TCA] cycle, and the pentose phosphate shunt). Precursor metabolites (see also Figure 2-11) that are produced are highlighted in red; production of energy in the form of ATP (~P) by substrate-level phosphorylation is highlighted in yellow; and reduced carrier molecules for transport of electrons used in oxidative phosphorylation are highlighted in green.
Oxidative Phosphorylation.
Biosynthesis
Polymerization and Assembly
Structure and Function of the Bacterial Cell
Eukaryotic and Prokaryotic Cells
Bacterial Morphology
Bacterial Cell Components
Figure 2-13 General structures of the gram-positive and gram-negative bacterial cell envelopes. The outer membrane and periplasmic space are present only in the gram-negative envelope. The murein layer is substantially more prominent in gram-positive envelopes.
Cell Envelope
Outer Membrane.
Cell Wall (Murein Layer).
Figure 2-14 Peptidoglycan sheet (A) and subunit (B) structure. Multiple peptidoglycan layers compose the murein structure, and different layers are extensively cross-linked by peptide bridges. Note that amino acid chains are only derived from NAM. NAG, N-acetylglucosamine; NAM, N-acetylmuramic acid.
Periplasmic Space.
Cytoplasmic (Inner) Membrane.
Cellular Appendages.
Cell Interior
Bibliography
Chapter Review
Chapter 3 Host-Microorganism Interactions
Objectives
The Encounter Between Host and Microorganism
The Human Host’s Perspective
Microbial Reservoirs and Transmission
Figure 3-1 General stages of microbial-host interaction.
Box 3-1 Definitions of Selected Epidemiologic Terms
Human and Microbe Interactions
Animals as Microbial Reservoirs
Figure 3-2 Summary of microbial reservoirs and modes of transmission to humans.
Insects as Vectors
The Environment as a Microbial Reservoir
The Microorganism’s Perspective
Microorganism Colonization of Host Surfaces
The Host’s Perspective
Skin and Skin Structures
Mucous Membranes
Figure 3-3 Skin and skin structures.
TABLE 3-1 Protective Characteristics of the Skin and Skin Structures
General Protective Characteristics.
Figure 3-4 General features of mucous membranes highlighting protective features such as ciliated cells, mucus production, tight intercellular junctions, and cell sloughing.
Figure 3-5 Protective characteristics associated with the mucosal linings of different internal body surfaces.
TABLE 3-2 Protective Characteristics of Mucous Membranes
Specific Protective Characteristics.
The Microorganism’s Perspective
Microbial Colonization
Box 3-2 Microbial Factors Contributing to Colonization of Host Surfaces
Survival Against Environmental Conditions
Achieving Attachment and Adherence to Host Cell Surfaces
Other Factors
Microorganism Entry, Invasion, and Dissemination
The Host’s Perspective
Disruption of Surface Barriers
Responses to Microbial Invasion of Deeper Tissues
Nonspecific Responses.
Phagocytes.
Box 3-3 Factors Contributing to Disruption of the Skin and Mucosal Surface
Figure 3-6 Overview of phagocyte activity and possible outcomes of phagocyte-bacterial interactions.
Inflammation.
TABLE 3-3 Components of Inflammation
Figure 3-7 Overview of the components, signs, and functions of inflammation.
Specific Responses—the Immune System
Components of the Immune System
Figure 3-8 General structure of the IgG class antibody molecule.
Box 3-4 Cells of the Immune System
B Lymphocytes (B Cells)
T Lymphocytes (T Cells)
Natural Killer Cells (NK Cells)
Two Branches of the Immune System.
Figure 3-9 Overview of B-cell activation that is central to antibody-mediated immunity.
Figure 3-10 Overview of T-cell activation that is central to cell-mediated immunity.
The Microorganism’s Perspective
Colonization and Infection
Pathogens and Virulence
Microbial Virulence Factors
Attachment.
Invasion.
Box 3-5 Microbial Strategies for Surviving Inflammation
Avoid Killing by Phagocytes (Polymorphonuclear Leukocytes)
Avoid Phagocyte-Mediated Killing
Avoid Effects of the Complement System
Survival Against Inflammation.
Box 3-6 Microbial Strategies for Surviving the Immune System
Survival Against the Immune System.
Microbial Toxins.
Box 3-7 Summary of Bacterial Toxins
Endotoxins
Exotoxins
Genetics of Virulence: Pathogenicity Islands
Biofilm Formation.
Box 3-8 Biofilms and Human Infections
Artificial Prosthetics and Indwelling Devices
Food-Borne Contamination
Outcome and Prevention of Infectious Diseases
Outcome of Infectious Diseases
Figure 3-11 Possible outcomes of infections and infectious diseases.
Figure 3-12 Host-microorganism interactions and stages of infection or disease.
Box 3-9 Signs and Symptoms of Infection and Infectious Diseases
Prevention of Infectious Diseases
Box 3-10 Strategies for Preventing Infectious Diseases
Preventing Transmission
Controlling Microbial Reservoirs
Minimizing Risk Before or Shortly After Exposure
Immunization
Epidemiology
Chapter Review
Case Study 3-1
Questions
Bibliography
Part II General Principles in Clinical Microbiology
Section 1 Safety and Specimen Management
Chapter 4 Laboratory Safety
Objectives
Sterilization and Disinfection
Methods of Sterilization
Figure 4-1 Gravity displacement type of autoclave. A, Typical Eagle Century Series sterilizer for laboratory applications. B, Typical Eagle 3000 sterilizer piping diagram. The arrows show the entry of steam into the chamber and the displacement of air.
Methods of Disinfection
Physical Methods of Disinfection
Chemical Methods of Disinfection
Chemical Safety
Figure 4-2 National Fire Protection Association diamond indicating a chemical hazard. This information can be customized (as shown here for isopropyl alcohol) by applying the appropriate self-adhesive polyester numbers to the corresponding color-coded hazard area.
Figure 4-3 Fume hood. A, Model ChemGARD. B, Schematics. Arrows indicate airflow through cabinet to outside vent.
Fire Safety
Electrical Safety
Figure 4-4 A, Gas cylinders chained to the wall. B, Gas cylinder chained to a dolly during transportation.
Handling of Compressed Gases
Biosafety
Exposure Control Plan
Employee Education and Orientation
Disposal of Hazardous Waste
Figure 4-5 Autoclave bags.
Figure 4-6 A, Various bench-top pipette discard containers. B, Bench-top serologic pipette discard container.
Figure 4-7 Cartons for broken glass.
Standard Precautions
Figure 4-8 Sharps containers.
Engineering Controls
Laboratory Environment
Figure 4-9 Class I biologic safety cabinet. A, Model BSC-100. B, Schematics showing airflow.
Figure 4-10 Class II biologic safety cabinet. A, Model SterilGARD II. B, Schematics showing airflow.
Biologic Safety Cabinet
Figure 4-11 Class III biologic safety cabinet. A, Custom-built class III system. B, Schematics with arrows showing airflow through cabinet.
Figure 4-12 Personal protective equipment. A, Microbiologist wearing a laboratory gown, gloves, goggles, and face mask. B, Microbiologist wearing a laboratory coat, gloves, and respirator with high-efficiency particulate air (HEPA) filters (pink cartridges) for cleaning up spills of Mycobacterium tuberculosis.
Personal Protective Equipment
Postexposure Control
Classification of Biologic Agents Based on Hazard
Figure 4-13 A, The Bio-Pouch (lower right) is made of laminated, low-density polyethylene, which is virtually unbreakable. The label for shipping a diagnostic specimen is shown (UN 3373). B, The Bio-Bottle is made of high-density polyethylene and is used as the secondary container. This packaging is used for both infectious substances (the class 6 label is shown) with the UN 3373 label.
Mailing Biohazardous Materials
TABLE 4-1 Examples of Infectious Substances Included in Category A
Bibliography
Chapter Review
Chapter 5 Specimen Management
Objectives
General Concepts for Specimen Collection and Handling
Appropriate Collection Techniques
TABLE 5-1 Collection, Transport, Storage, and Processing of Specimens Commonly Submitted to a Microbiology Laboratory*
Figure 5-1 Specimen bag with biohazard label, separate pouch for paperwork, and self-seal.
Specimen Transport
Specimen Preservation
Specimen Storage
Specimen Labeling
Specimen Requisition
Rejection of Unacceptable Specimens
Specimen Processing
Gross Examination of Specimen
Direct Microscopic Examination
Figure 5-2 Examples of various types of hemolysis on blood agar. A, Streptococcus pneumoniae showing alpha (α)-hemolysis (i.e., greening around colony). B, Staphylococcus aureus showing beta (β)-hemolysis (i.e., clearing around colony). C, Enterococcus faecalis showing gamma (γ)-hemolysis (i.e., no hemolysis around colony).
Selection of Culture Media
Figure 5-3 MacConkey agar. A, Escherichia coli, a lactose fermenter. B, Pseudomonas aeruginosa, a nonlactose fermenter.
Specimen Preparation
Inoculation on Solid Media
Incubation Conditions
Specimen Workup
Extent of Identification Required
Communication of Laboratory Findings
Critical (Panic) Values
Expediting Results Reporting: Computerization
Bibliography
Chapter Review
Section 2 Approaches to Diagnosis of Infectious Diseases
Chapter 6 Role of Microscopy
Objectives
Bright-Field (Light) Microscopy
Principles of Light Microscopy
Magnification
Resolution
Figure 6-1 Principles of bright-field (light) microscopy.
Box 6-1 Applications of Microscopy in Diagnostic Microbiology
TABLE 6-1 Microscopy for Diagnostic Microbiology
Contrast
Procedure 6-1 Kohler Illumination
Purpose
Principle
Method
Staining Techniques for Light Microscopy
Smear Preparation
Figure 6-2 Smear preparations by swab roll (A) and pipette deposition (B) of patient specimen on a glass slide.
Gram Stain
Procedure Overview.
Procedure 6-2 Gram Stain
Purpose
Principle
Method
Expected Results
Reporting Results
Direct Smear
Indirect Smear
Limitations
Quality Control
Principle.
Gram Stain Examination.
Figure 6-3 Gram stain procedures and principles. A, Gram-positive bacteria observed under oil immersion appear purple. B, Gram-negative bacteria observed under oil immersion appear pink.
Figure 6-4 Examples of common bacterial cellular morphologies, Gram staining reactions, and cellular arrangements.
Gram Stain of Bacteria Grown in Culture.
Acid-Fast Stains
Principle.
Figure 6-5 Gram stains of direct smears showing squamous cells and bacteria (A), proteinaceous debris (B), and proteinaceous debris with polymorphonuclear leukocytes and bacteria (C).
Procedure Overview.
Figure 6-6 Gram stain of direct smears showing polymorphonuclear leukocytes, proteinaceous debris, and bacterial morphologies (arrows), including gram-positive cocci in chains (A), gram-positive cocci in pairs (B), gram-positive cocci in clusters (C), gram-negative coccobacilli (D), gram-negative bacilli (E), gram-negative diplococci (F), and mixed gram-positive and gram-negative morphologies (G).
Figure 6-7 Gram stains of direct smears can reveal infectious etiologies other than bacteria, such as the yeast Candida tropicalis.
Figure 6-8 Example of a slide map for staining several bacterial colony samples on a single slide.
Figure 6-9 The Ziehl-Neelsen acid-fast stain procedures and principles. A, Acid-fast positive bacilli. B, Acid-fast negative bacilli.
Figure 6-10 Acid-fast stain of direct smear to show acid-fast bacilli staining deep red (arrow A) and non–acid-fast bacilli and host cells staining blue with the counterstain methylene blue (arrow B).
Procedure 6-3 Acid Fast (Ziehl-Neelsen or Hot Method)
Purpose
Principle
Method
Expected Results
Limitations
Safety Considerations
Procedure 6-4 Acid Fast (Kinyoun-Cold Method)
Purpose
Principle
Method
Expected Results
Limitations
Safety Considerations
Phase Contrast Microscopy
Fluorescent Microscopy
Principle of Fluorescent Microscopy
Staining Techniques for Fluorescent Microscopy
Figure 6-11 Principle of fluorescent microscopy. Microorganisms in a specimen are stained with a fluorescent dye. On exposure to excitation light, organisms are visually detected by the emission of fluorescent light by the dye with which they have been stained (i.e., fluorochroming) or “tagged” (i.e., immunofluorescence).
Figure 6-12 Principles of fluorochroming and immunofluorescence. Fluorochroming (A) involves nonspecific staining of any bacterial cell with a fluorescent dye. Immunofluorescence (B) uses antibodies labeled with fluorescent dye (i.e., a conjugate) to specifically stain a particular bacterial species.
Fluorochroming
Figure 6-13 Comparison of acridine orange fluorochroming and Gram stain. Gram stain of mycoplasma demonstrates the inability to distinguish cell wall-deficient organisms from amorphous gram-negative debris (A). Staining the same specimen with acridine orange confirms the presence of nucleic acid–containing organisms (B). Gram stain distinguishes between gram-positive and gram-negative bacteria (C), but all bacteria stain the same with the nonspecific acridine orange dye (D).
Figure 6-14 Comparison of the Ziehl-Neelsen–stained (A) and auramine-rhodamine–stained (B) Mycobacterium spp. (arrows).
Acridine Orange.
Procedure 6-5 Acridine Orange Stain
Purpose
Principle
Method
Expected Results
Limitations
Auramine-Rhodamine.
Figure 6-15 Immunofluorescence stains of Legionella spp. (A) and Bordetella pertussis (B) used for identification.
Figure 6-16 Dark-field microscopy. Principal (A) and dark-field photomicrograph showing the tightly coiled characteristics of the spirochete Treponema pallidum (B).
Calcofluor White.
Immunofluorescence
Dark-Field Microscopy
Electron Microscopy
Figure 6-17 A, Transmission electron micrograph showing Escherichia coli cells internalized by a human mast cell (arrows). B, Scanning electron micrograph of E. coli interacting with the surface of human mast cell (arrows).
Bibliography
Chapter Review
Chapter 7 Traditional Cultivation and Identification
Objectives
Principles of Bacterial Cultivation
Nutritional Requirements
General Concepts of Culture Media
Phases of Growth Media
Figure 7-1 A, Clear broth indicating no bacterial growth (left) and turbid broth indicating bacterial growth (right). B, Individual bacterial colonies growing on the agar surface following incubation.
Figure 7-2 Growth of Legionella pneumophila on the enrichment medium buffered charcoal-yeast extract (BCYE) agar, used specifically to grow this bacterial genus.
Media Classifications and Functions
Figure 7-3 A, Heavy mixed growth of the gram-negative bacillus Escherichia coli (arrow A) and the gram-positive coccus Enterococcus spp. (arrow B) on the nonselective medium sheep blood agar (SBA). B, The selective medium phenylethyl-alcohol agar (PEA) only allows the enterococci to grow (arrow).
Figure 7-4 Differential capabilities of MacConkey agar as gram-negative bacilli capable of fermenting lactose appear deep purple (arrow A), whereas those not able to ferment lactose appear light pink or relatively colorless (arrow B).
Summary of Artificial Media for Routine Bacteriology
TABLE 7-1 Plating Media for Routine Bacteriology
Figure 7-5 Different colony morphologies exhibited on sheep blood agar by various bacteria, including alpha-hemolytic streptococci (arrow A), gram-negative bacilli (arrow B), beta-hemolytic streptococci (arrow C), and Staphylococcus aureus (arrow D).
Brain-Heart Infusion.
Chocolate Agar.
Columbia CNA with Blood.
Gram-Negative (GN) Broth.
Hektoen Enteric (HE) Agar.
MacConkey Agar.
Figure 7-6 Differential capabilities of HE agar for lactose-fermenting, gram-negative bacilli (e.g., Escherichia coli, arrow A), non–lactose-fermenters (e.g., Shigella spp., arrow B), and H2S producers (e.g., Salmonella spp., arrow C).
Phenylethyl Alcohol (PEA) Agar.
Sheep Blood Agar.
Modified Thayer-Martin Agar.
Thioglycollate Broth.
Xylose-Lysine-Desoxycholate (XLD) Agar.
Preparation of Artificial Media
Figure 7-7 Growth characteristics of various bacteria in thioglycollate broth. A, Facultatively anaerobic gram-negative bacilli (i.e., those that grow in the presence or absence of oxygen) grow throughout broth. B, Gram-positive cocci demonstrating flocculation. C, Strictly aerobic organisms (i.e., those that require oxygen for growth), such as Pseudomonas aeruginosa, grow toward the top of the broth. D, Strictly anaerobic organisms (i.e., those that do not grow in the presence of oxygen) grow in the bottom of the broth.
Figure 7-8 Differential capabilities of xylose-lysine-desoxycholate (XLD) agar for lactose-fermenting, gram-negative bacilli (e.g., Escherichia coli, arrow A), non–lactose-fermenters (e.g., Shigella spp., arrow B), and H2S producers (e.g., Salmonella spp., arrow C).
Media Sterilization.
Cell Cultures.
Environmental Requirements
Oxygen and Carbon Dioxide Availability
Temperature
pH
Moisture
Methods for Providing Optimum Incubation Conditions
Bacterial Cultivation
Isolation of Bacteria From Specimens
Evaluation of Colony Morphologies
Type of Media Supporting Bacterial Growth.
Figure 7-9 A, Dilution streak technique for isolation and semiquantitation of bacterial colonies. B, Actual plates show sparse, or 1+ bacterial growth that is limited to the first quadrant. C, Moderate, or 2+ bacterial growth that extends to the second quadrant. D, Heavy, or 3+ to 4+ bacterial growth that extends to the fourth quadrant.
Figure 7-10 A, Streaking pattern using a calibrated loop for enumeration of bacterial colonies grown from a liquid specimen such as urine. B, Actual plate shows well-isolated and dispersed bacterial colonies for enumeration obtained with the calibrated loop streaking technique.
TABLE 7-2 Semi-Quantitation Grading Procedure for Bacterial Isolates on Growth Media
Relative Quantities of Each Colony Type.
Colony Characteristics.
Gram Stain and Subcultures.
Figure 7-11 Colony morphologic features and descriptive terms for commonly encountered bacterial colonies.
Principles of Identification
Figure 7-12 Mixed bacterial culture on sheep blood agar (A) requires subculture of individually distinct colonies (arrows) to obtain pure cultures of Staphylococcus aureus (beta hemolysis evident) (B) and Streptococcus pneumoniae (alpha hemolytic) (C).
Organism Identification Using Genotypic Criteria
Organism Identification Using Phenotypic Criteria
Figure 7-13 Flowchart example of a bacterial identification scheme (not applicable to anaerobic organisms).
Microscopic Morphology and Staining Characteristics
Figure 7-14 Microscopic examination of a wet preparation demonstrates the size difference between most yeast cells, such as those of Candida albicans (arrow A), and bacteria, such as Staphylococcus aureus (arrow B).
Macroscopic (Colony) Morphology
Environmental Requirements for Growth
Resistance or Susceptibility to Antimicrobial Agents
Figure 7-15 A, Zone of growth inhibition around the 5-µg vancomycin disk is indicative of a gram-positive bacterium. B, The gram-negative organism is not inhibited by this antibiotic, and growth extends to the edge of the disk.
Nutritional Requirements and Metabolic Capabilities
Establishing Enzymatic Capabilities.
Types of Enzyme-Based Tests.
Single Enzyme Tests.
Catalase Test.
Oxidase Test.
Indole Test.
Urease Test.
PYR Test.
Hippurate Hydrolysis.
Figure 7-16 Principle of glucose oxidative-fermentation (O-F) test. Fermentation patterns shown in O-F tubes including examples of oxidative, fermentative, and nonutilizing bacteria.
Tests for Presence of Metabolic Pathways.
Oxidation and Fermentation Tests.
Amino Acid Degradation.
Single Substrate Utilization.
Establishing Inhibitor Profiles.
Principles of Phenotype-Based Identification Schemes
Selection and Inoculation of Identification Test Battery
Figure 7-17 Four basic components of bacterial identification schemes and systems.
Type of Bacteria to Be Identified
Clinical Significance of the Bacterial Isolate
Availability of Reliable Testing Methods
Incubation for Substrate Utilization
Conventional Identification
Rapid Identification
Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF)
Detection of Metabolic Activity
Colorimetry
Fluorescence
Turbidity
Analysis of Metabolic Profiles
Identification Databases
TABLE 7-3 Generation and Use of Genus-Identification Database Probability: Percentage of Positive Reactions for 100 Known Strains
Figure 7-18 Example of converting a metabolic profile to an octal profile for bacterial identification.
Use of the Database to Identify Unknown Isolates
Confidence in Identification.
TABLE 7-4 Generation and Use of Genus-Identification Database Probability: Probability That Unknown Strain X Is a Member of a Known Genus Based on Results of Each Individual Parameter Tested
Commercial Identification Systems
Advantages and Examples of Commercial System Designs
Figure 7-19 Biochemical test panel (API; bioMérieux, Inc., Hazelwood, MO). The test results obtained with the substrates in each cupule are recorded, and an organism identification code is calculated by octal code conversion on the form provided. The octal profile obtained then is matched with an extensive database to establish organism identification.
Figure 7-20 Vitek cards composed of multiple wells containing dried substrates that are reconstituted by inoculation with a bacterial suspension (bioMérieux, Inc., Hazelwood, MO). Test results in the card wells are automatically read by the manufacturer’s reading device.
Overview of Commercial Systems
Bibliography
Chapter Review
Chapter 8 Nucleic Acid–Based Analytic Methods for Microbial Identification and Characterization
Objectives
Overview of Molecular Methods
Specimen Collection and Transport
Nucleic Acid Hybridization Methods
Figure 8-1 Principles of nucleic acid hybridization. Identification of an unknown organism is established by positive hybridization (i.e., duplex formation) between a nucleic acid strand from the known sequence (i.e., the probe) and a target nucleic acid strand from the organism to be identified. Failure to hybridize indicates lack of homology between the probe and the target nucleic acid.
Hybridization Steps and Components
Production and Labeling of Probe Nucleic Acid.
Preparation of Target Nucleic Acid.
Figure 8-2 Reporter molecule labeling of nucleic acid probes and principles of hybridization detection. Use of probes labeled with a radioactive reporter, with hybridization detected by autoradiography (A); probes labeled with biotin-avidin reporter, with hybridization detected by a colorimetric assay (B); probes labeled with chemiluminescent reporter (i.e., acridinium), with hybridization detected by a luminometer to detect emitted light (C).
Mixture and Hybridization of Target and Probe.
Detection of Hybridization.
Hybridization Formats
Solution Format.
Figure 8-3 Principle of the solution hybridization format.
Solid Support Format.
Figure 8-4 Principle of solid support hybridization formats. A, Filter hybridization. B, Southern hybridization. C, Sandwich hybridization.
In Situ Hybridization.
Figure 8-5 Peptide nucleic acid (PNA) probes. Structure of DNA compared to the structure of a synthetic PNA probe; the chemical modification of DNA allows for greater sensitivity and specificity of the PNA probes compared to the DNA probes.
Peptide Nucleic Acid (PNA) Probes.
Hybridization with Signal Amplification.
Amplification Methods—PCR Based
Figure 8-6 Using a fluorescent-tagged peptide nucleic acid (PNA) probe in conjunction with fluorescent in situ hybridization (FISH), Staphylococcus aureus (A) or Candida albicans (B) was directly identified in blood cultures. A drop from the positive blood culture bottle is added to a slide containing a drop of fixative solution, which keeps the cells intact. After fixation, the appropriate fluorescent-labeled PNA probe is added. The PNA probe penetrates the microbial cell wall and hybridizes to the ribosomal RNA (rRNA). Slides are examined under a fluorescent microscope. If the specific target is present, bright green, fluorescent-staining organisms are present. Blood cultures negative for either S. aureus (C) or C. albicans (D) by PNA FISH technology are shown.
Overview of PCR and Derivations
Extraction and Denaturation of Target Nucleic Acid.
Figure 8-7 The MagNaPure LC System from Roche Applied Science has been on the market since 1999. It is a fully automated nucleic acid extractor, capable of isolating DNA, RNA and viral nucleic acid from a variety of samples: blood, cells, plasma/serum, or tissue. Based on a magnetic bead technology, it is designed to automate nucleic acid purification and PCR set up. The new MagNaPure LC 2.0 is equipped with an integrated computer, LCD monitor with touch screen, and Laboratory Information Management System (LIMS) network compatibility.
Primer Annealing.
Extension of Primer-Target Duplex.
Figure 8-8 Overview of polymerase chain reaction. The target sequence is denatured to single strands, primers specific for each target strand sequence are added, and DNA polymerase catalyzes the addition of deoxynucleotides to extend and produce new strands complementary to each of the target sequence strands (cycle 1). In cycle 2, both double-stranded products of cycle 1 are denatured and subsequently serve as targets for more primer annealing and extension by DNA polymerase. After 25 to 30 cycles, at least 107 copies of target DNA may be produced.
Detection of PCR Products.
Derivations of the PCR Method.
Figure 8-9 Use of ethidium bromide–stained agarose gels to determine the size of PCR amplicons for identification. Lane A shows molecular-size markers, with the marker sizes indicated in base pairs. Lanes B, C, and D contain PCR amplicons typical of the enterococcal vancomycin-resistance genes vanA (783 kb), vanB (297 kb), and vanC1 (822 kb), respectively.
Figure 8-10 Ethidium bromide–stained gels containing amplicons produced by multiplex PCR. Lane A shows molecular-size markers, with the marker sizes indicated in base pairs. Lanes B and C show amplicons obtained with multiplex PCR consisting of control primers and primers specific for the staphylococcal methicillin-resistance gene mecA. The presence of only the control amplicon (370 bp) in Lane B indicates that PCR was successful, but the strain on which the reaction was performed did not contain mecA. Lane C shows both the control and the mecA (310 bp) amplicons, indicating that the reaction was successful and that the strain tested carries the mecA resistance gene.
Figure 8-11 Examples of real-time PCR instruments. A, Applied Biosystems. B, iCycler. C, Light Cycler. D, SmartCycler.
Real-Time PCR
TABLE 8-1 Examples of Automation and Instrumentation Available for the Molecular Microbiology Research and Clinical Laboratory
Figure 8-12 Fluorogenic probes (probes with an attached fluorophore, a fluorescent molecule that can absorb light energy and then be elevated to an excited state and released as fluorescence in the absence of a quencher) commonly used for detection of amplified product in real-time PCR assays. A, Hydrolysis probe. In addition to the specific primers for amplification, an oligonucleotide probe with a reporter fluorescent dye (R) and a quencher dye (Q) at its 5′ and 3′ ends, respectively, is added to the reaction mix. During the extension phase, the quencher (the molecule that can accept energy from a fluorophore and then dissipate the energy, resulting in no fluorescence) can quench the reporter fluorescence when the two dyes are close to each other (a). Once amplification occurs and the fluorogenic probe binds to amplified product, the bound probe is degraded by the 5′-3′ exonuclease activity of Taq polymerase; therefore, quenching is no longer possible, and fluorescence is emitted and then measured (b). B, Molecular beacon. Molecular beacons are hairpin-shaped molecules with an internally quenched fluorophore that fluoresces once the beacon probe binds to the amplified target and the quencher is no longer in proximity to the fluorophore. These probes are designed such that the loop portion of the molecule is a sequence complementary to the target of interest (a). The “stem” portion of the beacon probe is formed by the annealing of complementary arm sequences on the respective ends of the probe sequence. In addition, a fluorescent moiety (R) and a quencher moiety (Q) at opposing ends of the probe are attached (a). The stem portion of the probe keeps the fluorescent and quencher moieties in proximity to one another, quenching the fluorescence of the fluorophore. When it encounters a target molecule with a complementary sequence, the molecular beacon undergoes a spontaneous conformational change that forces the stem apart, thereby causing the fluorophore and quencher to move away from each other and leading to restoration of fluorescence (b). C, Fluorescent resonant energy transfer (FRET) or hybridization probes. Two different hybridization probes are used, one carrying a fluorescent reporter moiety at its 3′ end (designated R1) and the other carrying a fluorescent dye at its 5′ end (designated R2) (a). These two oligonucleotide probes are designed to hybridize to amplified DNA target in a head-to-tail arrangement in very close proximity to one another. The first dye (R1) is excited by a filtered light source and emits a fluorescent light at a slightly longer wavelength. Because the two dyes are so close to each other, the energy emitted from R1 excites R2 attached to the second hybridization probe, which emits fluorescent light at an even longer wavelength (b). This energy transfer is referred to as FRET. Selection of an appropriate detection channel on the instrument allows the intensity of light emitted from R2 to be filtered and measured.
Figure 8-13 Melting curve analyses performed using the LightCycler HSV1/2 Detection Kit. DNA was extracted and subjected to real-time PCR using the LightCycler to detect the presence of herpes simplex virus (HSV) DNA. After amplification, melting curve analysis was performed in which amplified product was cooled to below 55°C and the temperature then was raised slowly. The Tm is the temperature at which half of the DNA is single strand and is specific for the sequence of the particular DNA product. The specific melting temperature is determined at 640 nm (channel F2 on the cycler) for the clinical samples and the positive and negative controls. For illustration purposes, melting curve analyses are “overlaid” relative to one another in this figure for three clinical samples and the HSV-1 and HSV-2 positive or “template” control. The clinical specimens containing HSV-1 DNA (red line) or HSV-2 (green line) result in a melting peak at 54°C (the Tm) or 67°C (the Tm), respectively. The LightCycler positive or template control containing HSV-1 and HSV-2 DNA, displayed as a purple line, shows two peaks at 54°C and 67°C, respectively. The clinical sample that is negative (brown line) for both HSV-1 and HSV-2 shows no peaks.
Digital PCR
Figure 8-14 Quantitation using real-time PCR. A, In the example, four samples containing known amounts of target are amplified by real-time PCR. The inverse log of their fluorescence is plotted against the cycle number and their respective CT is determined; the fewer the number of targets, the greater the CT value. B, Similarly, the clinical specimen is also amplified by real-time PCR, and its CT value is determined. C, The log of the nucleic acid concentration and the respective CT value for each specimen containing a known amount of target or nucleic acid are plotted to generate a standard curve. Knowing the CT value of the clinical specimen allows the concentration of target in the original sample to be determined.
Amplification Methods: Non–PCR-Based
TABLE 8-2 Examples of Commercially Available Signal Amplification Methods
Isothermal Amplification
Probe Amplification
Sequencing and Enzymatic Digestion of Nucleic Acids
Nucleic Acid Sequencing
TABLE 8-3 Examples of Non-Polymerase Chain Reaction–Based Nucleic Amplification Tests
Post-Amplification and Traditional Analysis
Nucleic Acid Electrophoresis
Pyrosequencing
High-Density DNA Probes
Low- to Moderate-Density Arrays
Enzymatic Digestion and Electrophoresis of Nucleic Acids
Figure 8-15 Overview of high-density DNA probes. High-density oligonucleotide arrays are created using light-directed chemical synthesis that combines photolithography and solid-phase chemical synthesis. Because of this sophisticated process, more than 500 to as many as 1 million different oligonucleotide probes may be formed on a chip; an array is shown in A. Nucleic acid is extracted from a sample and then hybridized within seconds to the probe array in a GeneChip Fluidics Station. The hybridized array (B) is scanned using a laser confocal fluorescent microscope that looks at each site (i.e., probe) on the chip, and the intensity of hybridization is analyzed using imaging processing software.
Figure 8-16 DNA enzymatic digestion and gel electrophoresis to separate DNA fragments resulting from the digestion. An example of a nucleic acid recognition site and enzymatic cut produced by EcoR1, a commonly used endonuclease, is shown in the inset.
Figure 8-17 Restriction fragment length polymorphisms of vancomycin-resistant Enterococcus faecalis isolates in Lanes A through G as determined by pulsed-field gel electrophoresis. All isolates appear to be the same strain.
Figure 8-18 Although antimicrobial susceptibility profiles indicated that several methicillin-resistant S. aureus isolates were the same strain, restriction fragment length polymorphism analysis using pulsed-field gel electrophoresis (Lanes A through F) demonstrates that only isolates B and C were the same.
Figure 8-19 Restriction patterns generated by pulsed-field gel electrophoresis for two Streptococcus pneumoniae isolates, one that was susceptible to penicillin (Lane B) and one that was resistant (Lane C), from the same patient. Restriction fragment length polymorphism analysis indicates that the patient was infected with different strains. Molecular-size markers are shown in Lane A.
Applications of Nucleic Acid–based Methods
Direct Detection of Microorganisms
Advantages and Disadvantages
Specificity.
Sensitivity.
Amplification Techniques Enhance Sensitivity.
Applications for Direct Molecular Detection of Microorganisms
Identification of Microorganisms Grown in Culture
Characterization of Microorganisms Beyond Identification
Detection of Antimicrobial Resistance
Investigation of Strain Relatedness/Pulsed-Field Gel Electrophoresis
TABLE 8-4 Examples of Methods to Determine Strain Relatedness
Figure 8-20 Procedural steps for pulsed-field gel electrophoresis (PFGE).
Automation and Instrumentation
Bibliography
Chapter Review
Chapter 9 Immunochemical Methods Used for Organism Detection
Objectives
Production of Antibodies for Use in Laboratory Testing
Polyclonal Antibodies
Monoclonal Antibodies
Figure 9-1 Group A Streptococcus (Streptococcus pyogenes) contains many antigenic structural components and produces various antigenic enzymes, each of which may elicit a specific antibody response from the infected host.
Figure 9-2 Production of a monoclonal antibody.
Principles of Immunochemical Methods Used for Organism Detection
Precipitation Tests
Double Immunodiffusion
Figure 9-3 Exo-Antigen Identification System (Immuno-Mycologics, Inc., Norman, Okla.) The center well is filled with a 50× concentrate of an unknown mold. The arrow identifies well 1; wells 2 to 6 are shown clockwise. Wells 1, 3, and 5 are filled with anti-Histoplasma. anti-Blastomyces, and anti-Coccidioides reference antisera, respectively. Wells 2, 4, and 6 are filled with Histoplasma antigen, Blastomyces antigen, and Coccidioides antigen, respectively. The unknown organism can be identified as Histoplasma capsulatum based on the formation of line(s) of identity (arc) linking the control band(s) with one or more bands formed between the unknown extract (center well) and the reference antiserum well (well 1).
Particle Agglutination
Latex Agglutination
Figure 9-4 Alignment of antibody molecules bound to the surface of a latex particle and latex agglutination reaction.
Figure 9-5 Cryptococcal Antigen Latex Agglutination System (CALAS) (Meridian Diagnostics, Inc., Cincinnati, Ohio.) Patient 1 shows positive agglutination; patient 2 is negative.
Figure 9-6 Streptex (Remel, Inc., Lenexa, Kan.) Colony of beta-hemolytic Streptococcus agglutinates with group B Streptococcus (Streptococcus agalactiae) latex suspension.
Coagglutination
Immunofluorescent Assays
Figure 9-7 Coagglutination.
Figure 9-8 Legionella (Direct) Fluorescent Test System (Scimedx Corp., Denville, N.J.). Legionella pneumophila serogroup 1 in sputum.
Enzyme Immunoassays
Figure 9-9 Direct and indirect fluorescent antibody tests for antigen detection.
Figure 9-10 ProSpecT Giardia/Cryptosporidium Microplate Assay. A, Breakaway microwell cupules and kit components. B, Positive (yellow) changing to blue following the addition of the stop reagent. The negative reactions remain clear.
Solid-Phase Immunoassay
Membrane-Bound SPIA
Figure 9-11 Principle of direct solid-phase enzyme immunosorbent assay (SPIA). A, Solid phase is microtiter well. B, Solid phase is bead.
Figure 9-12 Principle of indirect solid-phase enzyme immunosorbent assay (SPIA).
Figure 9-13 Directigen respiratory syncytial virus (RSV) membrane-bound cassette. A, Positive reaction. B, Negative reaction.
Other Immunoassays
Bibliography
Chapter Review
Chapter 10 Serologic Diagnosis of Infectious Diseases
Objectives
Features of the Immune Response
Characteristics of Antibodies
Figure 10-1 Structure of immunoglobulin G. The heavy chains determine the antibody class (IgG, IgA, IgD, IgE, or IgM). The Fab fragment containing the variable regions determines the antibody binding specificity. The Fc portion (or function cells) binds to various immune cells to activate specific functions in the immune system.
Figure 10-2 Structure of immunoglobulin M.
Features of the Humoral Immune Response Useful in Diagnostic Testing
Figure 10-3 Relative humoral response to antigen stimulation over time.
Interpretation of Serologic Tests
Serodiagnosis of Infectious Diseases
TABLE 10-1 Noninclusive Overview of Tests Available for Serodiagnosis of Infectious Diseases
Principles of Serologic Test Methods
Separating IgM from IgG for Serologic Testing
Methods of Antibody Detection
Direct Whole Pathogen Agglutination Assays
Particle Agglutination Tests
Flocculation Tests
Figure 10-4 MACRO-VUE RPR card test. R, Reactive (positive) test indicated by the diffuse degree of clumping. NR, non-reactive (negative test), indicated by a smooth suspension or non-diffuse slight roughness as demonstrated here as a peripheral roughness in well 1 or somewhat centric roughness in well 2.
Immunodiffusion Assays
Hemagglutination Inhibition Assays
Neutralization Assays
Complement Fixation Assays
Figure 10-5 Complement fixation test.
Enzyme-Linked Immunosorbent Assays
Figure 10-6 Indirect fluorescent antibody tests for Toxoplasma gondii, IgG antibodies. A, Positive reaction. B, Negative reaction.
Indirect Fluorescent Antibody Tests and Other Immunomicroscopic Methods
Radioimmunoassays
Fluorescent Immunoassays
Figure 10-7 Diagram of Western blot immunoassay system.
Western Blot Immunoassays
Figure 10-8 Human immunodeficiency virus type 1 (HIV-1) Western blot immunoassay. Samples are characterized as positive, indeterminate, or negative based on the bands found to be present in significant intensity. A positive blot has any two or more of the following bands: p24, gp41, and/or gp120/160. An indeterminate blot contains some bands but not the definitive ones. A negative blot has no bands present. Lane 16 shows antibodies from a control serum binding to the virus-specific proteins (p) and glycoproteins (gp) transferred onto the nitrocellulose paper.
Chapter Review
Section 3 Evaluation of Antimicrobial Activity
Chapter 11 Principles of Antimicrobial Action and Resistance
Objectives
Antimicrobial Action
Principles
Figure 11-1 The basic steps required for antimicrobial activity and strategic points for bacterial circumvention or interference (marked by X) of antimicrobial action, leading to resistance.
TABLE 11-1 Anatomic Distribution of Some Common Antibacterial Agents
Box 11-1 Bacteriostatic and Bactericidal Antibacterial Agents*
Generally Bacteriostatic
Generally Bactericidal
TABLE 11-2 Summary of Mechanisms of Action for Commonly Used Antibacterial Agents
Mode of Action of Antibacterial Agents
Inhibitors of Cell Wall Synthesis
β-Lactam (Beta-Lactam) Antimicrobial Agents.
Figure 11-2 Basic structures and examples of commonly used β-lactam antibiotics. The core β-lactam ring is highlighted in yellow in each structure.
Figure 11-3 Structure of vancomycin, a non–β-lactam antibiotic that inhibits cell wall synthesis.
Glycopeptides and Lipopeptides.
Inhibitors of Cell Membrane Function
Inhibitors of Protein Synthesis
Aminoglycosides and Aminocyclitols.
Figure 11-4 Structure of the commonly used aminoglycoside gentamicin. Potential sites of modification by adenylating, phosphorylating, and acetylating enzymes produced by bacteria are highlighted.
Macrolide-Lincosamide-Streptogramin (MLS) Group.
Ketolides.
Oxazolidinones.
Chloramphenicol.
Tetracyclines.
Glycylglycines.
Inhibitors of DNA and RNA Synthesis
Fluoroquinolones.
Figure 11-5 Structures of the fluoroquinolones ciprofloxacin and ofloxacin.
Metronidazole.
Rifamycin.
Figure 11-6 Bacterial folic acid pathway indicating the target enzymes for sulfonamide and trimethoprim activity.
Inhibitors of Other Metabolic Processes
Sulfonamides.
Trimethoprim.
Nitrofurantoin.
Mechanisms of Antibiotic Resistance
Principles
Biologic Versus Clinical Resistance
Environmentally Mediated Antimicrobial Resistance
Figure 11-7 Cations (Mg++ and Ca++) and aminoglycosides (AG++) compete for the negatively charged binding sites on the outer membrane surface of Pseudomonas aeruginosa. Such competition is an example of the impact that environmental factors (e.g., cation concentrations) can have on the antibacterial activity of aminoglycosides.
Microorganism-Mediated Antimicrobial Resistance
TABLE 11-3 Examples of Intrinsic Resistance to Antibacterial Agents
Intrinsic Resistance
Figure 11-8 Overview of common pathways bacteria use to effect antimicrobial resistance.
Acquired Resistance
Common Pathways for Antimicrobial Resistance
Resistance to Beta-Lactam Antibiotics
Figure 11-9 Mode of β-lactamase enzyme activity. The enzyme cleaves the β-lactam ring, and the molecule can no longer bind to penicillin-binding proteins (PBPs) and is no longer able to inhibit cell wall synthesis.
TABLE 11-4 Summary of Resistance Mechanisms for Beta-Lactams, Vancomycin, Aminoglycosides, and Fluoroquinolones
Figure 11-10 Diagrammatic summary of β-lactam resistance mechanisms for gram-positive and gram-negative bacteria. A, Among gram-positive bacteria, resistance is mediated by β-lactamase production and altered PBP targets. B, In gram-negative bacteria, resistance can also be mediated by decreased uptake through the outer membrane porins.
Resistance to Glycopeptides
Resistance to Aminoglycosides
Resistance to Quinolones
Resistance to Other Antimicrobial Agents
Emergence and Dissemination of Antimicrobial Resistance
Figure 11-11 Factors contributing to the emergence and dissemination of antimicrobial resistance among bacteria.
Box 11-2 Bacterial Resistance Mechanisms for Miscellaneous Antimicrobial Agents
Chloramphenicol
Tetracyclines
Macrolides (i.e., Erythromycin) and Clindamycin
Sulfonamides and Trimethoprim
Rifampin
Case Study 11-1
Questions
Bibliography
Chapter Review
Chapter 12 Laboratory Methods and Strategies for Antimicrobial Susceptibility Testing
Objectives
Goal and Limitations
Standardization
Limitations of Standardization
Testing Methods
Principles
Methods That Directly Measure Antimicrobial Activity
Conventional Testing Methods: General Considerations
Inoculum Preparation.
Figure 12-1 Bacterial suspension prepared to match the turbidity of the 0.5 McFarland standard. Matching this turbidity provides a bacterial inoculum concentration of 1 to 2 × 108 CFU/mL. The McFarland standard on the right indicates the correct turbidity required for testing.
Selection of Antimicrobial Agents for Testing.
Box 12-1 Criteria for Antimicrobial Battery Content and Use
Organism Identification or Group
Acquired Resistance Patterns Common to Local Microbial Flora
Antimicrobial Susceptibility Testing Method Used
Site of Infection
Availability of Antimicrobial Agents in the Formulary
Conventional Testing Methods: Broth Dilution
TABLE 12-1 Summary of Broth Dilution Susceptibility Testing Conditions
Procedures.
Medium and Antimicrobial Agents.
Inoculation and Incubation.
Figure 12-2 Microtiter tray used for broth microdilution testing. Doubling dilutions of each antimicrobial agent in test broth occupies one vertical row of wells.
Reading and Interpretation of Results.
Advantages and Disadvantages.
Figure 12-3 Bacterial growth profiles in a broth microdilution tray. The wells containing the lowest concentration of an antibiotic that completely inhibits visible growth (arrow) are recorded in micrograms per milliliter (µg/mL) as the minimal inhibitory concentration (MIC).
Box 12-2 Definitions of Susceptibility Testing Interpretive Categories*
Susceptible
Intermediate
Resistant
Conventional Testing Methods: Agar Dilution
Figure 12-4 Growth pattern on an agar dilution plate. Each plate contains a single concentration of antibiotic. Growth is indicated by a spot on the agar surface. No spot is seen for isolates inhibited by the concentration of antibiotic incorporated into the agar of that particular plate.
TABLE 12-2 Summary of Agar Dilution Susceptibility Testing Conditions
Conventional Testing Methods: Disk Diffusion
Figure 12-5 A, Disk diffusion method: antibiotic disks are placed on the agar surface just after inoculation of the surface with the test organism. B, Zones of growth inhibition around various disks are apparent after 16 to 18 hours of incubation.
Figure 12-6 Example of a regression analysis plot to establish zone-size breakpoints to define the categorical limits for susceptible, intermediate, and resistant for an antimicrobial agent. In this example, the maximum achievable serum concentration of the antibiotic is 8 µg/mL. Disk inhibition zones less than or equal to 18 mm in diameter indicate resistance; zones greater than or equal to 26 mm in diameter indicate susceptibility; the intermediate category is indicated by zones ranging from 19 to 25 mm in diameter.
Procedures.
Medium and Antimicrobial Agents.
TABLE 12-3 Summary of Disk Diffusion Susceptibility Testing Conditions
Inoculation and Incubation.
Reading and Interpretation of Results.
Figure 12-7 Disk diffusion plate inoculated with a mixed culture, as evidenced by the various colonial morphologies (arrows) appearing throughout the lawn of growth.
Figure 12-8 Examination of a disk diffusion plate by transmitted and reflected light.
Figure 12-9 Bacterial growth is visible inside the zone of inhibition (arrows). This may indicate inoculation with a mixed culture. However, emergence of resistant mutants of the test isolate is a more likely reason for this growth pattern.
Advantages and Disadvantages.
Commercial Susceptibility Testing Systems
Broth Microdilution Methods.
Agar Dilution Derivations.
Diffusion in Agar Derivations.
Automated Antimicrobial Susceptibility Test Systems.
Figure 12-10 Growth patterns on a plate containing an antibiotic gradient (the concentration decreases from the center of the plate to the periphery) applied by the Spiral Gradient instrument. The distance from the point where growth is noted at the edge of the plate to the point where growth is inhibited toward the center of the plate is measured. This value is used in a formula to calculate the MIC of the antimicrobial agent against each of the bacterial isolates streaked on the plate.
Alternative Approaches for Enhancing Resistance Detection
Figure 12-11 The Etest® strip uses the principle of a predefined antibiotic gradient on a plastic strip to generate an MIC value. It is processed in the same way as the disk diffusion. A, Individual antibiotic strips are placed on an inoculated agar surface. B, After incubation, the MIC is read where the growth/inhibition edge intersects the strip graduated with an MIC scale across 15 dilutions (arrow). Several antibiotic strips can be tested on a plate.
Figure 12-12 The VITEK® 2 antimicrobial susceptibility test card contains 64 wells with multiple concentrations of up to 22 antibiotics. The antibiotic is rehydrated when the organism suspension is introduced into the card during the automated filling process.
Supplemental Testing Methods.
Figure 12-13 The components of the VITEK® 2 system consist of the instrument housing; the sample processing and reader/incubator; the computer workstation, which provides data analysis, storage, and epidemiology reports; the Smart Carrier Station, which is the direct interface between the microbiologist on the bench and the instrument; and a bar code scanner to facilitate data entry.
Figure 12-14 Microdilution tray format (A) used with the MicroScan WalkAway instrument (B) for automated incubation, reading, and interpretation of antimicrobial susceptibility tests.
TABLE 12-4 Supplemental Methods for Detection of Antimicrobial Resistance
Predictor Antimicrobial Agents.
Methods That Directly Detect Specific Resistance Mechanisms
Phenotypic Methods
Figure 12-15 The chromogenic cephalosporin test allows direct detection of β-lactamase production. When the β-lactam ring of the cephalosporin substrate in the disk is hydrolyzed by the bacterial inoculum, a deep pink color is produced (A). Lack of color production indicates the absence of β-lactamase (B).
β-Lactamase Detection.
Chloramphenicol Acetyltransferase Detection.
Genotypic Methods
Special Methods for Complex Antimicrobial/Organism Interactions
Bactericidal Tests
Minimal Bactericidal Concentration.
Time-Kill Studies.
Serum Bactericidal (Schlichter Test).
Tests for Activity of Antimicrobial Combinations
Figure 12-16 Goals of effective antimicrobial susceptibility testing strategies.
Laboratory Strategies for Antimicrobial Susceptibility Testing
Relevance
When to Perform a Susceptibility Test
Determining Clinical Significance
Predictability of Antimicrobial Susceptibility
TABLE 12-5 Categorization of Bacteria According to Need for Routine Performance of Antimicrobial Susceptibility Testing*
Availability of Reliable Susceptibility Testing Methods
Selection of Antimicrobial Agents for Testing
Accuracy
Use of Accurate Methodologies
TABLE 12-6 Selection of Antimicrobial Agents for Testing Against Common Bacterial Groups*
Review of Results
Components of Results Review Strategies
Data Review.
TABLE 12-7 Examples of Susceptibility Testing Profiles Requiring Further Evaluation
Resolution.
Accuracy and Antimicrobial Resistance Surveillance
Communication
Case Study 12-1
Questions
Reference
Bibliography
Chapter Review
Part III Bacteriology
Section 1 Principles of Identification
Chapter 13 Overview of Bacterial Identification Methods and Strategies
Objectives
Rationale for Approaching Organism Identification
Figure 13-1 Data demonstrating that more than 500 different bacterial species or taxa are reported from clinical laboratories across the United States. However, 95% of the isolates reported are distributed among only 27 different taxa, and more than 90% are represented by 16 different taxa.
Figure 13-2 Relative frequency with which the most common bacterial species and taxa are reported by clinical laboratories.
TABLE 13-1 Examples of Commercial Identification Systems for Various Organisms
Future Trends of Organism Identification
Procedure 13-1 Acetamide Utilization
Purpose
Principle
Method
Figure 13-3 Acetamide utilization. A, Positive. B, Negative.
Expected Results
Limitations
Quality Control
Procedure 13-2 Acetate Utilization
Purpose
Principle
Method
Figure 13-4 Acetate utilization. A, Positive. B, Negative.
Expected Results
Limitations
Quality Control
Procedure 13-3 Bacitracin Susceptibility
Purpose
Principle
Method
Expected Results
Limitations
Quality Control
Figure 13-5 Bacitracin (A disk) Susceptibility. Any zone of inhibition is positive (Streptococcus pyogenes); growth up to the disk is negative (Streptococcus agalactiae).
Procedure 13-4 Bile Esculin Test
Purpose
Principle
Method
Expected Results
Limitations
Quality Control
Figure 13-6 Bile esculin agar. A, Positive. B, Negative.
Procedure 13-5 Bile Solubility Test
Purpose
Principle
Method
Expected Results
Limitations
Quality Control
Figure 13-7 Bile solubility (desoxycholate) test. A, Colony lysed. B, Intact colony.
Procedure 13-6 Butyrate Disk
Purpose
Principle
Method
Expected Results
Limitations
Quality Control
Figure 13-8 Butyrate disk. A, Positive. B, Negative.
Procedure 13-7 CAMP Test
Purpose
Principle
Method
Expected Results
Limitations
Quality Control
Figure 13-9 CAMP test. A, Positive; arrowhead zone of beta-hemolysis (at arrow), typical of group B streptococci. B, Negative; no enhancement of hemolysis.
Procedure 13-8 Catalase Test
Purpose
Principle
Method
Expected Results
Limitations
Quality Control
Figure 13-10 Catalase test. A, Positive. B, Negative.
Procedure 13-9 Cetrimide Agar
Purpose
Principle
Method
Expected Results
Limitations
Quality Control
Figure 13-11 Cetrimide agar. A, Positive. B, Negative.
Procedure 13-10 Citrate Utilization
Purpose
Principle
Method
Expected Results
Limitations
Quality Control
Figure 13-12 Citrate utilization. A, Positive. B, Negative.
Procedure 13-11 Coagulase Test
Purpose
Principle
Method
A Slide Test (Detection of ?)
Expected Results
B Tube Test
Expected Results
Limitations
Slide Test
Tube Test
Quality Control
Figure 13-13 Coagulase test. A, Slide coagulase test for clumping factor. Left side is positive; right side is negative. B, Tube coagulase test for free coagulase. Tube on the left is positive, exhibiting clot. Tube on the right is negative.
Procedure 13-12 Decarboxylase Tests (Moeller’s Method)
Purpose
Principle
Method
A Glucose-Nonfermenting Organisms
B Glucose-Fermenting Organisms
Expected Results
Limitations
Quality Control
Figure 13-14 Decarboxylase tests (Moeller’s method). A, Positive. B, Negative. C, Uninoculated tube.
Procedure 13-13 DNA Hydrolysis (DNase Test Agar)
Purpose
Principle
Method
Expected Results
Limitations
Quality Control
Figure 13-15 DNA hydrolysis. A, Positive, Staphylococcus aureus. B, Positive, Serratia marcescens. C, Negative.
Procedure 13-14
Figure 13-16 Esculin hydrolysis. A, Positive, blackening of slant. B, Uninoculated tube.
Esculin Hydrolysis
Purpose
Principle
Method
Expected Results
Limitations
Quality Control
Procedure 13-15 Fermentation Media
Purpose
Principle
Method
A Peptone Medium with Andrade’s Indicator (for Enterics and Coryneforms)
Expected Results
B Broth (Brain Heart Infusion Broth May Be Substituted) with Bromcresol Purple Indicator (for Streptococci and Enterococci)
Expected Results
Limitations
Quality Control
A Peptone Medium with Andrade’s Indicator
B Brain-Heart Infusion Broth with Bromocresol Purple Indicator
Figure 13-17 Fermentation media. A, Peptone medium with Andrade’s indicator. The tube on the left ferments glucose with the production of gas (visible as a bubble [arrow] in the inverted [Durham] tube); the tube in the middle ferments glucose with no gas production; and the tube on the right does not ferment glucose. B, Heart infusion broth with bromocresol purple indicator. The tube on the left is positive; the tube on the right is negative.
Procedure 13-16 Flagella Stain (Wet Mount Technique)
Purpose
Principle
Method
Expected Results
Limitations
Quality Control
Figure 13-18 Flagella stain (wet mount technique). A, Alcaligenes spp., peritrichous flagella (arrows). B, Pseudomonas aeruginosa, polar flagella (arrows).
Procedure 13-17 Gelatin Hydrolysis
Purpose
Principle
Method
Expected Results
Limitations
Quality Control
Figure 13-19 Gelatin hydrolysis. A, Positive; note liquefaction at top of tube. B, Uninoculated tube.
Procedure 13-18 Growth at 42°C
Purpose
Principle
Method
Expected Results
Quality Control
Figure 13-20 Growth at 42°C. A, Positive; good growth. B, Negative; no growth.
Procedure 13-19 Hippurate Hydrolysis
Purpose
Principle
Method
Expected Results
Limitations
Quality Control
Figure 13-21 Hippurate hydrolysis. A, Positive. B, Negative.
Procedure 13-20 Indole Production
Purpose
Principle
Method
A Enterobacteriaceae
B Other Gram-Negative Bacilli
Expected Results
Limitations
Quality Control
A Kovac’s Method
B Ehrlich’s Method
C Ehrlich’s Method (Anaerobic)
Figure 13-22 Indole production. A, Positive. B, Negative.
Procedure 13-21 Leucine Aminopeptidase (LAP) Test
Purpose
Principle
Method
Expected Results
Limitations
Quality Control
Figure 13-23 LAP test. A, Positive. B, Negative.
Procedure 13-22 Litmus Milk Medium
Purpose
Principle
Method
Quality Control
Limitations
Expected Results
Appearance of Indicator (Litmus Dye)
Appearance of Milk
Figure 13-24 Litmus milk. A, Acid reaction. B, Alkaline reaction. C, No change. D, Reduction of indicator. E, Clot. (Note separation of clear fluid from clot at arrow.) F, Peptonization.
Procedure 13-23 Lysine Iron Agar (LIA)
Purpose
Principle
Method
Expected Results
Limitations
Quality Control
Figure 13-25 Lysine iron agar. A, Alkaline slant/alkaline butt (K/K). B, Alkaline slant/alkaline butt, H2S positive (K/K H2S+). C, Alkaline slant/acid butt (K/A). D, Red slant/acid butt (R/A). E, Uninoculated tube.
Procedure 13-24 Methyl Red/Voges-Proskauer (Mrvp) Tests
Purpose
Principle
Method
A MR (Methyl Red) Test
Expected Results
B VP (Voges-Proskauer) Test (Barritt’s Method) for Gram-Negative Rods
Expected Results
C VP (Voges-Proskauer) Test (Coblentz Method) for Streptococci
Limitations
Quality Control
Figure 13-26 Methyl red/Voges-Proskauer (MRVP) tests. A, Positive methyl red. B, Negative methyl red. C, Positive Voges-Proskauer. D, Negative Voges-Proskauer.
Procedure 13-25 Microdase Test (Modified Oxidase)
Purpose
Principle
Method
Expected Results
Limitations
Quality Control
Figure 13-27 Microdase test. A, Positive. B, Negative.
Procedure 13-26 Motility Testing
Purpose
Principle
Method
Expected Results
Limitations
Quality Control
Figure 13-28 Motility test. A, Positive. B, Negative.
Procedure 13-27 MRS Broth
Purpose
Principle
Method
Expected Results
Quality Control
Figure 13-29 MRS broth. A, Positive; gas production by Leuconostoc sp. (arrow). B, Positive: growth, no gas production by Lactobacillus sp.
Procedure 13-28 4-Methylumbelliferyl-β-D-Glucuronide (MUG) Test
Purpose
Principle
Method
Expected Results
Limitations
Quality Control
Figure 13-30 MUG test. A, Positive. B, Negative.
Procedure 13-29 Nitrate Reduction
Purpose
Principle
Method
Expected Results
Figure 13-31 Nitrate reduction. A, Positive, no gas. B, Positive, gas (arrow). C, Positive, no color after addition of zinc (arrow).D, Uninoculated tube.
Limitations
Quality Control
Procedure 13-30 Nitrite Reduction
Purpose
Principle
Method
Expected Results
Limitations
Quality Control
Figure 13-32 Nitrite reduction. A, Positive, no color change after addition of zinc dust and gas in Durham tube (arrow). B, Negative.
Procedure 13-31 o-Nitrophenyl-β-D-Galactopyranoside (ONPG) Test
Purpose
Principle
Method
Expected Results
Quality Control
Figure 13-33 OPNG test. A, Positive. B, Negative.
Procedure 13-32 Optochin (P disk) Susceptibility Test
Purpose
Principle
Method
Expected Results
Limitations
Quality Control
Figure 13-34 Optochin (TaxoP disk) test. A, Streptococcus pneumoniae showing zone of inhibition greater than 14 mm. B, Alpha-hemolytic Streptococcus sp. growing up to the disk.
Procedure 13-33 Oxidase Test (Kovac’s Method)
Purpose
Principle
Method
Expected Results
Limitations
Quality Control
Figure 13-35 Oxidase test. A, Positive. B, Negative.
Procedure 13-34 Oxidation/Fermentation (of) Medium (CDC Method)
Purpose
Principle
Method
Expected Results
Limitations
Quality Control
Figure 13-36 Oxidation/fermentation medium (CDC method). A, Fermenter. B, Oxidizer. C, Nonutilizer.
Procedure 13-35 Phenylalanine Deaminase Agar
Purpose
Principle
Method
Expected Results
Quality Control
Figure 13-37 Phenylalanine deaminase. A, Positive. B, Negative.
Procedure 13-36 L-Pyrrolidonyl Arylamidase (PYR) Test
Purpose
Principle
Method
Expected Results
Quality Control
Figure 13-38 PYR test. A, Positive. B, Negative.
Procedure 13-37 Pyruvate Broth
Purpose
Principle
Method
Expected Results
Quality Control
Figure 13-39 Pyruvate broth. A, Positive. B, Negative.
Procedure 13-38 Salt Tolerance Test
Purpose
Principle
Method
Expected Results
Quality Control
Figure 13-40 Salt tolerance (6.5% NaCl) test. A, Positive. B, Negative.
Procedure 13-39 Spot Indole Test
Purpose
Principle
Method
Expected Results
Limitations
Quality Control
Figure 13-41 Spot indole test. A, Positive. B, Negative.
Procedure 13-40 Triple Sugar Iron Agar (TSI)
Purpose
Principle
Method
Expected Results
Quality Control
Figure 13-42 Triple sugar iron agar. A, Acid slant/acid butt with gas, no H2S (A/A). B, Alkaline slant/acid butt, no gas, H2S-positive (K/A H2S+). C, Alkaline slant/alkaline butt, no gas, no H2S (K/K). D, Uninoculated tube.
Procedure 13-41 Urease Test (Christensen’s Method)
Purpose
Principle
Method
Expected Results
Limitations
Quality Control
Figure 13-43 Urea hydrolysis (Christensen’s method). A, Positive. B, Negative.
Procedure 13-42 X and V Factor Test
Purpose
Principle
Method
Expected Results
Quality Control
Figure 13-44 X (hemin) and V (nicotinamide adenine dinucleotide [NAD]) factor test. A, Positive: growth around XV disk only. B, Positive: growth around V disk. C, Negative: growth over entire plate.
Bibliography
Section 2 Catalase-Positive, Gram-Positive Cocci
Chapter 14 Staphylococcus, Micrococcus, and Similar Organisms
Objectives
Genera and Species to Be Considered
General Characteristics
Epidemiology
TABLE 14-1 Epidemiology
Pathogenesis and Spectrum of Disease
TABLE 14-2 Pathogenesis and Spectrum of Diseases
Laboratory Diagnosis
Specimen Collection and Transport
Specimen Processing
Direct Detection Methods
Microscopy
Nucleic Acid Testing
Figure 14-1 Gram stain of Staphylococcus aureus from blood agar.
Figure 14-2 A, Yellow colonies of S. aureus fermenting mannitol as evident by the yellow color of the agar. B, White colonies of S. epidermidis, no-mannitol fermenting, as evident by the original pink color of the agar.
Cultivation
Media of Choice
Figure 14-3 CHROMagar for the identification of MRSA isolates through the selective and differential formation of mauve-colored colonies.
Incubation Conditions and Duration
Colonial Appearance
Approach to Identification
Figure 14-4 Staphylococcus epidermis screening plate showing resistance to bacitracin (taxo A disk) and susceptible to furazolidone (FX disk).
TABLE 14-3 Colonial Appearance and Characteristics on 5% Sheep Blood Agar
Comments Regarding Specific Organisms
TABLE 14-4 Differentiation among Gram-Positive, Catalase-Positive Cocci
Figure 14-5 Staphylococcal identification to species.
Serodiagnosis
TABLE 14-5 Differentiation among the Most Clinically Significant Coagulase-Positive Staphylococci
TABLE 14-6 Differentiation among Coagulase-Negative, PYR-Negative, Novobiocin-Resistant Staphylococci
TABLE 14-7 Differentiation among Coagulase-Negative, PYR-Negative, Novobiocin-Susceptible, Alkaline Phosphatase-Negative Staphylococci
TABLE 14-8 Differentiation of Coagulase-Negative, PYR-Positive, Novobiocin-Susceptible, Alkaline Phosphatase-Negative Staphylococci
TABLE 14-9 Differentiation of Coagulase-Negative, PYR-Positive, Novobiocin-Susceptible, Alkaline Phosphatase–Positive Staphylococci
Antimicrobial Susceptibility Testing and Therapy
TABLE 14-10 Antimicrobial Therapy and Susceptibility Testing
Prevention
Chapter Review
Case Study 14-1
Questions
Figure 14-6 Gram stain clinical specimen demonstrating the presence of gram-positive cocci in clusters and infiltrated with white blood cells.
Case Study 14-2
Questions
Figure 14-7 Single microscopic field of vision of the Gram stain of the patient’s synovial fluid.
Bibliography
Section 3 Catalase-Negative, Gram-Positive Cocci
Chapter 15 Streptococcus, Enterococcus, and Similar Organisms
Objectives
Genera and Species to Be Considered
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
Beta-Hemolytic Streptococci
TABLE 15-1 Epidemiology
TABLE 15-2 Pathogenesis and Spectrum of Disease
Streptococcus Pneumoniae and Viridans Streptococci
Enterococci
Miscellaneous Other Gram-Positive Cocci
Laboratory Diagnosis
Specimen Collection and Transport
Specimen Processing
Direct Detection Methods
Antigen Detection
Molecular Diagnostic Testing
Gram Stain
Figure 15-1 S. pneumoniae lancet-shaped diplococci in Gram stain; note the encapsulated organisms as evident by the clear “halo.”
Cultivation
Media of Choice
Figure 15-2 Chains of streptococci seen in Gram stain prepared from broth culture.
TABLE 15-3 Differentiation of Catalase-Negative, Gram-Positive Coccoid Organisms Primarily in Chains
Incubation Conditions and Duration
Colonial Appearance
TABLE 15-4 Differentiation of Catalase-Negative, Gram-Positive, Coccoid Organisms Primarily in Clusters or Tetrads
Approach to Identification
Comments Regarding Specific Organisms
Figure 15-3 Stabbing the inoculating loop vertically into the agar after streaking the blood agar plate (A) allows subsurface colonies to display hemolysis caused by streptolysin O (B).*Based on the reactions of only one isolate.†S. bovis variant includes S. infantarius subsp. infantarius, S. lutetiensis, and S. gallolyticus subsp. pasteurianus. Most S. bovis variant strains will be positive for α-galactosidase and S. salivarius will be negative.‡Optochin test must be performed in CO2 to avoid misidentification with S. pseudopneumoniae.
Figure 15-4 Differentiation of gram-positive cocci.
Figure 15-5 Positive CAMP reaction as indicated by enlarged zone of hemolysis shaped like a tip of the arrow, S. agalactiae intersecting with S. aureus streak line.
TABLE 15-5 Colonial Appearance and Characteristics on 5% Sheep Blood Agar
TABLE 15-6 Differentiation of the Clinically Relevant Beta-Hemolytic Streptococci
Figure 15-6 Differentiation of clinically relevant viridans streptococcal groups. S. mitis group includes S. mitis, S. sanguinis, S. parasanguinis, S. gordonii, S. oralis, and S. cristatus. S. mutans group includes S. mutans and S. sobrinus. S. anginosus group includes S. anginosus, S. constellatus subsp. constellatus, and S. intermedius. S. salivarius group includes S. salivarius and S. vestibularis. β-gal, Beta-galactosidase; PYR, pyrrolidonyl arylamidase; R, resistant; S, sensitive; +, positive; =, negative.
Serodiagnosis
Figure 15-7 Species identification of clinically relevant enterococcal and enterococcal-like isolates. =, Signifies a negative result.
Antimicrobial Susceptibility Testing and Therapy
TABLE 15-7 Antimicrobial Therapy and Susceptibility Testing
Prevention
Chapter Review
Case Study 15-1
Questions
Case Study 15-2
Laboratory Results
Questions
Figure 15-8 Patient’s sputum Gram stain.
Bibliography
Section 4 Non-Branching, Catalase-Positive, Gram-Positive Bacilli
Chapter 16 Bacillus and Similar Organisms
Objectives
Genera and Species to Be Considered
General Characteristics
Bacillus Anthracis
Epidemiology
Figure 16-1 Red Line Alert Test. A red line appears on the cassette if the culture isolate is presumptive Bacillus anthracis.
TABLE 16-1 Epidemiology
Pathogenesis and Spectrum of Disease
Bacillus Cereus
Epidemiology
Pathogenesis and Spectrum of Disease
Figure 16-2 Lecithinase production by Bacillus cereus on egg yolk agar. The organism has been streaked down the center of the plate. The positive test for lecithinase is indicated by the opaque zone of precipitation around the bacterial growth (arrows).
TABLE 16-2 Pathogenesis and Spectrum of Disease
Bacillus Thuringiensis
Bacillus Subtilis, Brevibacillus Sp., and Paenibacillus Spp.
Epidemiology
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Specimen Processing
Direct Detection Methods
Figure 16-3 Gram stain of Bacillus cereus. The arrow is pointed at a spore, the clear area inside the gram-positive vegetative cell.
Figure 16-4 Spore stain of Bacillus cereus. The arrows are pointed at green spores in a pink vegetative cell.
Cultivation
Media of Choice
TABLE 16-3 Colonial Appearance and Other Characteristics
Incubation Conditions and Duration
Colonial Appearance
Approach to Identification
Serodiagnosis
Molecular Diagnostics
Antimicrobial Susceptibility Testing and Therapy
Prevention
Chapter Review
TABLE 16-4 Differentiation of Clinically Relevant Bacillus spp., Brevibacillus, and Paenibacillus
TABLE 16-5 Antimicrobial Therapy and Susceptibility Testing
Case Study 16-1
Questions
Bibliography
Chapter 17 Listeria, Corynebacterium, and Similar Organisms
Objectives
Genera and Species to Be Considered
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
TABLE 17-1 Epidemiology
TABLE 17-2 Pathogenesis and Spectrum of Diseases
Laboratory Diagnosis
Specimen Collection and Transport
Specimen Processing
Direct Detection Methods
Cultivation
Media of Choice
Figure 17-1 Gram stain of Corynebacterium diphtheriae. Note palisading and arrangements of cells in formations that resemble Chinese letters (arrows).
Incubation Conditions and Duration
TABLE 17-3 Gram Stain Morphology, Colonial Appearance, and Other Distinguishing Characteristics
Figure 17-2 A, Rothia dentocariosa from broth. B, R. dentocariosa from solid media.
Figure 17-3 Corynebacterium urealyticum on blood agar with Tween 80 (A) and blood agar (B) at 48 hours. This organism is lipophilic and grows much better on the lipid-containing medium.
Figure 17-4 Colony of Corynebacterium diphtheriae on Tinsdale agar. Note black colonies with brown halo.
Colonial Appearance
Approach to Identification
Comments on Specific Organisms
Figure 17-5 Diagram of an Elek plate for demonstration of toxin production by Corynebacterium diphtheriae. A filter paper strip impregnated with diphtheria antitoxin is buried just beneath the surface of a special agar plate before the agar hardens. Strains to be tested and known positive and negative toxigenic strains are streaked on the agar’s surface in a line across the plate and at a right angle to the antitoxin paper strip. After 24 hours of incubation at 37°C, the plates are examined with transmitted light for the presence of fine precipitin lines at a 45-degree angle to the streaks. The presence of precipitin lines indicates that the strain produced toxin that reacted with the homologous antitoxin. Line 1 is the negative control. Line 2 is the positive control. Line 3 is an unknown organism that is a nontoxigenic strain. Line 4 is an unknown organism that is a toxigenic strain.
Figure 17-6 Umbrella motility of Listeria monocytogenes grown at room temperature.
TABLE 17-4 Catalase-Positive, Non–Acid-Fast, Gram-Positive Rodsa
TABLE 17-5 Fermentative, Nonlipophilic, Tinsdale-Positive Corynebacterium spp.*
TABLE 17-6 Fermentative, Nonlipophilic, Tinsdale-Negative Clinically Relevant Corynebacterium spp.*†
TABLE 17-7 Strictly Aerobic, Nonlipophilic, Nonfermentative, Clinically Relevant Corynebacterium spp.a,b
TABLE 17-8 Strictly Aerobic, Lipophilic, Nonfermentative, Clinically Relevant Corynebacterium spp.*
TABLE 17-9 Lipophilic, Fermentative, Clinically Relevant Corynebacterium spp.*
TABLE 17-10 Antimicrobial Therapy and Susceptibility Testing
Serodiagnosis
Antimicrobial Susceptibility Testing and Therapy
Prevention
Case Study 17-1
Questions
Chapter Review
Bibliography
Section 5 Non-Branching, Catalase-Negative, Gram-Positive Bacilli
Chapter 18 Erysipelothrix, Lactobacillus, and Similar Organisms
Objectives
Genera and Species to Be Considered
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
TABLE 18-1 Epidemiology
TABLE 18-2 Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Specimen Collection and Transport
Specimen Processing
Direct Detection Methods
Figure 18-1 Gram stain of Lactobacillus spp. Note spiral forms (arrow).
Cultivation
Media of Choice.
Incubation Conditions and Duration
Figure 18-2 Gardnerella vaginalis on human blood bilayer Tween (HBT) agar. Note small colonies with diffuse zone of beta-hemolysis (arrow).
TABLE 18-3 Colonial Appearance on 5% Sheep Blood Agar and Other Characteristics
Colonial Appearance
Approach to Identification
TABLE 18-4 Biochemical and Physiologic Characteristics of Catalase-Negative, Gram-Positive, Aerotolerant, Non–Spore-Forming Rods
Figure 18-3 Reverse Christie, Atkins, Munch-Petersen (CAMP) test. Arcanobacterium haemolyticum is streaked on a blood agar plate. Staphylococcus aureus is then streaked perpendicular to the Arcanobacterium path. A positive reverse CAMP test result is indicated (arrow).
Comments Regarding Specific Organisms
Figure 18-4 H2S production by Erysipelothrix rhusiopathiae in TSI (B). A negative TSI (A) is included for comparison.
Serodiagnosis
Antimicrobial Susceptibility Testing and Therapy
TABLE 18-5 Antimicrobial Therapy and Susceptibility Testing
Prevention
Case Study 18-1
Questions
Chapter Review
Bibliography
Section 6 Branching or Partially Acid-Fast, Gram-Positive Bacilli
Chapter 19 Nocardia, Streptomyces, Rhodococcus, and Similar Organisms
Objectives
Genera and Species to Be Considered
General Characteristics
TABLE 19-1 Clinically Relevant Aerobic Actinomycetes*
Partially Acid-Fast Aerobic Actinomycetes
Nocardia spp.
TABLE 19-2 Species Included in the Genera Rhodococcus, Gordonia, and Tsukamurella
Box 19-1 Nocardia spp. Considered Human Pathogens or Have Been Implicated in Human Disease
Less Common or Prevalence Not Established
Rhodococcus, Gordonia, Tsukamurella spp.
TABLE 19-3 Non–Acid-Fast Aerobic Actinomycetes Associated with Human Disease
Non–acid-Fast Aerobic Actinomycetes: Streptomyces, Actinomadura, Dermatophilus, Nocardiopsis, and the Thermophilic Actinomycetes
Epidemiology and Pathogenesis
Partially Acid-Fast Aerobic Actinomycetes
Nocardia spp.
Rhodococcus, Gordonia, Tsukamurella spp.
Non–acid-Fast Aerobic Actinomycetes: Streptomyces, Actinomadura, Dermatophilus, Nocardiopsis, and the Thermophilic Actinomycetes
Spectrum of Disease
Partially Acid-Fast Aerobic Actinomycetes
Nocardia spp.
TABLE 19-4 Epidemiology of the Non–Acid-Fast Aerobic Actinomycetes
Rhodococcus, Gordonia, Tsukamurella spp.
TABLE 19-5 Infections Caused by Rhodococcus, Gordonia, and Tsukamurella spp.
Non–acid-Fast Aerobic Actinomycetes: Streptomyces, Actinomadura, Dermatophilus, Nocardiopsis, and the Thermophilic Actinomycetes
Figure 19-1 A, Gram stain of sputum obtained from a patient with pulmonary nocardiosis caused by Nocardia asteroides. B, The same sputum stained with a modified acid-fast stain. The organism is indicated by the arrow.
TABLE 19-6 Clinical Manifestations of Infections Caused by Non–Acid-Fast Aerobic Actinomycetes
Laboratory Diagnosis
Specimen Collection, Transport, and Processing
Direct Detection Methods
Procedure 19-1 Partially Acid-Fast Stain for Identification of Nocardia spp.
Principle
Method
Molecular Diagnostics
Cultivation
Approach to Identification
Procedure 19-2 Lysozyme Resistance for Differentiating Nocardia from Streptomyces spp.
Principle
Method
Figure 19-2 Gram stains of different aerobic actinomycetes. A, Nocardia asteroides grown on Löwenstein-Jensen medium. The arrows indicate branching rods. B, Rhodococcus equi from broth. C, R. equi grown on chocolate agar. D, Streptomyces spp. grown on Sabouraud dextrose agar.
Figure 19-3 Aerobic actinomycetes grown on solid media. A, Nocardia asteroides grown on Löwenstein-Jensen medium. B, Rhodococcus equi grown on chocolate agar.
Figure 19-4 Lysozyme (A) and glycerol (B) broths. The lysozyme broth demonstrates enhanced growth, which is typical of Nocardia asteroides.
TABLE 19-7 Typical Gram-Stain Morphology and Colonial Appearance
TABLE 19-8 Preliminary Grouping of the Clinically Relevant Aerobic Actinomycetes
TABLE 19-9 Key Tests for Differentiation of the Pathogenic Nocardia spp.
Serodiagnosis
Antimicrobial Susceptibility Testing and Therapy
TABLE 19-10 Primary Drugs of Choice for Infections Caused by Aerobic Actinomycetes
Prevention
Case Study 19-1
Questions
Chapter Review
Reference
Bibliography
Section 7 Gram-Negative Bacilli and Coccobacilli (MacConkey-Positive, Oxidase-Negative)
Chapter 20 Enterobacteriaceae
Objectives
Genera and Species to Be Considered
Opportunistic Pathogens
Pathogenic Organisms
Primary Intestinal Pathogens
Pathogenic Yersinia spp.
Rare Human Pathogens
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Diseases
TABLE 20-1 Epidemiology of Clinically Relevant Enterobacteriaceae
Specific Organisms
Opportunistic Human Pathogens
Citrobacter spp. (C. freundii, C. koseri, C. braakii)
Cronobacter sakazakii
TABLE 20-2 Pathogenesis and Spectrum of Disease for Clinically Relevant Enterobacteriaceae
TABLE 20-3 Biochemical Differentiation of Citrobacter Species
Edwardsiella tarda
Enterobacter spp. (E. aerogenes, E. cloacae, E. gergoviae, E. amnigenus, E. taylorae)
Escherichia coli (UPEC, MNEC, ETEC, EIEC, EAEC, EPEC and EHEC)
Ewingella americana
Hafnia alvei
Klebsiella spp. (K. pneumoniae, K. oxytoca)
Morganella spp. (M. morganii, M. psychrotolerans)
Pantoea agglomerans
Plesiomonas shigelloides
Proteus spp. (P. mirabilis, P. vulgaris, P. penneri) and Providencia spp. (P. alcalifaciens, P. heimbachae, P. rettgeri, P. stuartii, P. rustigianii)
Serratia spp. (S. marcescens, S. liquefaciens group)
Primary Intestinal Pathogens
Salmonella (All Serotypes)
Shigella spp. (S. dysenteriae, S. flexneri, S. boydii, S. sonnei)
Yersinia spp. (Y. pestis, Y. enterocolitica, Y. frederiksenii, Y. intermedia, Y. pseudotuberculosis)
Rare Human Pathogens
Laboratory Diagnosis
Specimen Collection and Transport
Specimen Processing
Direct Detection Methods
Cultivation
Media of Choice
Figure 20-1 Bull’s-eye colony of Yersinia enterocolitica (arrow) on cefsulodin-irgasan-novobiocin (CIN) agar.
Incubation Conditions and Duration
Colonial Appearance
TABLE 20-4 Biochemical Media used in the Differentiation and Isolation of Enterobacteriaceae
Approach to Identification
TABLE 20-5 Colonial Appearance and Characteristics of the Most Commonly Isolated Enterobacteriaceae*
Figure 20-2 Red-pigmented Serratia marcescens on MacConkey agar.
Figure 20-3 Proteus mirabilis swarming on blood agar (arrow points to swarming edge).
TABLE 20-6 Biochemical Differentiation of Unusual LDC-, ODC- and ADH-negative Enterobacteriaceae
Specific Considerations for Identifying Enteric Pathogens
Figure 20-4 Biochemical differentiation of representative Enterobacteriaceae. V, Variability can be equally either positive or negative; +(v), greater probability for positive reaction >50%; −(v), greater probability for negative reaction >50%; (+), positive > 80%; (−), negative > 80%. The pink squares indicate a pattern useful for preliminary recognition. The green squares indicate a key characteristic for biochemical identification.
Figure 20-5 Algorithm for the identification of Enterobacteriaceae. * Denotes variability in lactose fermentation reactions. LF, Late fermenter; C, indicates growth on Simmons citrate agar; U, indicates urease reaction; I, indicates indole reaction; MR, methyl red; +, positive > 90%; − indicates ≤ 10% negative; +/−, > 50%; +, −/+, indicates less than 5% positive]; KI, Kligler iron agar; OD, ornithine decarboxylase positive; AD, arginine decarboxylase positive; LD, lysine decarboxylase positive; PPA, phenylalanine deamination to phenylpyruvic acid; M, mannitol fermentation; ONPG, ortho-nitrophenyl-beta-galactoside test.
TABLE 20-7 TSI and LIA Reactions Used to Screen for Enteropathogenic Enterobacteriaceae and Aeromonas/Vibrio spp.*†
Serodiagnosis
Antimicrobial Susceptibility Testing and Therapy
TABLE 20-8 Therapy for Gastrointestinal Infections Caused by Enterobacteriaceae
Extended Spectrum β-Lactamase (ESBL)–Producing Enterobacteriaceae
TABLE 20-9 Antimicrobial Therapy and Susceptibility Testing of Clinically Relevant Enterobacteriaceae
Expanded-Spectrum Cephalosporin Resistance and carbapenemase resistance
TABLE 20-10 Intrinsic Antibiotic Resistance in Enterobacteriaceae*
TABLE 20-11 Extended Beta-Lactamase Antibiotic Resistance Pattern Based on Vitek 2 Gram-Negative Susceptibility AST-GN24 of an E. coli Isolate
Procedure 20-1 Modified Hodge Test (MHT)
Purpose
Principle
Method
Expected Results
Limitations
Quality Control
Multidrug-Resistant Typhoid Fever (MDRTF)
Prevention
Chapter Review
Case Study 20-1
Questions
Case Study 20-2
Patient Vital Signs
Initial Evaluation
Questions
Bibliography
Chapter 21 Acinetobacter, Stenotrophomonas, and Similar Organisms
Objectives
Genera and Species to Be Considered
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
TABLE 21-1 Epidemiology
TABLE 21-2 Pathogenesis and Spectrum of Diseases
Laboratory Diagnosis
Specimen Collection and Transport
Specimen Processing
Direct Detection Methods
Cultivation
Media of Choice
Incubation Conditions and Duration
Colonial Appearance
TABLE 21-3 Colonial Appearance and Characteristics
Approach to Identification
Comments Regarding Specific Organisms
Figure 21-1 Colony of Acinetobacter spp. on MacConkey agar. Note purple color.
TABLE 21-4 Key Biochemical and Physiologic Characteristics
Serodiagnosis
TABLE 21-5 Antimicrobial Therapy and Susceptibility Testing
Antimicrobial Susceptibility Testing and Therapy
Prevention
Case Study 21-1
Questions
Bibliography
Chapter Review
Section 8 Gram-Negative Bacilli and Coccobacilli (MacConkey-Positive, Oxidase-Positive)
Chapter 22 Pseudomonas, Burkholderia, and Similar Organisms
Objectives
Genera and Species to Be Considered
General Characteristics
Epidemiology
Burkholderia spp. and Ralstonia Pickettii
TABLE 22-1 Epidemiology
Pseudomonas spp. and Brevundimonas spp.
TABLE 22-2 Pathogenesis and Spectrum of Disease
Pathogenesis and Spectrum of Disease
Burkholderia spp. and Ralstonia Pickettii
Pseudomonas spp. and Brevundimonas spp.
Laboratory Diagnosis
Specimen Collection and Transport
Specimen Processing
Direct Detection Methods
Nucleic Acid Detection
Cultivation
Media of Choice
Figure 22-1 Burkholderia cepacia on chocolate agar. Note green pigment.
Incubation Conditions and Duration
Colonial Appearance
Approach to Identification
Figure 22-2 Pseudomonas aeruginosa on tryptic soy agar (B). Note bluish-green color. Uninoculated tube (A) is shown for comparison.
TABLE 22-3 Colonial Appearance and Other Characteristics of Pseudomonas, Brevundimonas, Burkholderia, Ralstonia, and Other Organisms
Comments Regarding Specific Organisms
Figure 22-3 Pseudomonas aeruginosa on MacConkey agar.
TABLE 22-4 Biochemical and Physiologic Characteristics
Serodiagnosis
Antimicrobial Susceptibility Testing and Therapy
Prevention
Chapter Review
TABLE 22-5 Antimicrobial Therapy and Susceptibility Testing
Case Study 22-1
Questions
Figure 22-4 Pseudomonas aeruginosa on blood agar.
Case Study 22-2
Questions
Figure 22-5 Gram stain result for the wound specimen obtained from the patient in Case Study 22-2. Note the cluster of organisms in the center of the photograph.
Bibliography
Chapter 23 Rhizobium, Ochrobactrum, and Similar Organisms
Objectives
Genera and Species to Be Considered
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
TABLE 23-1 Epidemiology
TABLE 23-2 Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Specimen Collection and Transport
Specimen Processing
Direct Detection Methods
Cultivation
Media of Choice
Incubation Conditions and Duration
Colonial Appearance
Figure 23-1 Paracoccus yeei; note doughnut-shaped organism on Gram stain (arrows).
Approach to Identification
Comments Regarding Specific Organisms
TABLE 23-3 Colonial Appearance and Characteristics
TABLE 23-4 Key Biochemical and Physiologic Characteristics
Serodiagnosis
Antimicrobial Susceptibility Testing and Therapy
TABLE 23-5 Antimicrobial Therapy and Susceptibility Testing
Prevention
Case Study 23-1
Questions
Bibliography
Chapter Review
Chapter 24 Chryseobacterium, Sphingobacterium, and Similar Organisms
Objectives
Genera and Species to Be Considered
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
TABLE 24-1 Epidemiology
TABLE 24-2 Pathogenesis and Spectrum of Diseases
Laboratory Diagnosis
Specimen Collection and Transport
Specimen Processing
Direct Detection Methods
Cultivation
Media of Choice
Incubation Conditions and Duration
TABLE 24-3 Colonial Appearance and Characteristics
Colonial Appearance
Approach to Identification
Comments Regarding Specific Organisms
Serodiagnosis
TABLE 24-4 Key Biochemical and Physiologic Characteristics
TABLE 24-5 Antimicrobial Therapy and Susceptibility Testing
Antimicrobial Susceptibility Testing and Therapy
Prevention
Case Study 24-1
Questions
Bibliography
Chapter Review
Chapter 25 Alcaligenes, Bordetella (Non-pertussis), Comamonas, and Similar Organisms
Objectives
Genera and Species to Be Considered
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
TABLE 25-1 Epidemiology
Laboratory Diagnosis
Specimen Collection and Transport
Specimen Processing
Direct Detection Methods
TABLE 25-2 Pathogenesis and Spectrum of Disease
Cultivation
Media of Choice
Incubation Conditions and Duration
Colonial Appearance
Approach to Identification
TABLE 25-3 Colonial Appearance and Characteristics
Comments Regarding Specific Organisms
TABLE 25-4 Key Biochemical and Physiologic Characteristics for Coccoid Species
TABLE 25-5 Key Biochemical and Physiologic Characteristics for Rod-Shaped Nonmotile Species
Serodiagnosis
Antimicrobial Susceptibility Testingand Therapy
TABLE 25-6 Key Biochemical and Physiologic Characteristics for Rod-Shaped Motile Species with Polar Flagella
TABLE 25-7 Key Biochemical and Physiologic Characteristics for Rod-Shaped Motile Species with Peritrichous Flagella
TABLE 25-8 Antimicrobial Therapy and Susceptibility Testing
Prevention
Case Study 25-1
Questions
Bibliography
Chapter Review
Chapter 26 Vibrio, Aeromonas, Chromobacterium, and Related Organisms
Objectives
Genera and Species to Be Considered
General Characteristics
Epidemiology
TABLE 26-1 Epidemiology
Pathogenesis and Spectrum of Disease
TABLE 26-2 Pathogenesis and Spectrum of Diseases
Laboratory Diagnosis
Specimen Collection and Transport
Specimen Processing
Figure 26-1 Gram stain of Vibrio parahaemolyticus.
Direct Detection Methods
Cultivation
Media of Choice
Incubation Conditions and Duration
Figure 26-2 Colonies of Chromobacterium violaceum on DNase agar. Note violet pigment.
TABLE 26-3 Colonial Appearance and Characteristics
Colonial Appearance
Figure 26-3 Colonies of Vibrio cholerae (A) and V. parahaemolyticus (B) on TCBS agar.
Approach to Identification
Figure 26-4 String test used to differentiate Vibrio spp. (positive) from Aeromonas spp. and P. shigelloides (negative).
Comments Regarding Specific Organisms
TABLE 26-4 Key Biochemical and Physiologic Characteristics of Vibrio spp. and Grimontia hollisae
TABLE 26-5 Key Biochemical and Physiologic Characteristics of Aeromonas spp., P. shigelloides, and C. violaceum
Serodiagnosis
Antimicrobial Susceptibility Testing and Therapy
Prevention
TABLE 26-6 Antimicrobial Therapy and Susceptibility Testing
Case Study 26-1
Questions
Chapter Review
Bibliography
Section 9 Gram-Negative Bacilli and Coccobacilli (MacConkey-Negative, Oxidase-Positive)
Chapter 27 Sphingomonas paucimobilis and Similar Organisms
Objectives
Organisms to Be Considered
General Considerations
Epidemiology, Spectrum of Disease, and Antimicrobial Therapy
Laboratory Diagnosis
Specimen Collection and Transport
Specimen Processing
Direct Detection Methods
Cultivation
Media of Choice
TABLE 27-1 Epidemiology, Spectrum of Disease, and Antimicrobial Therapy
Incubation Conditions and Duration
Colonial Appearance
Approach to Identification
Comments Regarding Specific Organisms
Acidovorax facilis.
CDC groups IIc, IIe, IIh, IIi, O-1, O-2, and O-3.
TABLE 27-2 Colonial Appearance and Characteristics
TABLE 27-3 Key Biochemical and Physiologic Characteristics
Sphingobacterium mizutaii.
TABLE 27-4 Specific Biochemical Characteristics for Differentiation of CDC groups IIc, IIe, and IIh*
Sphingobacterium multivorum.
TABLE 27-5 Specific Biochemical Characteristics for Differentiation of the Sphingobacterium spp.
TABLE 27-6 Specific Biochemical Characteristics for Differentiation of the Sphingomonas spp.
Sphingobacterium spiritivorum.
Figure 27-1 Sphingomonas paucimobilis growth on BAP.
Sphingomonas paucimobilis.
Sphingomonas parapaucimobilis.
Antimicrobial Susceptibility
Serodiagnosis
Prevention
Case Study 27-1
Questions
Case Study 27-2
Questions
Chapter Review
Bibliography
Chapter 28 Moraxella and Related Organisms
Objectives
Genera and Species to Be Considered
General Characteristics
Epidemiology, Spectrum of Disease, and Antimicrobial Therapy
TABLE 28-1 Epidemiology, Pathogenesis, and Spectrum of Disease
TABLE 28-2 Antimicrobial Therapy and Susceptibility Testing
Laboratory Diagnosis
Specimen Collection and Transport
Specimen Processing
Direct Detection Methods
Cultivation
Media of Choice
Incubation Conditions and Duration
Colonial Appearance
Approach to Identification
Comments Regarding Specific Organisms
TABLE 28-3 Colonial Appearance and Characteristics
Serodiagnosis
Prevention
Chapter Review
TABLE 28-4 Key Biochemical and Physiologic Characteristics
Case Study 28-1
Questions
Bibliography
Chapter 29 Eikenella and Similar Organisms
Objectives
Genera and Species to Be Considered
General Characteristics
Epidemiology, Spectrum of Disease, and Antimicrobial Therapy
Laboratory Diagnosis
Specimen Collection and Transport
TABLE 29-1 Epidemiology, Pathogenesis, and Spectrum of Disease
Specimen Processing
Direct Detection Methods
Cultivation
Media of Choice
TABLE 29-2 Antimicrobial Therapy and Susceptibility Testing
Incubation Conditions and Duration
Colonial Appearance
Approach to Identification
Comments Regarding Specific Organisms
TABLE 29-3 Colonial Appearance and Characteristics
Serodiagnosis
TABLE 29-4 Key Biochemical and Physiologic Characteristics
Prevention
Case Study 29-1
Questions
Bibliography
Chapter Review
Chapter 30 Pasteurella and Similar Organisms
Objectives
Genera and Species to Be Considered
General Characteristics and Taxonomy
Epidemiology, Spectrum of Disease, and Antimicrobial Therapy
TABLE 30-1 Epidemiology of Selected Pasteurella spp. and Similar Organisms
TABLE 30-2 Pathogenesis and Spectrum of Disease of Selected Pasteurella spp. and Similar Organisms
TABLE 30-3 Antimicrobial Therapy and Susceptibility Testing for Pasteurella spp. and Similar Organisms
Laboratory Diagnosis
Specimen Collection and Transport
Specimen Processing
Direct Detection Methods
Cultivation
Media of Choice
Incubation Conditions and Duration
Colonial Appearance
Approach to Identification
TABLE 30-4 Colonial Appearance and Characteristics of Selected Pasteurella spp. and Similar Organisms on Sheep Blood Agar
TABLE 30-5 Key Biochemical Characteristics of Selected Pasteurella spp. and Similar Organisms
Comments Regarding Specific Organisms
Serodiagnosis
Prevention
Case Study 30-1
Questions
Chapter Review
Bibliography
Chapter 31 Actinobacillus, Aggregatibacter, Kingella, Cardiobacterium, Capnocytophaga, and Similar Organisms
Objectives
Genera and Species to Be Considered
General Characteristics
Epidemiology, Pathogenesis, and Spectrum of Disease, and Antimicrobial Therapy
TABLE 31-1 Epidemiology
TABLE 31-2 Pathogenesis and Spectrum of Diseases
TABLE 31-3 Antimicrobial Therapy and Susceptibility Testing
Laboratory Diagnosis
Specimen Collection and Transport
Specimen Processing
Direct Detection Methods
Cultivation
Media of Choice
Incubation Conditions and Duration
Colonial Appearance
Approach to Identification
Comments Regarding Specific Organisms
TABLE 31-4 Colonial Appearance and Characteristics on 5% Sheep Blood Agar
TABLE 31-5 Biochemical and Physiologic Characteristics of Actinobacillus spp. and Related Organisms
TABLE 31-6 Biochemical and Physiologic Characteristics of Capnocytophaga spp., Dysgonomonas spp., and Similar Organisms
Serodiagnosis
Prevention
Case Study 31-1
Questions
Chapter Review
Bibliography
Section 10 Gram-Negative Bacilli and Coccobacilli (MacConkey-Negative, Oxidase-Variable)
Chapter 32 Haemophilus
Objectives
Organisms to Be Considered
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
TABLE 32-1 Epidemiology
TABLE 32-2 Pathogenesis and Spectrum of Diseases
Laboratory Diagnosis
Specimen Collection and Transport
Specimen Processing
Direct Detection Methods
Direct Observation
Figure 32-1 Gram stain of Haemophilus influenzae.
TABLE 32-3 Differentiation of Haemophilus influenzae and H. parainfluenzae Biotypes
Antigen Detection
Molecular Testing
Cultivation
Media of Choice
Figure 32-2 Haemophilus influenzae satellite phenomenon (arrow) around colonies of Staphylococcus aureus.
Incubation Conditions and Duration
TABLE 32-4 Colonial Appearance and Characteristics
Colonial Appearance
Approach to Identification
Serotyping
Serodiagnosis
Antimicrobial Susceptibility Testing and Therapy
TABLE 32-5 Key Biochemical and Physiologic Characteristics of Haemophilus spp.
TABLE 32-6 Antimicrobial Therapy and Susceptibility Testing
Prevention
Case Study 32-1
Questions
Bibliography
Chapter Review
Section 11 Gram-Negative Bacilli that are Optimally Recovered on Special Media
Chapter 33 Bartonella and Afipia
Objectives
Genera and Species to Be Considered
Bartonella
General Characteristics
Epidemiology and Pathogenesis
TABLE 33-1 Organisms Belonging to the Genus Bartonella and Recognized to Cause Disease in Humans*
Spectrum of Disease
Laboratory Diagnosis
Specimen Collection, Transport, and Processing
Direct Detection Methods
Figure 33-1 A, Colonies of Bartonella henselae on blood agar. B, Gram stain of a colony of B. henselae from blood agar.
Cultivation
Approach to Identification
Serodiagnosis
Antimicrobial Susceptibility Testing and Therapy
Prevention
Afipia felis
Chapter Review
Case Study 33-1
Questions
Bibliography
Chapter 34 Campylobacter, Arcobacter, and Helicobacter
Objectives
Genera and Species to be Considered
Campylobacter and Arcobacter
General Characteristics
Epidemiology and Pathogenesis
Figure 34-1 Identification scheme for the differentiation of the genera Helicobacter, Campylobacter, and Arcobacter. BAP, Blood agar plate.
Spectrum of Disease
TABLE 34-1 Campylobacter and Arcobacter spp., Their Source, and Spectrum of Disease in Humans
Laboratory Diagnosis
Specimen Collection, Transport, and Processing
Direct Detection
Antigen Detection
Media
Cultivation
Stool.
Figure 34-2 A, Gram stain appearance of Campylobacter jejuni subsp. jejuni from a colony on a primary isolation plate. Note seagull and curved forms (arrows). B, Appearance of Campylobacter jejuni subsp. jejuni in a direct Gram stain of stool obtained from a patient with campylobacteriosis. Arrows point to the seagull form.
TABLE 34-2 Selective Media and Incubation Conditions to Recover Campylobacter and Arcobacter spp. from Stool Specimens
Blood.
Figure 34-3 Colonies of Campylobacter jejuni following 48 hours of incubation on a selective medium in a microaerobic atmosphere.
Approach to Identification
Serodiagnosis
Antimicrobial Susceptibility Testing and Therapy
Prevention
Helicobacter
General Characteristics
TABLE 34-3 Differential Characteristics of Clinically Relevant Campylobacter, Arcobacter, and Helicobacter spp.
Epidemiology and Pathogenesis
TABLE 34-4 Genes and Their Possible Role in Enhancing Virulence of H. pylori
Spectrum of Disease
Laboratory Diagnosis
Specimen Collection, Transport, and Processing
Direct Detection
Cultivation
Approach to Identification
Serodiagnosis
Antimicrobial Susceptibility Testing and Therapy
Prevention
Case Study 34-1
Questions
Chapter Review
Bibliography
Chapter 35 Legionella
Objectives
Genus and Species to be Considered
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
Figure 35-1
Box 35-1 Some Legionella spp. Isolated from Humans and Environmental Sources
Species Isolated From Humans
Species Isolated From the Environment Only
TABLE 35-1 Disease Spectrum Associated with Legionella sp.
Box 35-2 Examples of L. pneumophila Factors Crucial for Intracellular Infection
Laboratory Diagnosis
Specimen Collection and Transport
Specimen Processing
Direct Detection Methods
Stains
Figure 35-2 Fluorescent antibody-stained Legionella pneumophila.
Antigens
Nucleic Acid Amplification
Cultivation
Figure 35-3 Colonies of Legionella pneumophila on buffered charcoal-yeast extract agar.
Figure 35-4 Gram stain of a colony of Legionella pneumophila showing thin, gram-negative bacilli (arrows).
Approach to Identification
Serodiagnosis
Antimicrobial Susceptibility Testing and Therapy
Prevention
Case Study 35-1
Questions
Chapter Review
Advanced Case Study 35-2
Laboratory Results
Questions
Bibliography
Chapter 36 Brucella
Objectives
Genera and Species to be Considered
General Characteristics
Epidemiology and Pathogenesis
TABLE 36-1 Brucella spp. and Their Respective Natural Animal Hosts
Spectrum of Disease
Laboratory Diagnosis
Specimen Collection, Transport, and Processing
Direct Detection Methods
Cultivation
Figure 36-1 Growth of Brucella spp. on chocolate agar after incubation for 2 days (A) and 4 days (B).
Figure 36-2 Brucella melitensis with traditional Gram stain (A) and Gram stain with 2-minute safranin counterstain (B) to allow easier visualization of the organism.
Approach to Identification
TABLE 36-2 Characteristics of Brucella spp. That Are Pathogenic for Humans
Serodiagnosis
Antimicrobial Susceptibility Testing and Therapy
Prevention
Case Study 36-1
Questions
Chapter Review
Bibliography
Chapter 37 Bordetella pertussis, Bordetella parapertussis, and Related Species
Objectives
Genera and Species to be Considered
General Characteristics
Epidemiology and Pathogenesis
Epidemiology
Pathogenesis
TABLE 37-1 Major Virulence Determinants of Bordetella pertussis
Spectrum of Disease
Box 37-1 Factors Known to Affect the Clinical Manifestation of Bordetella pertussis Infection
Laboratory Diagnosis
Specimen Collection, Transport, and Processing
TABLE 37-2 Examples of Selective Media for Primary Isolation of B. pertussis and B. parapertussis
Direct Detection Methods
Figure 37-1 Growth of Bordetella pertussis on Regan-Lowe agar.
Cultivation
Figure 37-2 Typical Gram stain appearance of Bordetella pertussis.
TABLE 37-3 Characteristics That Differentiate Bordetella spp.
Approach to Identification
Serodiagnosis
Antimicrobial Susceptibility Testing and Therapy
Prevention
Chapter Review
Case Study 37-1
Questions
Bibliography
Chapter 38 Francisella
Objectives
Genera and Species to be Considered
General Characteristics
Epidemiology and Pathogenesis
Spectrum of Disease
TABLE 38-1 Most Recent Taxonomy of the Genus Francisella and Key Characteristics
TABLE 38-2 Clinical Manifestations of Francisella tularensis Infection
Laboratory Diagnosis
Specimen Collection, Transport, and Processing
Direct Detection Methods
TABLE 38-3 Recommended Specimen Type Based on Clinical Manifestation
Cultivation
Box 38-1 Indications of a Possible Francisella Species
Approach to Identification
Serodiagnosis
Antimicrobial Susceptibility Testing and Therapy
Prevention
Case Study 38-1
Questions
Figure 38-1 Francisella tularensis growing on chocolate agar after 72 hours of incubation.
Chapter Review
Bibliography
Chapter 39 Streptobacillus moniliformis and Spirillum minus
Objectives
Genera and Species to be Considered
Streptobacillus Moniliformis
General Characteristics
Epidemiology and Pathogenesis
Spectrum of Disease
Laboratory Diagnosis
Specimen Collection, Transport, and Processing
Direct Detection Methods
Figure 39-1 Gram stain of Streptobacillus moniliformis from growth in thioglycollate broth with 20% serum.
Cultivation
Approach to Identification
Serodiagnosis
Antimicrobial Susceptibility Testing and Therapy
Prevention
Spirillum Minus
General Characteristics
Epidemiology and Pathogenesis
Spectrum of Disease
Laboratory Diagnosis
Specimen Collection, Transport, and Processing
Direction Detection Methods
Serodiagnosis
Antimicrobial Susceptibility Testing and Therapy
Prevention
Case Study 39-1
Questions
Bibliography
Chapter Review
Section 12 Gram-Negative Cocci
Chapter 40 Neisseria and Moraxella catarrhalis
Objectives
Genera and Species to be Considered
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
TABLE 40-1 Epidemiology
Figure 40-1 JEMBEC system. Plate contains modified Thayer-Martin medium. The CO2-generating tablet is composed of sodium bicarbonate and citric acid. After inoculation the tablet is placed in the well, and the plate is closed and placed in the zip-lock plastic pouch. The moisture in the agar activates the tablet, generating a CO2 atmosphere in the pouch.
Laboratory Diagnosis
Specimen Collection and Transport
TABLE 40-2 Pathogenesis and Spectrum of Disease
Specimen Processing
Direct Detection Methods
Gram Stain
Figure 40-2 Gram stain of Neisseria gonorrhoeae showing gram-negative diplococci (arrows).
Commercial Molecular Assays
Antigen Detection
Cultivation
Media of Choice
Figure 40-3 Candle jar.
Incubation Conditions and Duration
Colonial Appearance
TABLE 40-3 Colonial Appearance and Other Characteristics on Chocolate Agar*
Approach to Identification
Biochemical Identification
TABLE 40-4 Biochemical and Physiologic Characteristics of Moraxella catarrhalis and Coccoid Neisseria spp.
Comments About Specific Organisms
Procedure 40-1 Carbohydrate Utilization Method—CTA
Purpose
Method
Expected Results and Quality Control
Limitations
Immunoserologic Identification
Serotyping
Serodiagnosis
Antimicrobial Susceptibility Testing and Therapy
Prevention
TABLE 40-5 Antimicrobial Therapy and Susceptibility Testing
Case Study 40-1
Questions
Chapter Review
Bibliography
Section 13 Anaerobic Bacteriology: Laboratory Considerations
Chapter 41 Overview and General Considerations
Objectives
General Characteristics
Specimen Collection and Transport
Figure 41-1 Anaerobic transport system for liquid specimens. The specimen is injected into the tube through the rubber septum. Agar at the bottom contains an oxygen tension indicator.
Figure 41-2 Anaerobic transport system for swab specimens: Vacutainer Anaerobic Specimen Collector (BD Diagnostic Systems, Sparks, Md.). The sterile pack contains a sterile swab and an oxygen-free inner tube. After the specimen has been collected, the swab is inserted back into the inner tube. Agar on the bottom of the outer tube contains an oxygen tension indicator.
Figure 41-3 Anaerobic transport system for tissue specimens. Tissue is placed in a small amount of saline to keep it moist. It then is inserted into a self-contained, atmosphere-generating anaerobic bag for transportation. This system is called the GasPak Pouch.
Box 41-1 Clinical Specimens Suitable for Anaerobic Culture
Box 41-2 Clinical Specimens Unsuitable for Anaerobic Culture
Macroscopic Examination of Specimens
Direct Detection Methods
Antigen Detection
Gram Staining
Specimen Processing
Anaerobe Jars or Pouches
TABLE 41-1 Gram Stain Morphology, Colonial Appearance, and Other Distinguishing Features of Common Anaerobic Bacteria
Figure 41-4 GasPak anaerobe jar (BD Diagnostic Systems, Sparks, Md.). Inside the jar are inoculated plates, an activated gas-generating envelope, and an indicator strip. A wire-mesh basket attached to the lid of the jar contains palladium-coated alumina pellets that catalyze the reaction to remove oxygen. Newer models of the GasPak jar use reagent packs that simply require the addition of water to catalyze a reaction (see chapter text).
Holding Jars
Anaerobe Chamber
Anaerobic Media
Figure 41-5 Gloveless anaerobe chamber. Anaerobic chambers were developed more than 40 years ago, and a variety of new models are being used in cutting-edge research.
TABLE 41-2 Common Anaerobic Media
Incubation Conditions and Duration
Figure 41-6 Prereduced, anaerobically sterilized (PRAS) plated media.
Approach to Identification
Examination of Primary Plates
Figure 41-7 Algorithm for isolation and identification of anaerobic bacteria. BAP, Sheep blood agar; CHOC, chocolate agar; BBE, Bacteroides bile esculin agar; LKV, laked kanamycin-vancomycin agar; PEA, phenylethyl agar; EYA, egg yolk agar (for suspected Clostridium spp.), THIO, thioglycollate enrichment broth (should be examined daily and incubated for up to 7 days if no growth is identified on primary media; subculture to anaerobic media if growth is detected in broth culture).
Subculture of Isolates
Procedure 41-1 Antibiotic Identification Disks
Principle
Method
Expected Results
Quality Control
Figure 41-8 Special potency antibiotic disk and other disks. The disk that shows a red pigment contains Actinomyces odontolyticus.
TABLE 41-3 Preliminary Grouping of Anaerobic Bacteria Based on Minimal Criteria
Presumptive Identification of Isolates
Definitive Identification
TABLE 41-4 Abbreviated Identification of Gram-Negative Anaerobes
TABLE 41-5 Abbreviated Identification of Gram-Positive Anaerobes
Figure 41-9 Actual chromatogram of volatile acid standard. A, Acetic acid; P, propionic acid; iB, isobutyric acid; B, butyric acid; iV, isovaleric acid; V, valeric acid; iC, isocaproic acid; C, caproic acid.
Antimicrobial Susceptibility Testing and Therapy
TABLE 41-6 Antimicrobial Therapy and Susceptibility Testing of Anaerobic Bacteria
TABLE 41-7 Summary of Antimicrobial Susceptibility Testing Methods for Anaerobic Bacteria
Box 41-3 Indications for Performing Antimicrobial Susceptibility Testing with Anaerobic Bacteria
Bibliography
Procedure 41-2
Purpose
Principle
Method
Expected Results
Limitations
Quality Control
Chapter Review
Chapter 42 Overview of Anaerobic Organisms
Objectives
Genera and Species to be Considered
Epidemiology
TABLE 42-1 Incidence of Anaerobes as Normal Flora of Humans
TABLE 42-2 Acquisition of Anaerobic Infections and Diseases
Pathogenesis and Spectrum of Disease
TABLE 42-3 Pathogenesis and Spectrum of Disease for Anaerobic Bacteria
Gram-Positive, Spore-Forming Bacilli
Figure 42-1 Gram stain of Clostridium perfringens.
TABLE 42-4 Characteristics of Clinically Significant Clostridium species
Procedure 42-1 Ethanol Shock Spore Isolation
Purpose
Principle
Method
Expected Results
Limitations
Figure 42-2 Clostridium difficile on cycloserine cefoxitin fructose agar (CCFA).
Laboratory Diagnosis and Specimen Collection
Figure 42-3 Clostridium perfringens on anaerobic blood agar. Note double zone of beta-hemolysis. 1, First zone; 2, second zone.
TABLE 42-5 Differentiation of Representative Gram-Negative Bacilli and Gram-Positive Cocci
Gram-Positive, Non–spore-Forming Bacilli
Figure 42-4 Gram stain of Bacteroides fragilis.
Laboratory Diagnosis
Procedure 42-2 Aerotolerance Test
Purpose
Principle
Method
Expected Results
Limitations
Gram-Negative Rods
Bacteroides Fragilis Group
Nonpigmented Prevotella spp.
Figure 42-5 Bacteroides fragilis on anaerobic blood agar.
Figure 42-6 Bacteroides fragilis on a biplate containing anaerobic blood agar and Bacteroides bile esculin agar (BBE) (arrow).
Pigmented Porphyromonas and Prevotella spp.
Bacteroides ureolyticus
Figure 42-7 Prevotella disiens on laked kanamycin-vancomycin blood agar. Note black pigment (arrow).
Figure 42-8 Porphyromonas spp. on anaerobic blood agar. Red fluorescence is seen under ultraviolet light (365 nm).
Figure 42-9 Bacteroides ureolyticus on anaerobic blood agar. Note pitting of agar (arrow).
Figure 42-10 Fusobacterium nucleatum subsp. nucleatum on anaerobic blood agar. Note bread crumb–like colonies and greening of agar.
Figure 42-11 Gram stain of Fusobacterium nucleatum subsp. nucleatum. Note pointed ends.
Fusobacteriaceae
Protobacteria
Figure 42-12 Peptostreptococcus anaerobius on anaerobic blood agar.
Anaerobic Gram-Positive and Gram-Negative Cocci
Laboratory Diagnosis
Prevention
Chapter Review
Bibliography
Section 14 Mycobacteria and Other Bacteria with Unusual Growth Requirements
Chapter 43 Mycobacteria
Objectives
Major Genera and Species to be Considered
Mycobacterium tuberculosis Complex
Nontuberculous Mycobacteria
Early-Pigmented, Rapid-Growing Mycobacterium spp.
Mycobacterium Tuberculosis Complex
Box 43-1 Major Groupings of Organisms Belonging to the Genus Mycobacterium*
Mycobacterium tuberculosis Complex
Nontuberculous Mycobacteria
Slow-Growing Nonphotochromogens
Photochromogens
Scotochromogens
Noncultivatable
Rapid-Growing, Potentially Pathogenic
Rarely Pathogenic or Not Yet Associated with Infection
General Characteristics
Epidemiology and Pathogenesis
Epidemiology
Pathogenesis
Spectrum of Disease
TABLE 43-1 Epidemiology of Organisms Belonging to M. tuberculosis Complex That Cause Human Infections
Nontuberculous Mycobacteria
Box 43-2 Other Names That Have Been Used to Designate the Nontuberculous Mycobacteria
TABLE 43-2 Runyon Classification of Nontuberculous Mycobacteria (NTM)
TABLE 43-3 Characteristics of Nontuberculous Mycobacteria—Photochromogens
Slow-Growing Nontuberculous Mycobacteria
Photochromogens
Scotochromogens
Nonphotochromogens
TABLE 43-4 Characteristics of Nontuberculous Mycobacteria—Scotochromogens
TABLE 43-5 Characteristics of the Nontuberculous Mycobacteria—Nonphotochromogens and Species Considered Potential Pathogens
Mycobacterium avium Complex (MAC).
General Characteristics.
Epidemiology and Pathogenesis.
Clinical Spectrum of Disease.
Other Nonphotochromogens.
Rapidly Growing Nontuberculous Mycobacteria (RGM)
General Characteristics
Epidemiology and Pathogenesis
TABLE 43-6 Common Types of Infections Caused by Rapidly Growing Mycobacteria
Spectrum of Disease
Noncultivatable Nontuberculous Mycobacteria—mycobacterium Leprae
General Characteristics
Epidemiology and Pathogenesis
Epidemiology.
Pathogenesis.
Spectrum of Disease
Laboratory Diagnosis of Mycobacterial Infections
Specimen Collection and Transport
Pulmonary Specimens
Gastric Lavage Specimens
Urine Specimens
Fecal Specimens
Tissue and Body Fluid Specimens
Blood Specimens
Wounds, Skin Lesions, and Aspirates
Specimen Processing
Contaminated Specimens
Inadequate Specimens and Rejection Criteria
Overview.
Procedure 43-1 N-Acetyl-L-Cysteine–Sodium Hydroxide Method for Liquefaction and Decontamination of Specimens
Principle
Method
Figure 43-1 Flowchart for specimen processing to isolate mycobacteria.
Special Considerations.
Specimens Not Requiring Decontamination
Direct Detection Methods
Microscopy
Acid-Fast Stains
Figure 43-2 Gram staining of M. marinum demonstrates beaded appearance.
Procedure 43-2 Preparation of Smears for Acid-Fast Stain from Direct or Concentrated Specimens
Method
Figure 43-3 M. tuberculosis stained with (A) fluorochrome stain (×400) and (B) Kinyoun acid-fast stain (×1000).
Methods
Fluorochrome Stain.
Procedure 43-3 Auramine-Rhodamine Fluorochrome Stain
Principle
Method
Expected Results
Fuchsin Acid-Fast Stains.
Examination, Interpretation, and Reporting of Smears.
TABLE 43-7 Acid-Fast Smear Reporting
Antigen-Protein Detection
Immunodiagnostic Testing
Genetic Sequencing and Nucleic Acid Amplification
DNA Microarrays.
Chromatographic Analysis
Box 43-3 Suggested Media for Cultivation of Mycobacteria from Clinical Specimens*
Cultivation
Solid Media
Liquid Media
Interpretation
Approach to Identification
Conventional Phenotypic Tests
Growth Characteristics.
Procedure 43-4 Quality Control for Mycobacteriology
Reagents
Method
Interpreting and Recording Results
Example of Interpreting Quality Control Test Results of Decontamination and Concentration Procedure
Figure 43-4 Typical appearance of some mycobacteria on solid agar media. A, M. tuberculosis colonies on Löwenstein-Jensen agar after 8 weeks of incubation. B, A different colonial morphology is seen on culture of one strain of M. avium complex. C, M. kansasii colonies exposed to light. D, Scotochromogen M. gordonae showing yellow colonies. E, Smooth, multilobate colonies of M. fortuitum on Löwenstein-Jensen medium.
Growth Rate.
TABLE 43-8 Liquid Media Systems Commonly Used to Culture and Detect the Growth of Mycobacteria
Procedure 43-5 Determination of Pigment Production and Growth Rate
Principle
Method
Expected Results
Figure 43-5 Initial grouping of mycobacteria based on pigment production before and after exposure to light. In one test system, subcultures of each isolate are grown on two agar slants. One tube is wrapped in aluminum foil to prevent exposure of the organism to light, and the other tube is allowed light exposure. After sufficient growth is present, the wrapped tube is unwrapped, and the tubes are examined together. Photochromogens are nonpigmented when grown in the dark (tube A) and develop pigment after light exposure (tube B). Scotochromogens are pigmented in the dark (tube C); the color does not intensify after exposure to light (tube D). Nonphotochromogens are nonpigmented when grown in the dark (tube E) and remain so even after light exposure (tube F).
Pigment Production.
Biochemical Testing.
Niacin.
Figure 43-6 Niacin test performed with filter paper strips. With a positive test result (A), the liquid turns yellow. With a negative result (B), the liquid remains milky white or clear.
TABLE 43-9 Controls and Media Used for Biochemical Identification of Mycobacteria
TABLE 43-10 Distinctive Properties of Commonly Cultivable Mycobacteria Encountered in Clinical Specimens
TABLE 43-11 Key Biochemical Reactions to Help Differentiate Organisms Belonging to the Same Mycobacterial Group
Procedure 43-6 Niacin Test Performed with Commercially Available Filter Paper Test Strips*
Principle
Method
Expected Results
Nitrate Reduction.
Procedure 43-7 Nitrate Reduction Test Using Chemical Reagents
Principle
Method
Expected Results
Catalase.
Figure 43-7 Semiquantitative catalase test. The tube on the left contains a column of bubbles that has risen past the line (arrow), indicating 45-mm height (a positive test result). The tube on the right is the negative control.
Tween 80 Hydrolysis.
Tellurite Reduction.
Arylsulfatase.
Figure 43-8 A positive arylsulfatase test result is shown on the left; the tube containing the negative control is on the right.
Growth Inhibition by Thiophene-2-Carboxylic Acid Hydrazide (TCH).
Other Tests.
Antimicrobial Susceptibility Testing and Therapy
M. Tuberculosis Complex
Box 43-4 Antitubercular Agents Commonly Tested against M. tuberculosis
Primary Drugs
Secondary Drugs
Direct Versus Indirect Susceptibility Testing
Conventional Methods
New Approaches
TABLE 43-12 Overview of Conventional Methods to Determine Susceptibility of M. tuberculosis Isolates to Antimycobacterial Agents
Therapy
TABLE 43-13 CLSI Recommendations for Susceptibility Testing of Nontuberculous Mycobacteria
Nontuberculous Mycobacteria
Prevention
Case Study 43-1
Questions
Chapter Review
Bibliography
Chapter 44 Obligate Intracellular and Nonculturable Bacterial Agents
Objectives
Genera and Species to Be Considered
Chlamydia
Figure 44-1 The life cycle of chlamydiae. The entire cycle takes approximately 48 to 72 hours.
Chlamydia Trachomatis
TABLE 44-1 Differential Characteristics among Chlamydiae That Cause Human Disease
TABLE 44-2 Primary Syndromes Caused by C. trachomatis
General Characteristics
Epidemiology and Pathogenesis
Spectrum of Disease
Trachoma.
Lymphogranuloma Venereum.
Oculogenital Infections.
Perinatal Infections
Laboratory Diagnosis
Specimen Collection and Transport.
Cultivation.
Direct Detection Methods
Cytologic Examination.
Antigen Detection and Nucleic Acid Hybridization.
Figure 44-2 Appearance of fluorescein-conjugated, monoclonal antibody–stained elementary bodies in direct smear of urethral cell scraping from a patient with chlamydial urethritis.
TABLE 44-3 Use of Different Laboratory Tests to Diagnose C. trachomatis Infections
Nucleic Acid Amplification Tests.
Serodiagnosis.
Antibiotic Susceptibility Testing and Therapy
Prevention
Chlamydia Psittaci
General Characteristics
Epidemiology and Pathogenesis
Spectrum of Disease
Laboratory Diagnosis
Antibiotic Susceptibility Testing and Therapy
Figure 44-3 Electron micrograph of C. pneumoniae (A) and C. trachomatis (B) (bar = 50.5 µm). E, Elementary body; om, outer membrane; R, reticulate body; arrowhead, small electron-dense bodies of undetermined function.
Prevention
Chlamydia Pneumoniae
General Characteristics
Epidemiology and Pathogenesis
Spectrum of Disease
Laboratory Diagnosis
Direct Detection Methods.
Cultivation.
Serodiagnosis.
Antibiotic Susceptibility Testing and Therapy
Prevention
Rickettsia, Orientia, Anaplasma, and Ehrlichia
General Characteristics
TABLE 44-4 Characteristics of Prominent Rickettsia* Orientia, Anaplasma, and Ehrlichia spp.
Epidemiology and Pathogenesis
Spectrum of Disease
Laboratory Diagnosis
Direct Detection Methods
Cultivation
Serodiagnosis
Procedure 44-1 Weil-Felix Reaction
Purpose
Principle
Method
Expected Results
Limitations
Quality Control
Antibiotic Susceptibility Testing and Therapy
Prevention
Coxiella
General Characteristics
Epidemiology and Pathogenesis
Spectrum of Disease
Laboratory Diagnosis
Box 44-1 Clinical Manifestations of C. burnetii Infection
Antibiotic Susceptibility Testing and Therapy
Prevention
Tropheryma whipplei
General Characteristics
Epidemiology, Pathogenesis, and Spectrum of Disease
Laboratory Diagnosis
Antibiotic Susceptibility Testing and Therapy
Prevention
Klebsiella granulomatis
General Characteristics
Epidemiology and Pathogenesis
Spectrum of Disease
Laboratory Diagnosis
Antibiotic Susceptibility Testing and Therapy
Chapter Review
Case Study 44-1
Questions
Bibliography
Chapter 45 Cell Wall–Deficient Bacteria: Mycoplasma and Ureaplasma
Objectives
Genera and Species to Be Considered
General Characteristics
Epidemiology and Pathogenesis
Epidemiology
Figure 45-1 Taxonomy of the class Mollicutes.
TABLE 45-1 Mycoplasmas That Are Considered Normal Flora of the Oropharynx or Genital Tract
Pathogenesis
TABLE 45-2 Clinical Manifestations of Mycoplasma Infections Caused by Mycoplasma pneumoniae, Ureaplasma urealyticum, Ureaplasma parvum, M. hominis, and M. genitalium
Spectrum of Disease
Laboratory Diagnosis
Specimen Collection, Transport, and Processing
TABLE 45-3 Transport and Storage Conditions for Mycoplasma pneumoniae, Ureaplasma urealyticum, and M. hominis
TABLE 45-4 Cultivation of Mycoplasma pneumoniae, Ureaplasma spp., and M. hominis
Direct Detection Methods
Molecular Diagnostics
Cultivation
Procedure 45-1 Isolation of Mycoplasma Pneumoniae
Principle
Method
Procedure 45-2 Isolation of Ureaplasma urealyticum
Principle
Method
Procedure 45-3 Isolation of Mycoplasma hominis
Principle
Method A
Method B (Alternative Method)
Figure 45-2 Colonies of Mycoplasma pneumoniae visualized under 100× magnification. Note the variation in the size of the colonies (arrows).
TABLE 45-5 Basic Biochemical Differentiation of the Major Mycoplasma spp. and Ureaplasma urealyticum
Approach to Identification
Figure 45-3 Isolation of Mycoplasma hominis and Ureaplasma urealyticum (100× magnification). Note the “fried egg” appearance of the large M. hominis colony (arrow A) and the relatively small size of the U. urealyticum colony (arrow B).
Figure 45-4 Colonial growth characteristics of Mycoplasma in agar medium.
Serodiagnosis
Susceptibility Testing and Therapy
Prevention
Case Study 45-1
Questions
Chapter Review
Bibliography
Chapter 46 The Spirochetes
Objectives
Genera and Species to Be Considered
Treponema
General Characteristics
Epidemiology and Pathogenesis
Figure 46-1 Species designation of spirochetes based on morphology.
TABLE 46-1 Spirochetes Pathogenic for Humans
Spectrum of Disease
Laboratory Diagnosis
Specimen Collection
Direct Detection
TABLE 46-2 Epidemiology and Spectrum of Disease of the Treponemes Pathogenic for Humans
Molecular Diagnostics
Serodiagnosis
Figure 46-2 Appearance of Treponema pallidum in dark-field preparation.
Procedure 46-1 Rapid Plasma Reagin (RPR) Test
Purpose
Principle
Specimen
Method
Expected Results
Limitations
Quality Control
Procedure 46-2 Venereal Disease Research Laboratory (VDRL) Test
Purpose
Principle
Specimen
Method
Expected Results
Limitations
Quality Control
TABLE 46-3 Sensitivity of Commonly Used Serologic Tests for Syphilis
Antimicrobial Susceptibility Testing and Therapy
Prevention
Borrelia
General Characteristics
Figure 46-3 Traditional testing versus reverse testing.
Epidemiology and Pathogenesis
Relapsing Fever
Lyme Disease
Spectrum of Disease
Relapsing Fever
Figure 46-4 Appearance of the classic erythema migrans lesion of acute Lyme disease.
Lyme Disease
Laboratory Diagnosis
Specimen Collection, Transport, and Processing
Direct Detection Methods
Relapsing Fever.
Lyme Disease.
Cultivation
Serodiagnosis
Relapsing Fever.
Lyme Disease.
Figure 46-5 Two-step serodiagnostic procedure. *Note: If neuroborreliosis is suspected, a paired sera and CSF specimen is recommended for testing. Any disease of short duration. For Lyme disease, it is recommended that a follow-up serologic test be performed at a later date.
Molecular Diagnostics
Antibiotic Susceptibility Testing and Therapy
Prevention
Brachyspira
General Characteristics
Epidemiology and Pathogenesis
Laboratory Diagnosis
Specimen Collection and Direct Detection
Cultivation
Approach to Identification
Antibiotic Susceptibility and Therapy
Leptospira
General Characteristics
Epidemiology and Pathogenesis
Spectrum of Disease
Box 46-1 Potential Virulence Factors of Leptospira
Laboratory Diagnosis
Specimen Collection, Transport, and Processing
Direct Detection
Molecular Diagnostics
Cultivation
Approach to Identification
Serodiagnosis
Molecular Testing
Antibiotic Susceptibility and Therapy
Prevention
Case Study 46-1
Questions
Bibliography
Chapter Review
Part IV Parasitology
Section 1 Principles of Identification
Chapter 47 Laboratory Methods for Diagnosis of Parasitic Infections: Overview
Objectives
Epidemiology
Pathogenesis and Spectrum of Disease
TABLE 47-1 Description of the More Common Groups of Human Parasites
TABLE 47-2 Body Sites and Parasite Recovery (Trophozoites, Cysts, Oocysts, Spores, Adults, Larvae, Eggs, Amastigotes, Trypomastigotes)
Laboratory Diagnosis
TABLE 47-3 Specimens and/or Body Site: Specimen Options, Collection and Transport Methods, and Processing
TABLE 47-4 Epidemiology of the More Common Groups of Human Parasites
Specimen Collection and Transport
Specimen Processing
Procedure 47-1 Formalin-Ether (Formalin–Ethyl Acetate) Sedimentation Techniques
Principle
Method
Procedure 47-2 Wheatley’s Trichrome Stain for Fecal Specimens (Modification of Gomori’s Trichrome Stain for Tissue)
Principle
Reagents
A. Formula
B. Stain Preparation
Method
Expected Results
Procedure 47-3 Modified Iron-Hematoxylin Stain (with Carbolfuchsin Step)
Principle
Reagents
A. Mayer’s Albumin
B. Stock Solution of Hematoxylin Stain
C. Mordant
D. Working Solution of Hematoxylin Stain
E. Picric Acid
F. Acid-Alcohol Decolorizer
G. 70% Alcohol and Ammonia
H. Carbolfuchsin
Method
Procedure Notes
Procedure 47-4 Modified Acid-Fast Stain for Coccidia
Reagents
A. Carbolfuchsin
B. Decolorizer
C. Counterstain
“Cold” Modified Acid-Fast Stain Method (Kinyoun)
“Hot” Modified Acid-Fast Stain Method
Procedure 47-5 Modified Trichrome Stain for Microsporidia (Weber-Green)
Principle
Reagents
A. Modified Trichrome Stain
B. Acid-Alcohol
Method
Expected Results
Procedure 47-6 Modified Trichrome Stain for Microsporidia (Ryan-Blue)
Principle
Reagents
Method
Expected Results
Procedure Notes for Modified Trichrome Staining Methods (Weber or Ryan)
Procedure Limitations for Modified Trichrome Staining Methods (Weber or Ryan)
Procedure 47-7 Modified Trichrome Stain for Microsporidia (Kokoskin—Hot Method)
Principle
Method
Procedure 47-8 Staining Thin Films: Giemsa Stain
Principle
Method
Expected Results
Procedure 47-9 Staining Thick Films: Giemsa Stain
Principle
Method
TABLE 47-5 Parasitic Infections: Clinical Findings in Normal and Compromised Hosts
TABLE 47-6 Pathogenesis and Spectrum of Parasitic Diseases
Table 47-7 Recommendations for Stool Testing
TABLE 47-8 Stool Specimen Collection and Testing Options
Approach to Identification
Microscopic Examination
TABLE 47-9 Fecal Fixatives Used in Diagnostic Parasitology (Intestinal Tract Specimens)
Intestinal Tract
TABLE 47-10 Common Human Parasites: Diagnostic Specimens, Tests, and Positive Findings
Box 47-1 Direct Smear: Review
Principle
Specimen
Reagents
Examination
Results
Notes and Limitations
O&P Examination.
Box 47-2 Concentration: Review
Principle
Specimen
Reagents
Examination
Results
Notes and Limitations
Box 47-3 Permanent Stained Smear: Review
Principle
Specimen
Reagents
Examination
Results
Notes and Limitations
Important Reminder
Recovery of the Tapeworm Scolex.
Examination for Pinworm.
Sigmoidoscopy Material.
Figure 47-1 Stool material stained with Wheatley’s trichrome stain. A, Charcot-Leyden crystals. B, Polymorphonuclear leukocytes. C, Blastocystis hominis central body forms (larger objects) and yeast cells (smaller, more homogeneous objects).
Duodenal Drainage.
Duodenal Capsule Technique (Entero-Test).
Urogenital Tract Specimens
Box 47-4 Modified Acid-Fast Permanent Stained Smear: Review
Principle
Specimen
Reagents
Examination
Results
Notes and Limitations
Box 47-5 Modified Trichrome Permanent Stained Smear: Review
Principle
Specimen
Reagents
Examination
Results
Notes and Limitations
Commercial Suppliers
Figure 47-2 Processing of fecal specimens for ova and parasites (modified according to diagnostic guidelines established by the Centers for Disease Control and Prevention). Asterisk (*) indicates a special test procedure.
Sputum
Aspirates
Figure 47-3 Method of collecting a cellophane (Scotch) tape preparation for pinworm diagnosis. This method dispenses with the tongue depressor, requiring only tape and a glass microscope slide. The tape must be pressed deep into the anal crack.
Figure 47-4 Trichomonas vaginalis trophozoite.
Biopsy Specimens
Figure 47-5 A, Rapid identification kit for Trichomonas vaginalis. B, Culture system for T. vaginalis.
Figure 47-6 Echinococcus granulosus, hydatid sand (×300). Inset. Two individual hooklets (×1000).
Figure 47-7 Naegleria fowleri in brain tissue. (Hematoxylin and eosin stain.)
Figure 47-8 Trichinella spp. larvae encysted in muscle.
Box 47-6 Thin Blood Films: Review
Principle
Specimen
Reagents
Examination
Results
Notes and Limitations
Blood
Thin Blood Films.
TABLE 47-11 Examination of Impression Smears
Thick Blood Films.
Blood Film Stains.
Box 47-7 Thick Blood Films: Review
Principle
Specimen
Reagents
Examination
Results
Notes and Limitations
Buffy Coat Films.
Direct Detection Methods
Intestinal Parasites
Blood Parasites
Cultivation
Larval-Stage Nematodes
Protozoa
Serodiagnosis
TABLE 47-12 Antigen Detection Kits for Stool or Vaginal Discharge Specimens (may not be all-inclusive)*
Prevention
Chapter Review
Bibliography
Chapter 48 Intestinal Protozoa
Objectives
Parasites to Be Considered
Protozoa
Amebae
Entamoeba histolytica
General Characteristics
TABLE 48-1 Intestinal Protozoa: Trophozoites of Common Amebae
TABLE 48-2 Intestinal Protozoa—Cysts of Common Amebae
TABLE 48-3 Intestinal Protozoa—Trophozoites of Flagellates
TABLE 48-4 Intestinal Protozoa—Cysts of Flagellates
TABLE 48-5 Intestinal Protozoa—Ciliates
TABLE 48-6 Morphologic Criteria Used to Identify Intestinal Protozoa (Coccidia, Blastocystis hominis)
Epidemiology
Pathogenesis and Spectrum of Disease
TABLE 48-7 Microsporidia That Cause Human Infection
Figure 48-1 Various structures that may be seen in stool preparations. 1, 2, and 4, Blastocystis hominis. 3 and 5 to 8, Various yeast cells. 9, Macrophage with nucleus. 10 and 11, Deteriorated macrophage without nucleus. 12 and 13, Polymorphonuclear leukocytes. 14 and 15, Pollen grains. 16, Charcot-Leyden crystals.
Figure 48-2 Entamoeba histolytica trophozoite containing ingested red blood cells.
Figure 48-3 1, Trophozoite of Entamoeba histolytica (note ingested red blood cells). 2, Trophozoite of Entamoeba histolytica/Entamoeba dispar (morphology does not allow differentiation between the two species). 3 and 4, Early cysts of E. histolytica/E. dispar. 5 to 7, Cysts of E. histolytica/E. dispar. 8 and 9, Trophozoites of Entamoeba coli. 10 and 11, Early cysts of E. coli. 12 to 14, Cysts of E. coli. 15 and 16, Trophozoites of Entamoeba hartmanni. 17 and 18, Cysts of E. hartmanni.
Asymptomatic Infection.
Intestinal Disease.
Hepatic Disease.
Figure 48-4 Entamoeba histolytica/Entamoeba dispar cyst.
Figure 48-5 Entamoeba dispar trophozoite; no ingested red blood cells are present.
Laboratory Diagnosis
Routine Methods.
Antigen Detection.
Antibody Detection.
Histology.
Nucleic Acid-Based Techniques
Therapy
Asymptomatic Infection.
Mild to Moderate Disease.
Severe Intestinal Disease.
Hepatic Disease.
Prevention
Entamoeba coli
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Therapy
Figure 48-6 A to C, Trophozoites of Entamoeba histolytica (note ingested red blood cells). D, Trophozoite of E. histolytica/E. dispar. E, Early cyst of E. histolytica/E. dispar. F to H, Trophozoites of Entamoeba coli. I and J, Cysts of E. coli.
Prevention
Entamoeba hartmanni
General Characteristics
Figure 48-7 Entamoeba coli trophozoite.
Figure 48-8 Entamoeba coli cyst (trichrome stain) (poor preservation; typical appearance of some E. coli cysts).
Epidemiology
Figure 48-9 A, Entamoeba hartmanni trophozoite. B, E. hartmanni cyst.
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Endolimax nana
General Characteristics
Figure 48-10 A to C, Trophozoites of Entamoeba hartmanni. D and E, Cysts of E. hartmanni.
Epidemiology
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Iodamoeba bütschlii
General Characteristics
Figure 48-11 1 to 5, Trophozoites of Endolimax nana. 6 to 10, Cysts of E. nana. 11 to 13, Trophozoites of Iodamoeba bütschlii. 14 to 16, Cysts of I. bütschlii.
Epidemiology
Pathogeneis and Spectrum of Disease
Laboratory Diagnosis
Therapy
Figure 48-12 A to C, Trophozoites of Endolimax nana. D and E, Cysts of E. nana.
Figure 48-13 A, Endolimax nana trophozoite. B, E. nana cyst, iodine stain. C, E. nana cyst. D, E. nana cyst.
Prevention (E. hartmanni, E. nana, I. bütschlii)
Blastocystis hominis
General Characteristics
Figure 48-14 A, Trophozoites of Iodamoeba bütschlii. B and C, Cysts of I. bütschlii.
Figure 48-15 A, Iodamoeba bütschlii trophozoites. B, I. bütschlii cyst. C, I. bütschlii cyst. D, I. bütschlii cyst.
Epidemiology
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Routine Methods.
Antigen Detection.
Antibody Detection.
Therapy
Prevention
Flagellates
Giardia lamblia
General Characteristics
Epidemiology
Figure 48-16 1, Trophozoite of Pentatrichomonas hominis. 2, Trophozoite of Chilomastix mesnili. 3, Cyst of C. mesnili. 4, Trophozoite of Giardia lamblia (front view). 5, Trophozoite of G. lamblia (side view). 6, Cyst of G. lamblia. 7, Trophozoite of Enteromonas hominis. 8 to 10, Cysts of E. hominis. 11, Trophozoite of Retortamonas intestinalis. 12 to 13, Cysts of R. intestinalis.
Pathogenesis and Spectrum of Disease
Asymptomatic Infection.
Figure 48-17 A to C, Trophozoites of Giardia lamblia. D to F, Cysts of G. lamblia.
Figure 48-18 A, Giardia lamblia trophozoite. B, G. lamblia trophozoite, iodine stain. C, G. lamblia cysts.
Intestinal Disease.
Figure 48-19 A to C, Trophozoites of Chilomastix mesnili (A, silver stain). D and E, Cysts of C. mesnili.
Figure 48-20 A, Chilomastix mesnili trophozoite. B, Chilomastix mesnili cyst (both top figures are iodine stain). C, Chilomastix mesnili trophozoite (silver stain). D, C. mesnili cyst.
Figure 48-21 Trophozoites of Dientamoeba fragilis.
Figure 48-22 A and B, Trophozoites of Dientamoeba fragilis.
Figure 48-23 A, Dientamoeba fragilis, two nuclei. B, D. fragilis, one nucleus.
Chronic Disease.
Antigenic Variation.
Laboratory Diagnosis
Routine Methods.
Antigen Detection.
Antibody Detection.
Histology.
Nucleic Acid-Based Techniques
Prevention
Chilomastix mesnili
General Characteristics
Epidemiology
Pathogeneis and Spectrum of Disease
Laboratory Diagnosis
Therapy
Prevention
Dientamoeba fragilis
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Routine Methods.
Antigen Detection.
Antibody Detection.
Therapy
Prevention
Pentatrichomonas hominis
Epidemiology
Pathogeneis and Spectrum of Disease
Laboratory Diagnosis
Therapy
Prevention
Ciliates
Balantidium coli
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
Figure 48-24 A, Trophozoite of Balantidium coli. B, Cyst of B. coli.
Figure 48-25 A, Balantidium coli trophozoite. B, B. coli trophozoite.
Laboratory Diagnosis
Therapy
Prevention
Sporozoa (Apicomplexa)
Cryptosporidium spp.
General Characteristics
Figure 48-26 Life cycle of Cryptosporidium. (a) Sporulated oocyst in feces. (b) Excystation in intestine. (c) Free sporozoite in intestine. (d) Type I meront (six or eight merozoites). (e) Recycling of type I merozoite. (f) Type II meront (four merozoites). (g) Microgametocyte with approximately 16 microgametes. (h) Microgamete fertilizes macrogamete (i) to form zygote (j). Approximately 80% of the zygotes form thick-walled oocysts (k), which sporulate within the host cell. About 20% of the zygotes do not form an oocyst wall; their sporozoites are surrounded only by a unit membrane (l). Sporozoites in “autoinfective,” thin-walled oocysts (l) are released into the intestinal lumen (m) and reinitiate the endogenous cycle (at c).
Figure 48-27 Cryptosporidium. A, Oocysts recovered from a Sheather’s sugar flotation; organisms measure 4 to 6 µm. B, Scanning electron microscopy view of organisms at brush border of epithelial cells.
Figure 48-28 Cryptosporidium oocysts and Giardia cysts stained with monoclonal antibody–conjugated fluorescent reagent.
Epidemiology
Pathogenesis and Spectrum of Disease
Immunocompetent Individuals.
Immunocompromised Individuals.
Laboratory Diagnosis
Routine Methods.
Antigen Detection.
Nucleic Acid-Based Methods.
Antibody Detection.
Histology.
Therapy
Prevention
Cyclospora cayetanensis
General Characteristics
Figure 48-29 A, Cyclospora cayetanensis oocysts after modified acid-fast staining. Note the variability in the intensity of the stain. These oocysts measure 8 to 10 µm, twice the size of Cryptosporidium spp. (oil immersion, ×1000). B and C, Cyclospora cayetanensis oocysts exhibiting autofluorescence (high dry power, ×400).
Epidemiology
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Special Stains.
Flow Cytometry.
Other Diagnostic Methods.
Therapy
Prevention
Figure 48-30 A, Immature concept of Isospora belli. B, Mature oocyst of I. belli.
Isospora (cystoisospora) belli
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Histology.
Therapy
Prevention
Sarcocystis spp.
General Characteristics
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Therapy
Prevention
Microsporidia
General Characteristics
Figure 48-31 Life cycle diagram of the microsporidia. A to G, Asexual development of sporoblasts. H, Release of spores.
Figure 48-32 Diagram illustrating the polar tubule in a microsporidian spore.
Epidemiology
Pathogenesis and Spectrum of Disease
Enterocytozoon bieneusi
Figure 48-33 Microsporidial spores stained with Ryan-blue modified trichrome stain. A, Nasopharyngeal aspirate. B, Stool (enlarged image). C, Urine.
Figure 48-34 Routine histology micrograph of microsporidian spores in enterocytes (Giemsa stain). A, Note the small size. B, The spores are more easily seen; note the position between the nucleus and the brush border of the cell. C, In these spores, the granule is easily seen (stains positive with periodic acid-Schiff [PAS] stain).
Figure 48-35 Routine histology micrograph of microsporidian spores in enterocytes (Giemsa stain). A, Note the fully formed spores. B, These spores are not fully mature.
Encephalitozoon spp.
Encephalitozoon (Septata) intestinalis
Other Microsporidia
Laboratory Diagnosis
Antigen Detection
Antibody Detection
Molecular Methods
Histology
Therapy
Prevention
Chapter Review
Case Study 48-1
Figure 48-36 A, Cyst identified in stool specimen, B, Cyst identified in stool specimen
Figure 48-37 Rectal biopsy.
Questions
Bibliography
Chapter 49 Blood and Tissue Protozoa
Objectives
Parasites to be Considered
Protozoa
Plasmodium spp.
Figure 49-1 Life cycle of Plasmodium.
Plasmodium Vivax (Benign Tertian Malaria)
General Characteristics
Pathogenesis and Spectrum of Disease
Plasmodium Ovale
General Characteristics
TABLE 49-1 Plasmodium spp.: Clinical Characteristics of the Five Human Infections
Pathogenesis and Spectrum of Disease
Plasmodium Malariae (Quartan Malaria)
General Characteristics
TABLE 49-2 Plasmodia in Giemsa-Stained Thin Blood Smears
TABLE 49-3 Malaria Characteristics with Fresh Blood or Blood Collected Using EDTA with No Extended Lag Time*
Pathogenesis and Spectrum of Disease
Plasmodium Falciparum (Malignant Tertian Malaria)
General Characteristics
Pathogenesis and Spectrum of Disease
Figure 49-2 The morphology of malaria parasites. Plasmodium vivax: 1, Early trophozoite (ring form). 2, Late trophozoite with Schüffner’s dots (note enlarged red blood cell). 3, Late trophozoite with ameboid cytoplasm (very typical of P. vivax). 4, Late trophozoite with ameboid cytoplasm. 5, Mature schizont with merozoites (18) and clumped pigment. 6, Microgametocyte with dispersed chromatin. 7, Macrogametocyte with compact chromatin. Plasmodium malariae: 1, Early trophozoite (ring form). 2, Early trophozoite with thick cytoplasm. 3, Early trophozoite (band form). 4, Late trophozoite (band form) with heavy pigment. 5, Mature schizont with merozoites (9) arranged in rosette. 6, Microgametocyte with dispersed chromatin. 7, Macrogametocyte with compact chromatin. Plasmodium ovale: 1, Early trophozoite (ring form) with Schüffner’s dots. 2, Early trophozoite (note enlarged red blood cell). 3, Late trophozoite in red blood cell with fimbriated edges. 4, Developing schizont with irregularly shaped red blood cell. 5, Mature schizont with merozoites (8) arranged irregularly. 6, Microgametocyte with dispersed chromatin. 7, Macrogametocyte with compact chromatin. Plasmodium falciparum: 1, Early trophozoite (accolé or appliqué form). 2, Early trophozoite (one ring is in headphone configuration/double chromatin dots). 3, Early trophozoite with Maurer’s dots. 4, Late trophozoite with larger ring and Maurer’s dots. 5, Mature schizont with merozoites (24). 6, Microgametocyte with dispersed chromatin. 7, Macrogametocyte with compact chromatin. Note: Without the appliqué form, Schüffner’s dots, multiple rings/cell, and other developing stages, differentiation among the species can be difficult. It is obvious that the early rings of all four species can mimic one another very easily. Remember: One set of negative blood films cannot rule out a malarial infection.
Plasmodium Knowlesi (Simian Malaria, the Fifth Human Malaria)
General Characteristics
Pathogenesis and Spectrum of Disease
Figure 49-3 Morphology of malaria parasites. Column 1 (left to right): Plasmodium vivax (note enlarged infected RBCs). (1) Early trophozoite (ring form) (note one RBC contains 2 rings—not that uncommon); (2) older ring, note ameboid nature of rings; (3) late trophozoite with Schüffner’s dots (note enlarged RBC); (4) developing schizont; (5) mature schizont with 18 merozoites and clumped pigment; (6) microgametocyte with dispersed chromatin. Column 2: Plasmodium ovale (note enlarged infected RBCs). (1) Early trophozoite (ring form) with Schüffner’s dots (RBC has fimbriated edges); (2) early trophozoite (note enlarged RBC, Schüffner’s dots, and RBC oval in shape); (3) late trophozoite in RBC with fimbriated edges; (4) developing schizont with irregular-shaped RBC; (5) mature schizont with 8 merozoites arranged irregularly; (6) microgametocyte with dispersed chromatin. Column 3: Plasmodium malariae (note normal or smaller than normal infected RBCs). (1) Early trophozoite (ring form); (2) early trophozoite with thick cytoplasm; (3) late trophozoite (band form); (4) developing schizont; (5) mature schizont with 9 merozoites arranged in a rosette; (6) microgametocyte with compact chromatin. Column 4: Plasmodium falciparum. (1) Early trophozoites (the rings are in the headphone configuration with double chromatin dots); (2) early trophozoite (accolé or appliqué form); (3) early trophozoites (note the multiple rings/cell); (4) late trophozoite with larger ring (accolé or appliqué form); (5) crescent-shaped gametocyte; (6) crescent-shaped gametocyte. Column 5: Plasmodium knowlesi—with the exception of image 5, these were photographed at a higher magnification (note normal or smaller than normal infected RBCs). (1) Early trophozoite (ring form); (2) early trophozoite with slim band form; (3) late trophozoite (band form); (4) developing schizont; (5) mature schizont with merozoites arranged in a rosette; (6) microgametocyte with dispersed chromatin. Note: Without the appliqué form, Schüffner’s dots, multiple rings per cell, and other developing stages, differentiation among the species can be very difficult. It is obvious that the early rings of all five species can mimic one another very easily. Remember: One set of negative blood films cannot rule out a malaria infection.
Laboratory Diagnosis (All Species)
Routine Methods
Figure 49-4 Plasmodium falciparum. A, Ring forms; B, oocyte; and C, sporozoites.
Serologic Methods
Figure 49-5 Plasmodium vivax in thick smear. 1, Ameboid trophozoites. 2, Schizont, two divisions of chromatin. 3, Mature schizont. 4, Microgametocyte. 5, Blood platelets. 6, Nucleus of neutrophil. 7, Eosinophil. 8, Blood platelet associated with cellular remains of young erythrocytes.
Molecular Diagnostics
Automated Instruments
Therapy
Figure 49-6 A, Plasmodium malariae schizont. B, Plasmodium vivax schizont.
Babesia Spp.
General Characteristics
Organism
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Routine Methods
Molecular Diagnostics
Therapy
Prevention
Figure 49-7 Babesia in red blood cells.
Trypanosoma Spp.
Figure 49-8 Characteristic stages of species of Leishmania and Trypanosoma in human and insect hosts.
Figure 49-9 Trypanosoma cruzi trypomastigote.
TABLE 49-4 Characteristics of American Trypanosomiasis
TABLE 49-5 Characteristics of East and West African Trypanosomiasis
African Trypanosomiasis
Figure 49-10 A, Trypanosoma cruzi in blood film (1600×). B, Trypanosoma cruzi parasites in cardiac muscle (2500×).
General Characteristics
Pathogenesis and Spectrum of Disease
Trypanosoma brucei gambiense.
Trypanosoma brucei rhodesiense.
Laboratory Diagnosis (All Species)
Routine Methods.
Antigen Detection.
Antibody Detection.
Molecular Diagnostics.
Therapy
American Trypanosomiasis
Trypanosoma cruzi
General Characteristics.
Pathogenesis and Spectrum of Disease.
Laboratory Diagnosis
Routine Methods.
Molecular Diagnostics.
Xenodiagnosis.
Antigen Detection.
Antibody Detection.
Histology.
Therapy.
Leishmania Spp.
TABLE 49-6 Features of Human Leishmanial Infectionsa
General Characteristics
Pathogenesis and Spectrum of Disease
Figure 49-11 A, Leishmania donovani parasites in Küpffer cells of liver (2000×). B, Leishmania sp.
Laboratory Diagnosis
Therapy
Chapter Review
Bibliography
Chapter 50 Other Protozoa
Objectives
Parasites to be Considered
Amebae, Flagellates (Other Body Sites)
Coccidia (Other Body Sites)
Free-Living Amebae
Naegleria Fowleri
General Characteristics
Pathogenesis and Spectrum of Disease
Figure 50-1 Naegleria fowleri, Acanthamoeba spp. Diagram of trophozoites and cysts (upper row). Flagellate and cyst forms of Naegleria fowleri; (lower row) trophozoite and cyst of Acanthamoeba spp.
Laboratory Diagnosis
Routine Methods
Other Methods
Therapy
Acanthamoeba Spp.
General Characteristics
TABLE 50-1 Free-Living Amebae Causing Disease in Humans
Figure 50-2 Naegleria fowleri in brain tissue. Hematoxylin and eosin stain. Note the large karyosome.
Pathogenesis and Spectrum of Disease
GAE
Keratitis
Laboratory Diagnosis
Routine Methods
Other Methods
Therapy
Disseminated Infections
Acanthamoeba Keratitis
Balamuthia Mandrillaris
General Characteristics
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Therapy
Trichomonas Vaginalis
General Characteristics
TABLE 50-2 Characteristics of Trichomonas vaginalis
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Wet Mounts
Stained Smears
Culture
Antigen Detection
Molecular Diagnostics
Therapy
Pentatrichomonas Hominis
General Characteristics
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Therapy
Toxoplasma Gondii
Figure 50-3 Life cycle of Toxoplasma gondii.
General Characteristics
Pathogenesis and Spectrum of Disease
TABLE 50-3 Morphology of Toxoplasma gondii Stages Found in Humans
TABLE 50-4 People at Risk for Severe Toxoplasmosis
Immunocompetent Individuals
Immunocompromised Individuals
Congenital Infections
Ocular Infections
Laboratory Diagnosis
Therapy
Case Study 50-1
Questions
Figure 50-4 Patient’s specimen.
Chapter Review
Bibliography
Chapter 51 Intestinal Nematodes (Roundworms)
Objectives
Parasites to be Considered
Helminths
Nematodes
Ascaris Lumbricoides
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
Figure 51-1 Life cycle of Ascaris lumbricoides (indirect life cycle).
Figure 51-2 Bile-stained mammillated A. lumbricoides ovum.
Laboratory Diagnosis
Figure 51-3 A. lumbricoides adult male worm. Note the curved posterior end.
Figure 51-4 Life cycles of Enterobius vermicularis and Trichuris trichiura (direct life cycle).
Therapy
Prevention
Enterobius Vermicularis
General Characteristics
TABLE 51-1 Pathogenesis and Spectrum of Associated Diseases
Epidemiology
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Figure 51-5 Enterobius vermicularis eggs (cellophane [Scotch] tape preparation).
Figure 51-6 Enterobius vermicularis gravid female.
Figure 51-7 Strongyloides stercoralis life cycle.
Therapy
Prevention
Strongyloides Stercoralis
General Characteristics
Figure 51-8 Strongyloides stercoralis rhabditiform larva, iodine stain.
Epidemiology
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Figure 51-9 Rhabditiform larvae. A, Strongyloides. B, Hookworm. C, Trichostrongylus. bc, Buccal cavity; cb, beadlike swelling of caudal tip; es, esophagus; gp, genital primordia.
Therapy
Prevention
Trichostrongylus spp.
General Characteristics
Epidemiology
Figure 51-10 Trichostrongylus sp. egg.
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Therapy
Prevention
Trichuris Trichiura
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Therapy
Figure 51-11 Trichuris trichiura egg. Note the clearly evident polar hyaline plugs.
Prevention
Capillaria Philippinensis
General Characteristics
Epidemiology
Figure 51-12 Adult female Trichuris trichiura.
Figure 51-13 A, Ancylostoma duodenale head. B, Tail; note the appearance of the pointed tail.
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Therapy
Prevention
Hookworms
Figure 51-14 A, Necator americanus head; note the clearly evident rounded cutting plates protruding from the head. B, Copulatory bursa.
Epidemiology
Ancylostoma Duodenale
General Characteristics
Pathogenesis and Spectrum of Disease
Necator Americanus
General Characteristics
Figure 51-15 Hookworm egg, iodine stain.
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Figure 51-16 Hookworm rhabditiform larvae.
Therapy
Prevention
Case Study 51-1
Questions
Figure 51-17 Nematode eggs imbedded in liver tissue.
Bibliography
Chapter Review
Chapter 52 Tissue Nematodes (Roundworms)
Objectives
Parasites to be Considered
Helminths
Nematodes (Roundworms)
Trichinella Spiralis
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
Figure 52-1 Life cycle of Trichinella spiralis.
Figure 52-2 Trichinosis. Encysted larvae within tissue.
Laboratory Diagnosis
Therapy
Prevention
Toxocara Canis (Visceral Larva Migrans) and Toxocara Cati (Ocular Larva Migrans)
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
Figure 52-3 Toxocara canis egg. Note the rough appearance on the outer surface of the egg. The egg also contains an infectious L2 larvae.
Laboratory Diagnosis
Therapy
Prevention
Ancylostoma Braziliense or Ancylostoma Caninum (Cutaneous Larva Migrans)
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
TABLE 52-1 Pathogenesis and Spectrum of Associated Diseases
Laboratory Diagnosis
Therapy
Dracunculus Medinensis
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Therapy
Parastrongylus Cantonensis (Cerebral Angiostrongyliasis)
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Therapy
Parastrongylus Costaricensis (Abdominal Angiostrongyliasis)
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Therapy
Gnathostoma Spinigerum
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Therapy
Bibliography
Chapter Review
Chapter 53 Blood and Tissue (Filarial) Nematodes
Objectives
Parasites to be Considered
Nematodes
Wuchereria Bancrofti
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
Figure 53-1 Life cycle of human filarial worms.
Figure 53-2 Identification of microfilariae.
Endosymbiont
Figure 53-3 Anterior and posterior ends of microfilariae found in humans. A, Wuchereria bancrofti. B, Brugia malayi. C, Loa loa. D, Onchocerca volvulus. E, Mansonella perstans. F, Mansonella streptocerca. G, Mansonella ozzardi.
Figure 53-4 Microfilaria of Wuchereria bancrofti in thick blood film.
Laboratory Diagnosis
Direct Detection
Serologic Detection
Molecular Diagnostics
Brugia Malayi and Brugia Timori
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Therapy
Prevention
Loa Loa
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Direct Detection
Serologic Detection
Molecular Diagnostics
Therapy
Prevention
Onchocerca Volvulus
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Direct Detection
Figure 53-5 Microfilaria of Onchocerca volvulus.
Serologic Detection
Molecular Diagnostics
Therapy
Prevention
Mansonella Spp. (M. ozzardi, M. streptocerca, M. perstans)
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Therapy
Prevention
Case Study 53-1
Questions
Figure 53-6 Organism identified from patient.
Bibliography
Chapter Review
Chapter 54 Intestinal Cestodes
Objectives
Parasites to be Considered
Intestinal Cestodes (Tapeworms)
Diphyllobothrium Latum
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
Figure 54-1 A, Diphyllobothrium latum scolex. B, D. latum scolex, bothria visible. C, D. latum ovum.
Figure 54-2 Life cycle of Diphyllobothrium latum.
Figure 54-3 Proglottid demonstrating rosette-shaped uterus in D. latum.
Laboratory Diagnosis
Antiparasitic Susceptibility Testing and Therapy
Prevention
TABLE 54-1 Epidemiology of the Intestinal Cestodes That Cause Disease in Humans
TABLE 54-2 Common Human Parasites, Diagnostic Specimens, Tests, and Positive Findings
Dipylidium Caninum
General Characteristics
Figure 54-4 Dipylidium caninum tapeworm.
TABLE 54-3 Cestode Parasites of Humans (Intestinal)
Epidemiology
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Antiparasitic Susceptibility Testing and Therapy
Prevention
Hymenolepis Nana
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Figure 54-5 A, Taenia spp. egg. B, Diphyllobothrium latum egg. C, Hymenolepis diminuta egg. D, Hymenolepis nana egg. E, Dipylidium caninum egg packet.
Figure 54-6 Dipylidium caninum egg packet.
Figure 54-7 D. caninum scolex demonstrating the armed rostellum.
Figure 54-8 Life cycle of Hymenolepis nana.
Antiparasitic Susceptibility Testing and Therapy
Prevention
Hymenolepis Diminuta
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Antiparasitic Susceptibility Testing and Therapy
Prevention
Taenia Solium
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
Figure 54-9 Life cycle of Taenia saginata and Taenia solium.
Laboratory Diagnosis
Figure 54-10 Gravid proglottids. A, Taenia saginata. B, Taenia solium. C, Diphyllobothrium latum. D, Dipylidium caninum.
Figure 54-11 T. saginata scolex with suckers.
Antiparasitic Susceptibility Testing and Therapy
Prevention
Taenia Saginata
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Antiparasitic Susceptibility and Therapy
Prevention
Case Study 54-1
Figure 54-12 Scolex.
Questions
Figure 54-13 Proglottid.
Bibliography
Chapter Review
Chapter 55 Tissue Cestodes
Objectives
Parasites to be Considered
Cestodes (Tapeworms)
Taenia Solium
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
TABLE 55-1 Common Human Parasites, Diagnostic Specimens, Tests, and Positive Findings
Therapy
Prevention
Echinococcus Granulosus
General Characteristics
Epidemiology
Figure 55-1 Life cycle of Echinococcus granulosus (hydatid disease).
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Figure 55-2 A, Echinococcus granules. B, Ovum. C, Scolex.
Figure 55-3 Echinococcus granulosus, hydatid sand (300×). (Inset) Two individual hooklets (1000×).
Therapy
Prevention
Echinococcus Multilocularis
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Therapy
Prevention
Taenia Multiceps
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Therapy
Prevention
Spirometra Mansonoides
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Therapy
Prevention
Case Study 55-1
Questions
Figure 55-4 Scolex collected from patient’s liver biopsy.
Bibliography
Chapter Review
Chapter 56 Intestinal Trematodes
Objectives
Parasites to be Considered
Fasciolopsis Buski
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
Prevention
Heterophyes Heterophyes and Metagonimus Yokogawai
General Characteristics
Figure 56-1 Life cycle of trematodes acquired by humans through ingestion of raw fish, crabs, or crayfish and vegetation.
Figure 56-2 Whole mount of Fasciolopsis buski.
Figure 56-3 Fasciola egg. The eggs of F. buski and F. hepatica are indistinguishable morphologically.
Epidemiology
Pathogenicity and Spectrum of Disease
Prevention
Laboratory Diagnosis
Treatment
Chapter Review
TABLE 56-1 Diagnostic Characteristics of Intestinal Trematodes
Case Study 56-1
Questions
Bibliography
Chapter 57 Liver and Lung Trematodes
Objectives
Parasites to be Considered
Trematodes (Flukes)
The Liver Flukes
General Characteristics
Epidemiology and Life Cycle
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Figure 57-1 Cercaria of a liver fluke.
Figure 57-2 Life cycle of the liver and lung flukes.
TABLE 57-1 Characteristics of Liver and Lung Trematodes
Figure 57-3 Clonorchis sinensis egg.
Therapy and Prevention
The Lung Flukes
General Characteristics
Epidemiology and Life Cycle
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Treatment and Prevention
Figure 57-4 Paragonimus westermani egg.
Case Study 57-1
Questions
Bibliography
Chapter Review
Chapter 58 Blood Trematodes
Objectives
Parasites to be Considered
Trematodes
General Characteristics
Epidemiology
Figure 58-1 Mating of Schistosoma mansoni male and female worms.
Figure 58-2 A, Schistosoma mansoni egg. B, Schistosoma japonicum egg. C, Schistosoma haematobium egg.
TABLE 58-1 Diagnostic Characteristics of the Blood Trematodes
Pathology and Spectrum of Disease
Figure 58-3 S. japonicum egg.
Figure 58-4 S. mansoni miracidium.
Figure 58-5 S. mansoni cercaria.
Figure 58-6 Life cycle of human schistosomes.
Laboratory Diagnosis
Therapy
Prevention
Case Study 58-1
Questions
Bibliography
Chapter Review
Part V Mycology
Chapter 59 Overview of Fungal Identification Methods and Strategies
Objectives
Epidemiology
General Features of the Fungi
Taxonomy of the Fungi
Figure 59-1 A cleistothecium of Pseudallescheria boydii that has opened and is releasing numerous ascospores (×750).
Figure 59-2 Scedosporium apiospermum showing asexually produced conidia borne singly on conidiophores (anellophores [arrows]) (×430).
Figure 59-3 Graphium anamorph of P. boydii (×500).
Clinical Classification of the Fungi
TABLE 59-1 Phylogenetic Position of Medically Significant Fungi
TABLE 59-2 General Clinical Classification of Pathogenic Fungi
Practical Working Schema
Pathogeneis and Spectrum of Disease
TABLE 59-3 Most Commonly Encountered Fungi of Clinical Laboratory Importance: a Practical Working Schema
Box 59-1 Phenotypic Classification of Medically Important Fungi
Morphologic Classification of Medically Important Fungi, Monomorphic Yeasts, and Yeastlike Organisms
Thermally Dimorphic Fungi
Thermally Monomorphic Molds
Laboratory Diagnosis
Collection, Transport, and Culturing of Clinical Specimens
TABLE 59-4 Summary of Common Pathogens
TABLE 59-5 Virulence Factors of Medically Important Fungi
Respiratory Tract Secretions
Cerebrospinal Fluid
Blood
Hair, Skin, and Nail Scrapings
Vaginal
Urine
Tissue, Bone Marrow, and Sterile Body Fluids
Culture Media and Incubation Requirements
TABLE 59-6 Fungal Culture Media: Indications for Use
Direct Microscopic Examination
Procedure 59-1 Calcofluor White–Potassium Hydroxide Preparation
Method
Reagents
Limitations
Serodiagnosis
Molecular Detection
MALDI-TOF (Matrix-Assisted Laser Desorption Ionization)
General Considerations for the Identification of Yeasts
TABLE 59-7 Summary of Methods Available for Direct Microscopic Detection of Fungi in Clinical Specimens
TABLE 59-8 Summary of Characteristic Features of Fungi Seen in Direct Examination of Clinical Specimens
Figure 59-4 Fungi commonly seen in clinical specimens. A, This potassium hydroxide preparation of a skin scraping from a patient with a dermatophyte infection shows septate hyphae intertwined among epithelial cells. (Phase-contrast microscopy; ×500.) B, This calcofluor white stain of urine demonstrates Candida albicans. C, The deeply staining, small, uniform yeast cells in this histologic section of lung tissue are typical of Histoplasma capsulatum. (Methenamine silver stain; ×430.)
General Considerations for the Identification of Molds
Figure 59-5 Identification of yeasts in clinical specimens.
Procedure 59-2 Cellophane (Scotch) Tape Preparation
Method
Figure 59-6 Identification of filamentous fungi from clinical specimens.
Figure 59-7 Cellophane tape preparation showing placement of tape on slide containing lactophenol cotton or aniline blue.
Procedure 59-3 Wet Mount
Method
Figure 59-8 Performance of a wet mount showing agar positioned under coverslip before pressure is applied to disperse growth.
Limitations
Procedure 59-4 Tease Mount
Method
Procedure 59-5 Microslide Culture
Method
Figure 59-9 Microslide culture showing inoculation of agar plug (arrow).
Limitations
General Morphologic Features of the Molds
Figure 59-10 Antler hyphae showing swollen hyphal tips, resembling antlers, with lateral and terminal branching (favic chandeliers) (×500).
Figure 59-11 Racquet hyphae showing swollen areas (arrows) resembling a tennis racquet.
Figure 59-12 Spiral hyphae (arrow) showing corkscrewlike turns (×430).
Figure 59-13 Ascocarp showing dark-appearing ascospores (×430).
Figure 59-14 Conidia (asexual spores [A]) produced on specialized structures (conidiophores [B]) of Aspergillus (×430).
Figure 59-15 Arthroconidia formation (A) produced by the breaking down of a hyphal strand (B) into individual rectangular units (×430).
Figure 59-16 Chlamydoconidia composed of thick-walled spherical cells (arrows) (×430).
Figure 59-17 Simple tubular phialide with a cluster of conidia at its tip (arrow) characteristic of Acremonium (×430).
Figure 59-18 Complex method of sporulation in which conidia are borne on phialides produced on secondary branches (metulae [arrow]) characteristic of Penicillium (×430).
Figure 59-19 In this preparation of a Trichophyton species, the numerous, small, spherical microconidia (A) are contrasted with a large, elongated macroconidium (B) (×430).
Figure 59-20 Large, saclike sporangia that contain sporangiospores (arrow) characteristic of the mucorales (×250).
Clinical Relevance for Fungal Identification
TABLE 59-9 Fungi Most Commonly Recovered from Clinical Specimens
Laboratory Safety
TABLE 59-10 Common Filamentous Fungi Implicated in Human Mycotic Infections
TABLE 59-11 Common Yeastlike Organisms Implicated in Human Infection*
Prevention
Case Study 59-1
Questions
Chapter Review
Bibliography
Chapter 60 Hyaline Molds, Mucorales (Zygomycetes), Dermatophytes, and Opportunistic and Systemic Mycoses
Objectives
Hyaline Molds to be Considered
The Mucorales
General Characteristics
Epidemiology and Pathogenesis
Spectrum of Disease
Figure 60-1 Rhizopus spp. showing sporangium (A) on long sporangiophore (B) arising from pauciseptate hyphae. Note the characteristic rhizoids (C) at the base of the sporangiophore (×250).
Laboratory Diagnosis
Specimen Collection and Transport
Specimen Processing
Direct Detection Methods
Stains.
Antigen-Protein.
Nucleic Acid Amplification.
Cultivation.
Figure 60-2 Phase-contrast microscopy of a potassium hydroxide preparation of sputum. Note the fragmented portions (arrows) of broad, predominantly nonseptate hyphae of Rhizopus spp.
Figure 60-3 Rhizopus colony.
Approach to Identification
Figure 60-4 Mucor spp. showing numerous sporangia without rhizoids (×430).
Figure 60-5 Lichtheimia spp. (A) showing sporangia on long sporangiophores arising from pauciseptate hyphae (B). Note that rhizoids are produced between sporangiophores and not at their bases (×250).
Serodiagnosis
The Dermatophytes
General Characteristics
Epidemiology and Pathogenesis
Spectrum of Disease
Trichophyton spp.
Laboratory Diagnosis
Specimen Collection and Transport
Specimen Processing
Direct Detection Methods
Stains.
Figure 60-6 Calcofluor white stain of sputum showing intracellular yeast cells of H. capsulatum (arrows). The cells are 2 to 5 µm in diameter.
Antigen-Protein.
Nucleic Acid Amplification.
Cultivation.
Approach to Identification
Trichophyton spp.
TABLE 60-1 Characteristics of Dermatophytes Commonly Recovered in the Clinical Laboratory
Figure 60-7 Dermatophyte identification schema.
Figure 60-8 Trichophyton rubrum showing numerous pyriform microconidia borne singly on hyphae (×750).
Figure 60-9 A, Trichophyton mentagrophytes showing numerous microconidia in grapelike clusters. B, Several thin-walled macroconidia also are present (×500).
Figure 60-10 Hair perforation by Trichophyton mentagrophytes. Wedge-shaped areas (arrow) illustrate hair perforation (×100).
Figure 60-11 Trichophyton tonsurans showing numerous microconidia (A) that are borne singly or in clusters. A single macroconidium (B) (rare) is also present (×600).
Figure 60-12 Trichophyton verrucosum showing microconidia, which are rarely seen (×500).
Figure 60-13 Trichophyton schoenleinii showing swollen hyphal tips with lateral and terminal branching (favic chandeliers). Microconidia and macroconidia are absent (×500).
Figure 60-14 Large, rough-walled macroconidia of Microsporum canis (×430).
Microsporum spp.
Epidermophyton sp.
Figure 60-15 Microsporum gypseum showing ellipsoidal, multicelled macroconidia (×750).
Figure 60-16 Epidermophyton floccosum showing numerous smooth, multiseptate, thin-walled macroconidia that appear club shaped (×1000).
Serodiagnosis
The Opportunistic Mycoses
General Characteristics
Epidemiology and Pathogenesis
Aspergillus spp.
TABLE 60-2 Species of Aspergillus Recovered from Clinical Specimens During a 10-Year Period at the Mayo Clinic
Pathogenesis and Spectrum of Disease
Aspergillus spp.
Fusarium spp. and Other Hyaline Septate Opportunistic Molds
Laboratory Diagnosis
Specimen Collection and Transport
Specimen Processing
Direct Detection Methods
Stains.
Antigen-Protein.
Nucleic Acid Amplification.
MALDI-TOF (Matrix-Assisted Laser Desorption Ionization).
Figure 60-17 Papanicolaou staining of sputum shows the dichotomously branching septate hyphae (arrows) of Aspergillus fumigatus.
Cultivation.
Approach to Identification
Aspergillus spp.
Figure 60-18 Aspergillus fumigatus conidiophore and conidia (×400).
Figure 60-19 Aspergillus flavus showing spherical vesicles (A) that give rise to metulae (B) and phialides (C) that produce chains of conidia (×750).
Figure 60-20 Aspergillus niger showing larger spherical vesicle that gives rise to metulae, phialides, and conidia (×750).
Fusarium spp.
Geotrichum candidum.
Figure 60-21 Aspergillus terreus showing typical head of Aspergillus and aleurioconidia (arrow) found on submerged hyphae of this species (×500).
Figure 60-22 Fusarium spp. showing characteristic multicelled, sickle-shaped macroconidia (×500).
Figure 60-23 Geotrichum candidum showing numerous arthroconidia. Note that arthroconidia do not alternate with a clear (dysjunctor) cell as in the case of Coccidioides immitis (×430).
Acremonium spp.
Figure 60-24 Mycelial form of Coccidioides immitis showing numerous thick-walled, rectangular or barrel-shaped (arrows) alternate arthroconidia (×500).
Figure 60-25 Penicillium spp. showing typical brushlike conidiophores (penicilli) (×430).
Penicillium spp.
Paecilomyces spp.
Scopulariopsis spp.
Figure 60-26 Paecilomyces spp. showing long, tapering, delicate phialides (arrow).
Figure 60-27 Scopulariopsis spp. showing a large penicillus (A) with echinulate conidia (B) (×430).
Serodiagnosis
Systemic Mycoses
General Characteristics
Epidemiology
Blastomyces dermatitidis
Coccidioides immitis
Histoplasma capsulatum
Paracoccidioides brasiliensis
Penicillium marneffei
Sporothrix schenckii
Pathogenesis and Spectrum of Disease
Blastomyces dermatitidis
Coccidioides immitis
Histoplasma capsulatum
Paracoccidioides brasiliensis
Penicillium marneffei
Sporothrix schenckii
Laboratory Diagnosis
Specimen Collection and Transport
Specimen Processing
Direct Detection Methods
Stains.
Blastomyces dermatitidis.
Figure 60-28 Blastomyces dermatitidis yeast form showing thick-walled, oval to round, single-budding, yeastlike cells (×500).
Figure 60-29 Potassium hydroxide preparation of exudate shows a large budding yeast cell with a distinct broad base (arrow) between the cells, which is characteristic of Blastomyces dermatitidis. (Phase-contrast microscopy.)
Coccidioides immitis.
Figure 60-30 Auramine-rhodamine preparation of specimen material from a bone lesion demonstrates the characteristic broad-based budding yeast (arrow) of Blastomyces dermatitidis.
Figure 60-31 Tissue form of Coccidioides immitis (i.e., the spherule). The external wall of the spherule does not stain with the silver stain, whereas the internal endospores do stain (arrowhead). Also note how the juxtaposed endospores, which have been released from a spherule that has burst, resemble budding yeast (arrow). (GMS stain; ×400).
Histoplasma capsulatum.
Figure 60-32 Potassium hydroxide preparation of sputum demonstrates two spherules of Coccidioides immitis filled with endospores. When these lie adjacent to each other, they may be mistaken for Blastomyces dermatitidis. (Bright-field microscopy.)
Figure 60-33 Histologic section showing a well-developed spherule of Coccidioides immitis that is filled with endospores.
Figure 60-34 These small, oval yeast cells that are relatively uniform in size are characteristic of Histoplasma capsulatum (×2000).
Paracoccidioides brasiliensis.
Figure 60-35 Paracoccidioides brasiliensis in a bone marrow aspirate shows a yeast cell with multiple buds (arrow).
Figure 60-36 Penicillium marneffei and binary fission (arrows) (×500).
Penicillium marneffei.
Sporothrix schenckii.
Figure 60-37 The deeply staining bodies in this mouse testis are the yeast forms of Sporothrix schenckii.
Figure 60-38 Periodic acid-Schiff (PAS) staining of exudate shows the cigar-to-oval–shaped yeast cells (arrows) of Sporothrix schenckii.
Antigen-Protein.
Nucleic Acid Amplification.
Cultivation.
Blastomyces dermatitidis.
Coccidioides immitis.
Procedure 60-1 Hair Perforation Test
Method
Procedure 60-2
Nucleic Acid Probe Testing
Principle
Specimen
Method
Frequency and Tolerance of Controls
Expected Values
Limitations
Procedure 60-3 In Vitro Conversion of Dimorphic Molds
Principle
Method
Quality Control
Procedure 60-4
Exoantigen Test
Principle
Method
Quality Control
Histoplasma capsulatum.
Paracoccidioides brasiliensis.
Penicillium marneffei.
Sporothrix schenckii.
Figure 60-39 Mycelial form of Blastomyces dermatitidis shows oval conidia borne laterally on branching hyphae (×1000).
Approach to Identification
Blastomyces dermatitidis.
Coccidioides immitis.
Histoplasma capsulatum.
Figure 60-40 Trichosporon spp. produce arthroconidia (A) and an occasional blastoconidium (B).
Figure 60-41 Mycelial form of Histoplasma capsulatum produces characteristic tuberculate macroconidia (×1000).
Paracoccidioides brasiliensis.
Figure 60-42 Mycelial form of Paracoccidioides brasiliensis shows septate hyphae and pyriform conidia singly borne (arrow) (×430).
Penicillium marneffei.
Sporothrix schenckii.
Figure 60-43 Mycelial form of Sporothrix schenckii shows pyriform-to-ovoid microconidia in a flowerette morphology at the tip of the conidiophore (arrow) (×750).
Figure 60-44 Yeast form of Sporothrix schenckii consists of cigar-shaped and oval budding cells (×500).
Serodiagnosis
TABLE 60-3 Summary of the Characteristic Features of Fungi Known to Be Common Causes of Selected Fungal Infection in Humans
Case Study 60-1
Questions
Chapter Review
Bibliography
Chapter 61 Dematiaceous (Melanized) Molds
Objectives
Septate Dematiaceous Molds to be Considered
Superficial Infections
Mycetoma
Chromoblastomycosis
Phaeohyphomycosis
General Characteristics
Epidemiology and Pathogenesis
Superficial Infections (Tinea Nigra and Black Piedra)
Mycetoma
Chromoblastomycosis
Phaeohyphomycosis
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Specimen Collection and Transport
Specimen Processing
Direct Detection Method
Stains
Superficial Infections.
TABLE 61-1 Dematiaceous Fungi
Chromoblastomycosis.
Mycetoma and Phaeohyphomycosis.
Figure 61-1 Sclerotic bodies from the tissue of a patient with chromoblastomycosis (×400).
Antigen-Protein
Nucleic Acid Amplification
Cultivation
Figure 61-2 Yeast forms of Hortaea werneckii.
Superficial Infections.
Mycetoma
White Grain Mycetoma.
Black Grain Mycetoma.
Chromoblastomycosis.
Phaeohyphomycosis.
Approach to Identification
Superficial Infections
Mycetoma
White Grain Mycetoma: Pseudallescheria boydii and Acremonium spp.
Black Grain Mycetoma: Exophiala jeanselmei, Curvularia spp., and Madurella mycetomatis.
Chromoblastomycosis: Cladosporium, Phialophora, and Fonsecaea spp.
Figure 61-3 Phialophora richardsiae showing phialides with prominent, saucerlike collarette (arrows) (×500).
Figure 61-4 Cladosporium spp. showing Cladosporium type of sporulation (arrows) with chains of elliptical conidia (×430).
Figure 61-5 Phialophora verrucosa showing flask-shaped phialide (A) with distinct collarette (B) and conidia (C) near its tip (×750).
Figure 61-6 Both the Rhinocladiella and Phialophora types of sporulation may be produced by Fonsecaea pedrosoi and are demonstrated here (×430).
Phaeohyphomycosis: Alternaria, Bipolaris, Cladosporium, Curvularia, Drechslera, Exophiala, Exserohilum, and Phialophora spp.
Figure 61-7 Alternaria spp. showing chaining multiform dematiaceous conidia with horizontal and longitudinal septa.
Figure 61-8 Bipolaris spp. showing dematiaceous, multicelled conidia produced sympodially from geniculate conidiophores (×430).
Alternaria spp.
Bipolaris spp.
Figure 61-9 Cladosporium spp. showing branching chains of dematiaceous blastoconidia that are easily dislodged during preparation of a microscopic mount (×430).
Figure 61-10 Curvularia spp. showing twisted conidiophore and curved conidia with a swollen central cell (arrows) (×500).
Cladosporium spp.
Curvularia spp.
Drechslera spp.
Figure 61-11 Drechslera spp. showing dematiaceous, multicelled conidia. Most isolates produce only a few conidia.
Figure 61-12 Exophiala dermatitidis showing dematiaceous, yeastlike cells from a young culture. These forms asexually reproduce via annellides rather than through true budding (blastoconidiation) (×500).
Exophiala spp.
Figure 61-13 A, Exophiala jeanselmei showing elongated conidiophore (annellophore) with a narrow, tapered tip (×500). B, Exophiala dermatitidis showing elongated tubular annellophores (arrow); morphologically very similar to E. jeanselmei (×500).
Exserohilum spp.
Phialophora spp.
Serodiagnosis
Figure 61-14 Exserohilum spp. showing elongated, multicelled conidia with prominent hila (arrows).
Case Study 61-1
Questions
Bibliography
Chapter Review
Chapter 62 Opportunistic Atypical Fungus: Pneumocystis jiroveci
Objectives
Genus and Species to be Considered
General Characteristics
Epidemiology
Pathogenesis and Spectrum of Disease
Laboratory Diagnosis
Specimen Collection and Transport
Specimen Processing
Direct Detection Methods
Stains
Figure 62-1 Cystic forms of Pneumocystis jiroveci (arrows) stain well with methenamine silver and hematoxylin and eosin stain (×500).
Antigen-Protein
Nucleic Acid Amplification
Cultivation
Approach to Identification
Serodiagnosis
Chapter Review
Case Study 62-1
Questions
Bibliography
Chapter 63 The Yeasts
Objectives
Genera and Species to be Considered
General Characteristics
Figure 63-1 Blastoconidia (budding cells [arrow]) characteristic of the yeasts.
Figure 63-2 Germ tube test for C. albicans showing yeast cells with germ tubes.
Figure 63-3 Pseudohyphae consisting of elongated cells (arrow) with constrictions at attachment.
Epidemiology
Candida spp.
Cryptococcus spp.
Trichosporon and Malassezia spp.
Pathogenesis and Spectrum of Disease
Candida Albicans
Non-Albicans Candida
Cryptococcus Neoformans
Genus Cryptococcus
Cryptococcus Gattii
Trichosporon spp.
Malassezia spp.
Laboratory Diagnosis
Specimen Collection, Transport, and Processing
Figure 63-4 Periodic acid-Schiff (PAS) staining of urine demonstrates blastoconidia and pseudohyphae of Candida albicans.
Stains
Candida spp.
Cryptococcus spp.
Figure 63-5 Potassium hydroxide preparation of pleural fluid shows the encapsulated, variably sized, spherical yeast cells (arrow) of Cryptococcus neoformans (phase-contrast microscopy).
Trichosporon spp.
Malassezia spp.
Figure 63-6 Potassium hydroxide preparation of a skin scraping from a patient with tinea versicolor demonstrates spherical yeast cells (A) and short hyphal fragments (B) of Malassezia furfur. (Phase-contrast microscopy; ×500.)
Antigen Detection
Molecular Assays
Figure 63-7 Colonies of Cryptococcus neoformans appear shiny and mucoid because of the presence of a polysaccharide capsule.
Cultivation
Candida spp.
Cryptococcus spp.
Trichosporon spp.
Malassezia spp.
Approach to Identification
Candida spp.
Figure 63-8 Chlamydoconidia of Candida albicans (arrows).
Germ Tube Test
TABLE 63-1 Characteristic Microscopic Features of Commonly Encountered Yeasts on Cornmeal Tween 80 Agar
Procedure 63-1 Germ Tube Test
Principle
Method
Quality Control
Expected Results
Performance Schedule
Cryptococcus neoformans
Figure 63-9 Cryptococcus neoformans colonies are brown when grown on niger seed agar.
Rapid Urease Test
Procedure 63-2 Rapid Urease Test
Principle
Method
Quality Control
Expected Results
Performance Schedule
Procedure 63-3 Rapid Selective Urease Test
Principle
Method
Quality Control
Trichosporon spp.
Malassezia spp.
Commercially Available Yeast Identification Systems
API-20C AUX Yeast System
Microscan Yeast Identification Panel
Vitek Biochemical Cards
Chromagar Candida
Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF)
Conventional Yeast Identification Methods
Cornmeal Agar Morphology
Procedure 63-4 Cornmeal Agar Morphology
Principle
Method
Quality Control
Expected Results
Performance Schedule
Carbohydrate Utilization
Procedure 63-5 Conventional Carbohydrate Utilization Tests
Principle
Method
Quality Control
Phenoloxidase Detection Using Niger Seed Agar
Procedure 63-6 Phenoloxidase Detection Using Niger Seed Agar
Principle
Method
Quality Control
Expected Results
Performance Schedule
Chapter Review
Case Study 63-1
Questions
Bibliography
Chapter 64 Antifungal Susceptibility Testing, Therapy, and Prevention
Objectives
Antifungal Susceptibility Testing
Antifungal Therapy and Prevention
Polyene Macrolide Antifungals
Amphotericin B
Nystatin
Griseofulvin
5-Fluorocytosine (Flucytosine)
Azole Antifungal Drugs
Clotrimazole and Miconazole
Fluconazole
Ketoconazole
Itraconazole
Voriconazole
Posaconazole
Anidulafungin
Micafungin
Other
Terbinafine (Lamisil)
Echinocandins
Caspofungin
Selenium Sulfide
Potassium Iodide
Bibliography
Chapter Review
Part VI Virology
Chapter 65 Overview of the Methods and Strategies in Virology*
Objectives
General Characteristics
Viral Structure
Virus Taxonomy
Figure 65-1 Illustration of a viral particle. Enveloped and nonenveloped virions have an icosahedral or irregular (usually helical) shape.
Figure 65-2 Relative sizes of representative viruses, bacteriophages (bacterial viruses), and bacteria, including chlamydia.
Viral Replication
Figure 65-3 Illustration of the viral infectious cycle.
Epidemiology
Pathogenesis and Spectrum of Disease
Prevention and Therapy
Antiviral Agents
Figure 65-4 Viral pathogenesis is illustrated by the mechanisms through which the measles virus spreads in the body.
Viruses That Cause Human Diseases
Laboratory Diagnosis
Setting Up a Clinical Virology Laboratory
TABLE 65-1 Viral Syndromes and Common Viral Pathogens
Specimen Selection and Collection
General Principles
Figure 65-5 Roller drum used to hold cell culture tubes during incubation. Slow rotation continually bathes the cells in the medium.
Figure 65-6 Inverted microscope used to examine cell monolayers growing attached to the inside surface beneath the liquid medium. Note that the objective is under the glass test tube, facilitating observation of the cell monolayer.
Figure 65-7 Class II biological safety cabinet used in a clinical virology laboratory.
TABLE 65-2 Viruses Detected by Culture, PCR, or Assay for Antigen in a Community Hospital Virology Laboratory*
TABLE 65-3 Specimens for Diagnosis of Viral Diseases*
Throat, Nasopharyngeal Swab, or Aspirate
Bronchial and Bronchoalveolar Washes
Rectal Swabs and Stool Specimens
Urine
Skin and Mucous Membrane Lesions
Sterile Body Fluids Other Than Blood
Blood
Bone Marrow
Tissue
Genital Specimens
Serum for Antibody Testing
Specimen Transport and Storage
Specimen Processing
General Principles
Processing Based on Specimen Type
Lip and Genital Specimens.
Urine.
Stool.
TABLE 65-4 Laboratory Processing of Viral Specimens
Respiratory Tract.
Specimens from Neonatal Patients.
Cerebrospinal Fluid.
Blood.
Processing Based on Requests for Specific Viruses
Arboviruses.
Procedure 65-1 Processing Blood for Viral Culture: Leukocyte Separation Using Polymorphprep
Purpose
Specimen
Materials
Method
Troubleshooting
TABLE 65-5 Virus Detection or Quantitation Tests
Cytomegalovirus.
TABLE 65-6 Viral Serology Tests
Procedure 65-2 Cytomegalovirus Antigenemia Stain
Purpose
Specimen
Materials
Method
Quality Control
Enteroviruses.
Epstein-Barr Virus.
Hepatitis Viruses.
Figure 65-8 Algorithm for the processing of viral specimens based on specimen type and suspected virus. Virus detection implies viral culture, antigen detection, or molecular testing (e.g., polymerase chain reaction [PCR] assay).
Herpes Simplex Virus.
TABLE 65-7 Serology Tests for Hepatitis Viruses
Human Immunodeficiency Virus and Other Retroviruses.
Influenza A and B Viruses.
Pediatric Respiratory Viruses.
Gastroenteritis Viruses.
Figure 65-9 Fluorescent antibody staining of virus-infected cells. A, Influenza virus. B, Adenovirus. C, Varicella-zoster virus. D, Herpes simplex virus. E, Respiratory syncytial virus. F, Parainfluenza virus. G, Mumps virus. H, Measles virus.
TORCH.
Box 65-1 Overview of Respiratory Virus Detection by R-Mix Shell Vials
Purpose
Principle
Specimen
Materials
Methods
Interpretation
Procedure Notes
Figure 65-10 Flowchart for the detection and identification of pediatric respiratory viruses.
Varicella-Zoster Virus.
TABLE 65-8 Tests for Human Gastroenteritis Viruses
TABLE 65-9 Laboratory Diagnosis of Viral Diseases in the Newborn
Virus Detection Methods
Cytology and Histology
Figure 65-11 Viral inclusions. A, Pap-stained smear showing multinucleated giant cells typical of herpes simplex or varicella-zoster viruses. B, Hematoxylin and eosin (HE)–stained lung tissue containing intranuclear inclusion within enlarged cytomegalovirus (CMV)–infected cells. C, HE-stained lung tissue containing epithelial cells with intranuclear inclusions characteristic of adenovirus. D, HE-stained liver from stillborn fetus showing intranuclear inclusions in erythroblasts (extramedullary hematopoiesis) resulting from parvovirus infection. E, Pap stain of exfoliated cervicovaginal epithelial cells showing perinuclear vacuolization and nuclear enlargement characteristic of human papillomavirus infection. F, HE-stained epidermis filled with molluscum bodies, which are large, eosinophilic, cytoplasmic inclusions resulting from infection with molluscum contagiosum virus. G, HE-stained cells infected with measles virus. H, HE-stained brain tissue showing oval, eosinophilic rabies cytoplasmic inclusion (Negri body).
Figure 65-12 Electron micrographs of viruses. A, Rotavirus. B, Adenovirus. C, Norwalk agent virus. D, Coronavirus. E, Herpes simplex virus. F, Measles virus. G, Negatively stained preparation of JC virus in brain tissue.
Box 65-2 Varicella-Zoster Virus Detection by Polymerase Chain Reaction Assay
Purpose
Specimen
Materials
Method
Interpretation
Procedure Notes
Electron Microscopy
Immunodiagnosis (Antigen Detection)
TABLE 65-10 Interpretation of Fluorescence Intensity Using FITC
Enzyme-Linked Virus-Inducible System
Molecular Detection Using Nucleic Acid Probes and Polymerase Chain Reaction Assays
Figure 65-13 A, RSV-infected RMK (rhesus monkey kidney) cells at 400×, stained with Light Diagnostics RSV MoAb. Fluorescence is seen in the cytoplasm and associated with syncytia. Cytoplasmic staining is often punctuate with small inclusions. B, HSV I: HSV I infected Vero cell control slide 200×. Stained with Pathfinder HSV 1 MoAb DFA assay. Fluorescent staining is cytoplasmic. C, Influenza B infected RMK cells at 400×. Stained with Light Diagnostics Influenza B MoAb. Fluorescence is nuclear, cytoplasmic, or both. Nuclear staining is uniformly bright and the cytoplasmic staining is often punctuate with l large inclusions. D, Herpes Simplex II infected A549 cells at 200×. Stained with Pathfinder HSV II MoAb DFA assay. Fluorescence may stain the cytoplasm, the nucleus, or both depending on the stage of the infection cycle. When infected cells are rounded, staining may appear nuclear due to cytoplasm covering the nucleus. E, HSV II Infected A549 cells at 200×. F, HSV II Infected A549 cells at 400×. Picture shows the multinucleated “giant” cells characteristic of HSV II CPE (cytopathogenic effect).
Figure 65-14 Solid-phase enzyme immunoassay for detection of rotavirus with breakaway strips of microtiter wells for small-batch testing.
Figure 65-15 Positive- (top) and negative-membrane enzyme-linked immunoassays (ELISAs) for detection of rotavirus. The red line in the reaction area on the left represents a positive test result. A red line in the reaction area on the right represents an internal test control ensuring that the test has been carried out correctly. If the test control line is not present, the test is invalid and must be repeated.
Figure 65-16 Smear of cervical cells stained with probe for papillomavirus DNA. Dark-staining cells contain viral DNA.
Cell Culture
Conventional Cell Culture.
Figure 65-17 Real-time polymerase chain reaction (PCR) detection of herpes simplex virus (HSV). Black, red, and light green lines represent three different HSV type 1 (HSV-1) viruses. Pink and dark green lines represent two different HSV type 2 (HSV-2) viruses. A, Cycle crossover detection of HSV-1 and HSV-2 amplicons, with all viruses detected between cycles 34 and 40. B, Melt curve confirmation of the presence of HSV-1 and HSV-2 viruses. HSV-1 amplicons melt at approximately 54°C (three HSV-1 viruses confirmed), and HSV-2 amplicons melt at approximately 68°C (one HSV-2 virus confirmed).
Figure 65-18 Cell culture tubes incubating on their sides in a stationary rack. Tubes are oriented with the same glass surface facing downward, because an emblem printed on one side of the glass near the neck is used for correct positioning in the rack.
Shell Vial Cell Culture.
Procedure 65-3 Shell Vial Culture for Cytomegalovirus
Purpose
Specimen
Materials
Methods
Quality Control
Identification of Viruses Detected in Cell Culture.
Figure 65-19 Shell vial cell culture tubes and stained coverslips. At the bottom of each shell vial tube under the culture medium is a round coverslip with a cell monolayer on the top surface. After incubation, the coverslip is removed, stained, and placed on a microscope slide for fluorescence viewing. Note that two stained coverslips are on the glass slide.
Figure 65-20 Typical fluorescing nuclei of human diploid fibroblast cells infected with cytomegalovirus as seen in the shell vial assay.
TABLE 65-11 Isolation and Identification of Common Clinically Encountered Viruses
TABLE 65-12 Quantitation of Cell Culture Cytopathic Effects
Procedure 65-4 Culture Confirmation by Fluorescent Antibody Staining
Purpose
Materials
Specimen
Method
Interpretation of Results
Quality Control
Procedure 65-5 Hemadsorption of Primary Monkey Kidney Monolayers to Detect Influenza, Parainfluenza, and Mumps Viruses
Purpose
Specimen
Materials
Method
Interpretation
Quality Control
Viral Serology
General Principles
Figure 65-21 Cell culture morphology and viral cytopathic effects (CPE). A, Normal human diploid lung fibroblast cells (HDF). B, Normal HEp-2 cells. C, Normal primary monkey kidney cells (PMK). D, HEp-2 cells infected with adenovirus. E, HDF cells infected with cytomegalovirus. F, HDF cells infected with herpes simplex virus. G, PMK cells infected with hemadsorbing virus, such as influenza, parainfluenza, or mumps, plus guinea pig erythrocytes. H, HEp-2 cells infected with respiratory syncytial virus. I, HDF cells infected with rhinovirus. J, PMK cells infected with echovirus. K, HDF cells infected with varicella-zoster virus.
Figure 65-22 IgM is separated from human serum by passing the serum through an ion exchange column.
Immune Status Testing
TABLE 65-13 Serology Panels and Immune Status Testing for Common Viral Syndromes
Serology Panels
Preservation and Storage of Viruses
Case Study 65-1
Questions
Procedure 65-6 Preservation and Storage of Viruses by Freezing
Purpose
Materials
Methods
Chapter Review
Bibliography
Chapter 66 Viruses in Human Disease
Objectives
Viruses to be Considered
DNA Viruses
Family
RNA Viruses
Family
Viruses in Human Disease
Adenoviruses
TABLE 66-1 DNA and RNA Viruses That Cause Serious Disease in Humans
Arenaviruses
TABLE 66-2 Viral Syndromes and Common Viral Pathogens
TABLE 66-3 Adenoviruses
TABLE 66-4 Arenaviruses
Bunyaviruses
TABLE 66-5 Bunyaviruses
Caliciviruses
TABLE 66-6 Caliciviruses
Coronaviruses
TABLE 66-7 Coronaviruses
Filoviruses
TABLE 66-8 Filoviruses
Flaviviruses
TABLE 66-9 Flaviviruses
Figure 66-1 Time course of immune response and disease caused by hepatitis C virus.
Hepevirus
TABLE 66-10 Hepevirus
TABLE 66-11 Hepadnaviruses
Hepadnaviruses
Figure 66-2 Time course of antigenemia and immune response in a patient demonstrating recovery from acute hepatitis B infection.
Herpes Viruses
TABLE 66-12 Herpesviruses
Figure 66-3 Time course of immune response to Epstein-Barr (EBV) infection.
Box 66-1 Basic Outline for Epstein-Barr Virus PCR Amplification
Principle
Specimen
Method
Limitations of Procedure
Figure 66-4 Typical fluorescing white blood cells containing cytomegalovirus (CMV) antigen, as seen in the CMV antigenemia stain.
Orthomyxoviruses
TABLE 66-13 Orthomyxoviruses
Papillomaviruses
TABLE 66-14 Papillomaviruses
Paramyxoviruses
TABLE 66-15 Paramyxoviruses
Parvoviruses
TABLE 66-16 Parvoviruses
Picornaviruses
TABLE 66-17 Picornaviruses
TABLE 66-18 Enterovirus Infections
Polyomaviruses
Figure 66-5 Time course of disease and immune response to hepatitis A virus.
TABLE 66-19 Polyomaviruses
Poxviruses
TABLE 66-20 Poxviruses
Reoviruses
TABLE 66-21 Reoviruses
Retroviruses
TABLE 66-22 Retroviruses
Rhabdoviruses
Figure 66-6 Usual time course of immune response, viremia, and disease resulting from untreated human immunodeficiency virus type 1 (HIV-1) infection.
Figure 66-7 Western blot test detecting specific human immunodeficiency virus (HIV) antibody. Lane 1 is the high-positive control; lane 2 is the low-positive control; lane 3 is the negative control; lanes 4 through 8 are positive sera; lane 9 is an indeterminate serum. Numbers at left refer to approximate molecular weights of HIV antigens.
TABLE 66-23 Rhabdoviruses
Togaviruses
Miscellaneous Viruses
TABLE 66-24 Togaviruses
Interpretation of Laboratory Test Results
Viruses in Tissue and Body Fluids
Viruses in the Respiratory Tract
Viruses in the Eye
Detection of Epstein-Barr Virus
TABLE 66-25 Interpretation of Serology Results for Epstein-Barr Virus Infection
Detection of Enteroviruses
Detection of Hepatitis Viruses
TABLE 66-26 Serologic Profiles After Typical Hepatitis B Virus (HBV) Infection
Detection of Varicella-Zoster Virus and Herpes Simplex Virus
Detection of Cytomegalovirus
Detection of Human Immunodeficiency Virus
Chapter Review
Case Study 66-1
Questions
Bibliography
Chapter 67 Antiviral Therapy, Susceptibility Testing, and Prevention
Objectives
Antiviral Therapy
Antiviral Resistance
Methods of Antiviral Susceptibility Testing
Phenotypic Assays
Plaque Reduction Assay
DU Assay
DNA Hybridization
Enzyme Immunoassay
Flow Cytometry
Neuraminidase Inhibition Assay
Genotypic Susceptibility Assays
Pyrosequencing
Human Immunodeficiency Virus
Influenza
TABLE 67-1 Antiviral Agents
TABLE 67-2 Examples of Vaccines for Preventing Viral Diseases
Prevention of Other Viral Infections
Vaccination
TABLE 67-3 Immune Prophylaxis or Therapy for Viral Diseases
Immune Prophylaxis and Therapy
Eradication
Chapter Review
Bibliography
Part VII Diagnosis by Organ System
Chapter 68 Bloodstream Infections
Objectives
General Considerations
Etiology
Bacteria
Box 68-1 Organisms Commonly Isolated from Blood Cultures
Fungi
Parasites
Viruses
Types of Bacteremia
Types of Bloodstream Infections
Box 68-2 Agents of Infective Endocarditis
Intravascular Infections
Infective Endocarditis.
Figure 68-1 Vegetations of bacterial endocarditis. Arrow indicates the vegetations.
Mycotic Aneurysm and Suppurative Thrombophlebitis.
Intravenous Catheter–Associated Bacteremia.
Figure 68-2 Short-term, triple-lumen central venous catheter. The ends from which the catheter is accessed are usually referred to as the hubs. After the catheter is inserted, the tip resides within the bloodstream.
Figure 68-3 Possible routes by which microorganisms gain access to the bloodstream to cause intravenous catheter–associated bacteremias.
Box 68-3 Common Agents of IV Catheter–Associated Bacteremia
Extravascular Infections
TABLE 68-1 Organisms Commonly Associated with Bloodstream Invasion from Extravascular Sites of Infection
Clinical Manifestations
Immunocompromised Patients
Detection of Bacteremia
Specimen Collection
Preparation of the Site
Antisepsis.
Procedure 68-1 Drawing Blood for Culture
Principle
Method
Precautions.
Specimen Volume
Adults.
TABLE 68-2 Suggested Blood Volumes for Cultures from Infants and Children
Children.
Number of Blood Cultures
Timing of Collection
Miscellaneous Matters
Anticoagulation.
Dilution.
Blood Culture Media.
Types of Blood Culture Bottle
Culture Techniques
Conventional Blood Cultures
Incubation Conditions.
Self-Contained Subculture System
Lysis Centrifugation
Figure 68-4 Becton Dickinson Septi-Chek pediatric-size biphasic blood culture bottle. The medium-containing base bottle is inoculated with blood, and the top piece containing agar paddles is added in the laboratory. The agar is inoculated by tipping the bottle to allow the blood-containing medium to flow over the agar.
Figure 68-5 Lysis centrifugation blood culture (Isolator System, Alere, Waltham, MA) uses vacuum-draw collection tubes with a lysing agent and special apparatus (Isostat Press) to facilitate removal of the supernatant without use of needles.
Instrument-Based Systems
BACTEC Systems.
BacT/ALERT Microbial Detection System.
Versa TREK System.
Techniques to Detect IV Catheter–Associated Infections
Figure 68-6 A, Blood culture bottles for the BACTEC 9240, 9120, and 9050 continuous monitoring instruments. B, The BD BACTEC FX continuous monitoring blood culture system. C, Blood culture bottles for the BacT/Alert continuous monitoring blood culture instruments. D, The BacT/Alert continuous monitoring blood culture system. E, Blood culture bottles for Trek Diagnostic Systems, Inc., ESP Culture System II continuous monitoring instrument.
Handling Positive Blood Cultures
TABLE 68-3 Summary Characteristics of the More Commonly Used Continuous-Monitoring Blood Culture Systems
Interpretation of Blood Culture Results
Special Considerations for Other Relevant Organisms Isolated From Blood
HACEK (AACEK) Bacteria
Box 68-4 Microorganisms That Cause Bloodstream Infections but Do Not Grow on Artificial Media
Campylobacter and Helicobacter
Fungi
Mycobacteria
Brucella
Spirochetes
Borrelia
Leptospira
Vitamin B6-Dependent Streptococci
Mycoplasma Hominis
Bartonella
Case Study 68-1
Questions
Chapter Review
Bibliography
Chapter 69 Infections of the Lower Respiratory System
Objectives
General Considerations
Anatomy
Pathogenesis of the Respiratory Tract: Basic Concepts
Host Factors
Figure 69-1 Anatomy of the respiratory tract, including upper and lower respiratory tract regions.
Microorganism Factors
Box 69-1 Organisms Present in the Nasopharynx and Oropharynx of Healthy Humans
Possible Pathogens
Rarely Pathogens
Adherence.
Box 69-2 Respiratory Tract Pathogens
Definite Respiratory Tract Pathogens
Rare Respiratory Tract Pathogens
Toxins.
Microorganism Growth.
Avoiding the Host Response.
Diseases of the Lower Respiratory Tract
Bronchitis
Acute
Chronic versus Acute
TABLE 69-1 Major Causes of Acute Bronchitis
Box 69-3 Viral Agents That Cause Bronchiolitis
Bronchiolitis
Pneumonia
Pathogenesis
Clinical Manifestations
Epidemiology/Etiologic Agents
Community-Acquired Pneumonia.
Children.
Young Adults.
Adults (Viral pneumonia).
Adults (Fungal pneumonia).
Chronic Lower Respiratory Tract Infections.
Immunocompromised Patients.
Patients with Neoplasms.
Transplant Recipients.
HIV-Infected Patients.
Pleural Infections
TABLE 69-4 Examples of Infectious Agents Frequently Associated with Certain Malignancies
Laboratory Diagnosis of Lower Respiratory Tract Infections
Specimen Collection and Transport
Sputum
Expectorated.
Induced.
Figure 69-2 Tracheal secretions received in the laboratory in a Lukens trap.
Endotracheal or Tracheostomy Suction Specimens
Bronchoscopy.
Transtracheal Aspirates.
Other Invasive Procedures.
Figure 69-3 Overview for obtaining a protected catheter bronchial brush during a bronchoscopy examination. A, The bronchoscope is introduced into the nose and advanced through the nasopharyngeal passage into the trachea. The bronchoscope is then inserted into the lung area of interest. B, A small brush that holds 0.001 to 0.01 mL of secretions is placed within a double cannula. The end of the outermost tube or cannula is closed with a displaceable plug made of absorbable gel. The cannula is inserted to the proper area. C, Once in the correct area, the inner cannula is pushed out, dislodging the protective plug as it is extruded. D, The brush is then extended beyond the inner cannula, and the specimen is collected by “brushing” the involved area. The brush is withdrawn into the inner cannula, which is withdrawn into the outer cannula to prevent contamination by upper airway organisms as it is removed.
Figure 69-4 Gram stain of sputum specimens. A, This specimen contains numerous polymorphonuclear leukocytes and no visible squamous epithelial cells, indicating that the specimen is acceptable for routine bacteriologic culture. B, This specimen contains numerous squamous epithelial cells and rare polymorphonuclear leukocytes, indicating an inadequate specimen for routine sputum culture.
Specimen Processing
Direct Visual Examination
Routine Culture
Chapter Review
Case Study 69-1
Questions
Bibliography
Chapter 70 Upper Respiratory Tract Infections and Other Infections of the Oral Cavity and Neck
Objectives
General Considerations
Anatomy
Pathogenesis
Diseases of the Upper Respiratory Tract, Oral Cavity, and Neck
Upper Respiratory Tract
Laryngitis
Laryngotracheobronchitis
Figure 70-1 The pharynx, including its three divisions and nearby structures.
Epiglottitis
Pharyngitis, Tonsillitis, and Peritonsillar Abscesses
Pharyngitis and Tonsillitis.
Clinical Manifestations.
Pathogenesis.
Epidemiology/Etiologic Agents.
TABLE 70-1 Examples of Bacteria That Can Cause Acute Pharyngitis and/or Tonsillitis
Box 70-1 Viral Agents That Can Cause Rhinitis
Peritonsillar Abscesses.
Rhinitis
Miscellaneous Infections Caused by Other Agents.
Corynebacterium diphtheriae.
Bordetella pertussis.
Klebsiella spp.
Oral Cavity
Stomatitis
Thrush
Periodontal Infections
Types.
Etiologic Agents.
Salivary Gland Infections
Neck
Diagnosis of Upper Respiratory Tract Infections
Collection and Transport of Specimens
Direct Visual Examination or Detection
Culture
Streptococcus pyogenes (Beta-Hemolytic Group A Streptococci)
TABLE 70-2 Medium and Atmosphere for Incubation of Cultures to Recover Group A Streptococci from Pharyngeal Specimens
Corynebacterium diphtheriae
Bordetella pertussis
Neisseria
Epiglottitis
Diagnosis of Infections in the Oral Cavity and Neck
Collection and Transport
Direct Visual Examination
Culture
Case Study 70-1
Questions
Bibliography
Chapter Review
Chapter 71 Meningitis, Encephalitis, and Other Infections of the Central Nervous System
Objectives
General Considerations
Anatomy
Coverings and Spaces of the CNS
Cerebrospinal Fluid
Routes of Infection
Figure 71-1 Cross section of the brain shows the important membrane coverings and spacing and other key structures.
Figure 71-2 Flow of CSF through the brain. CSF originates in the choroid plexus and then flows through the ventricles and subarachnoid space and into the bloodstream.
TABLE 71-1 Inner Coverings (Meninges) of the Brain, Spinal Cord, and Surrounding Spaces
Diseases of the Central Nervous System
Meningitis
Purulent Meningitis.
Pathogenesis.
TABLE 71-2 Guidelines for Interpretation of Results Following Hematologic and Chemical Analysis of Cerebrospinal Fluid (CSF) from Children and Adults (Excluding Neonates)
Clinical Manifestations.
Acute.
Chronic.
Epidemiology/Etiologic Agents-Acute Meningitis.
Box 71-1 Etiologic Agents of Chronic Meningitis
Viral
Aseptic Meningitis.
Encephalitis/Meningoencephalitis
Viral.
TABLE 71-3 Final 2010 West Nile Virus Human Infections in the United States*
Parasitic
Brain Abscess
Shunt Infections
Laboratory Diagnosis of Central Nervous System Infections
Meningitis
Specimen Collection and Transport
Initial Processing
CSF Laboratory Results
Figure 71-3 Gram stain of cerebrospinal fluid showing white blood cells and many gram-positive diplococci. This specimen subsequently grew Streptococcus pneumoniae.
Visual Detection of Etiologic Agents
Stained Smear of Sediment.
Figure 71-4 A, Cytocentrifuge. B, Device used to prepare the concentrated smears of material from body fluid specimens such as CSF by cytocentrifugation.
Wet Preparation.
India Ink Stain.
Direct Detection of Etiologic Agents
Antigen.
Bacteria.
Procedure 71-1 Rapid Extraction of Antigen Procedure (REAP)
Principle
Method
Expected Results
Cryptococcus neoformans.
Molecular Methods.
Miscellaneous Tests
Culture
Bacteria and Fungi.
Parasites and Viruses.
Brain Abscess/Biopsies
Specimen Collection, Transport and Processing.
Chapter Review
Case Study 71-1
Questions
Bibliography
Chapter 72 Infections of the Eyes, Ears, and Sinuses
Objectives
Eyes
Anatomy
Resident Microbial Flora
Diseases
Pathogenesis
Figure 72-1 Key anatomic structures of the eye.
Epidemiology and Etiology of Disease
Blepharitis
Conjunctivitis
TABLE 72-1 Infections of the Eye
Keratitis
Endophthalmitis
Periocular
Other Infections
Laboratory Diagnosis
Specimen Collection and Transport
Direct Visual Examination
Culture
Nonculture Methods
Ears
Anatomy
Resident Microbial Flora
Diseases, Epidemiology, and Etiology of Disease
Otitis Externa (External Ear Infections)
Figure 72-2 The ear.
Otitis Media (Middle Ear Infections)
TABLE 72-2 Major Infectious Causes of Ear Disease
Pathogenesis
Laboratory Diagnosis
Specimen Collection and Transport
Direct Visual Examination
Culture and Nonculture Methods
Sinuses
Anatomy
Figure 72-3 Location of the paranasal sinuses.
Diseases
Pathogenesis
Epidemiology and Etiology of Disease
TABLE 72-3 Major Infectious Causes of Acute Sinusitis
Laboratory Diagnosis
Case Study 72-1
Questions
Bibliography
Chapter Review
Chapter 73 Infections of the Urinary Tract
Objectives
General Considerations
Anatomy
Resident Microorganisms of the Urinary Tract
Infections of the Urinary Tract
Epidemiology
Figure 73-1 Overview of the anatomy of the urinary tract.
Box 73-1 Resident Microflora of the Urethra
Etiologic Agents
Community-Acquired
Hospital-Acquired
Miscellaneous
Pathogenesis
Routes of Infection
The Host-Parasite Relationship
Box 73-2 Examples of Probable Virulence Factors of Uropathogenic E. coli
Box 73-3 Risk Factors Associated with Complicated Urinary Tract Infections
Types of Infection and Their Clinical Manifestations
Urethritis
Ureteritis
Asymptomatic Bacteriuria
Cystitis
Acute Urethral Syndrome
Pyelonephritis
Urosepsis
Laboratory Diagnosis of Urinary Tract Infections
Specimen Collection
Clean-Catch Midstream Urine
Figure 73-2 Collection device to obtain urine by “in and out” or “straight” catheterization.
Straight Catheterized Urine
Suprapubic Bladder Aspiration
Indwelling Catheter
Specimen Transport
Screening Procedures
Gram Stain
Pyuria
Indirect Indices
Nitrate Reductase (Greiss) Test.
Leukocyte Esterase Test.
Catalase.
Automated and Semiautomated Systems
General Comments Regarding Screening Procedures
Urine Culture
Inoculation and Incubation of Urine Cultures
Figure 73-3 Method for inserting a calibrated loop into urine to ensure that the proper amount of specimen adheres to the loop.
Figure 73-4 Method for streaking with calibrated urine loop to produce isolated colonies and countable colony-forming units.
Procedure 73-1 Inoculating Urine with a Calibrated Loop
Purpose
Principle
Method
Interpretation and Results
Interpretation of Urine Cultures
TABLE 73-1 Criteria for Classification of Urinary Tract Infections by Clinical Syndrome
Figure 73-5 Culture results illustrating some of the various interpretative guidelines. A, Growth of ≥105 CFU/mL of a lactose-fermenting gram-negative rod in a clean-catch midstream (CCMS) urine from a patient with pyelonephritis; complete workup would be done. B, Growth of ≥105 CFU/mL of a lactose-fermenting gram-negative rod (arrow A) and <104 CFU/mL of another organism type (arrow B) from a CCMS urine; only the organism with a colony count of >104 to 105 CFU/mL would be worked up completely.
TABLE 73-2 General Interpretative Guidelines for Urine Cultures
Case Study 73-1
Questions
Chapter Review
Bibliography
Chapter 74 Genital Tract Infections
Objectives
General Considerations
Anatomy
Resident Microbial Flora
Sexually Transmitted Diseases and Other Genital Tract Infections
Figure 74-1 Location of key anatomic structures of the female (A) and male (B) genital tracts in relation to other major anatomic structures.
Genital Tract Infections
Sexually Transmitted Diseases and Other Lower Genital Tract Infections
Epidemiology/Etiologic Agents
Routes of Transmission
TABLE 74-1 Major Causes of Genital Tract Infections and Sexually Transmitted Diseases
Sexually Transmitted.
Other Routes.
Clinical Manifestations
Asymptomatic.
Figure 74-2 Genital lesions of the skin and mucous membranes that are sexually transmitted. A, Genital herpes showing vesicular lesions. B, Typical chancre of primary syphilis. C, Early chancroid lesion of the penis. D, Condyloma acuminatum.
Dysuria.
Urethral Discharge.
Lesions of the Skin and Mucous Membranes.
TABLE 74-2 Summary of Common Causes of Genital Lesions of the Skin and Mucous Membranes
Vaginitis.
Figure 74-3 Gram stain of vaginal secretions from a patient with desquamate inflammatory vaginitis. A, Numerous polymorphonuclear cells (PMNs), a squamous epithelial cell (SEC), a parabasal cell (PB), and the absence of lactobacilli are observed. B, Numerous PMNs, several PBs, and the absence of lactobacilli are observed.
Cervicitis.
Anorectal Lesions.
Bartholinitis.
Infections of the Reproductive Organs and Other Upper Tract Infections
Females.
Pelvic Inflammatory Disease.
Infections after Gynecologic Surgery.
Infections Associated with Pregnancy.
TABLE 74-3 Common Etiologic Agents of Prenatal and Neonatal Infections
Box 74-1 Organisms Frequently Isolated in Chorioamnionitis
Males.
Gonorrhea.
Syphilis.
Laboratory Diagnosis of Genital Tract Infections
Lower Genital Tract Infections
Urethritis, Cervicitis, and Vaginitis
Specimen Collection.
Urethral.
Cervical/Vaginal.
Figure 74-4 InPouch TV diagnostic system for wet mount examination and culture of Trichomonas vaginalis. The swab collected from the patient is inserted into liquid medium in the upper chamber of the plastic pouch and swirled. The top of the pouch is folded over and then sealed with the tabs. Once received in the laboratory, the upper chamber is examined for motile organisms; if no motile organisms are observed by microscopy, the upper chamber material is inoculated into the lower chamber and then incubated for up to 5 days. The lower chamber is similarly examined daily by microscopy for the presence of motile T. vaginalis.
Figure 74-5 Gram-negative intracellular diplococci, which are diagnostic for gonorrhea in urethral discharge and presumptive for gonorrhea in vaginal discharge.
Transport.
Direct Microscopic Examination.
Figure 74-6 Clue cells in vaginal discharge suggestive of bacterial vaginosis.
Figure 74-7 A, Predominance of lactobacilli in Gram stain from healthy vagina. B, Absence of lactobacilli and presence of Gardnerella vaginalis (A arrows) and Mobiluncus spp. (B arrows) morphologies.
Culture.
Figure 74-8 JEMBEC plate containing modified Thayer-Martin medium in a plastic, snap-top box with a self-contained CO2-generating tablet, all sealed inside a Zip-Lok plastic envelope after inoculation.
Figure 74-9 Method of cross-streaking JEMBEC plate after original specimen has been inoculated by rolling the swab over the surface of the agar in a W pattern.
Procedure 74-1 Preparing and Scoring Vaginal Gram Stains for Bacterial Vaginosis
Smear Preparation
Nonculture Methods.
Figure 74-10 Affirm VP III Microbial Identification Test used to differentiate the three major causes of vaginitis/bacterial vaginosis from a single sample within 1 hour.
Genital Skin and Mucous Membrane Lesions
Procedure 74-2 Collection of Material from Suspected Herpetic Lesions
Principle
Method
Bubo
Infections of the Reproductive Organs
Pelvic Inflammatory Disease
Miscellaneous Infections
Infections of Neonates and Human Products of Conception
Case Study 74-1
Questions
Bibliography
Chapter Review
Chapter 75 Gastrointestinal Tract Infections
Objectives
Anatomy
Resident Microbial Flora
Figure 75-1 General anatomy of the gastrointestinal tract.
Box 75-1 Components of the Gastrointestinal Tract
Figure 75-2 Wall of the small intestine. Villi cover the folds of the mucosal layer; in turn, each villus is covered with epithelial cells.
Gastroenteritis
Pathogenesis
Host Factors
Microbial Factors
Primary Pathogenic Mechanisms.
Toxins
Enterotoxins.
TABLE 75-1 Examples of Microorganisms That Cause GI Infection for Each Primary Pathogenic Mechanism
Cytotoxins.
Neurotoxins.
Figure 75-3 Diagrammatic representation of the structure and action of cholera toxin.
TABLE 75-2 Overview of the Primary Groups of E. coli That Cause Diarrhea in Humans
Attachment.
Figure 75-4 It appears that the presence of EHEC/VTEC strain O157:H7 has actually increased in recent years and was not simply overlooked before 1982. E. coli O157:H7 strains are closely related to a Shiga toxin–negative EPEC strain O55:H7. It is proposed that this EPEC strain O55:H7 became infected by a bacteriophage that encoded Shiga toxin (SLT); it is now recognized that more than 100 different E. coli serotypes can express Shiga toxin.
Invasion.
TABLE 75-3 Types of Enteric Infections
Figure 75-5 The invasion of Shigella and Salmonella into intestinal epithelial cells.
Figure 75-6 Wright’s stain of stool from a patient with shigellosis showing moderate numbers of polymorphonuclear cells.
Miscellaneous Virulence Factors.
Clinical Manifestations
Epidemiology
Institutional Settings
Traveler’s Diarrhea
Food- and Water-Borne Outbreaks
Immunocompromised Hosts
Etiologic Agents
Other Infections of the Gastrointestinal Tract
Esophagitis
Gastritis
Proctitis
Miscellaneous
TABLE 75-4 General Characteristics of the Common Agents of Enteric Infections
Laboratory Diagnosis of Gastrointestinal Tract Infections
Specimen Collection and Transport
General Comments
Stool Specimens for Bacterial Culture
Stool Specimens for Ova and Parasites
Stool Specimens for Viruses
Miscellaneous Specimen Types
Direct Detection of Agents of Gastroenteritis in Feces
Wet Mounts
Stains
Antigen Detection
Molecular Biologic Techniques
Culture of Fecal Material for Isolation of Etiologic Agents
Bacteria
Organisms for Routine Culture.
Routine Culture Methods.
Salmonella/Shigella.
Campylobacter.
Enrichment Broths.
Laboratory Diagnosis of Clostridium Difficile–associated Diarrhea
Figure 75-7 Colonies of a lactose-positive organism growing on xylose-lysine deoxycholate (XLD) agar (A) and Hektoen enteric (HE) agar (B). Colonies of Salmonella enteritidis (lactose-negative) growing on XLD (C) and HE agar (D). (Note how both agars detect H2S production.) Colonies of Shigella (lactose-negative) growing on XLD (E) and HE agar (F).
Case Study 75-1
Questions
Case Study 75-2
Questions
Bibliography
Chapter Review
Chapter 76 Skin, Soft Tissue, and Wound Infections
Objectives
General Considerations
Anatomy of the Skin
Function of the Skin
Prevalence, Etiology, and Pathogenesis
Figure 76-1 Diagram of the skin.
Box 76-1 Resident Microbial Flora of the Skin
Skin and Soft Tissue Infections
Infections of the Epidermis and Dermis
Infections in or around Hair Follicles
Infections in the Keratinized Layer of the Epidermis
Infections in the Deeper Layers of the Epidermis and Dermis
TABLE 76-1 Manifestations of Skin Infections
TABLE 76-2 Infections Involving Hair Follicles
Infections of the Subcutaneous Tissues
Figure 76-2 A, Viral maculopapular rash. B, Furuncle. C, Folliculitis caused by Staphylococcus aureus showing numerous pustules. D, Desquamation (shedding or scaling) of the skin resulting from scarlet fever caused by group A streptococci.
TABLE 76-3 Infections of the Epidermal and Dermal Layers of the Skin
Infections of the Muscle Fascia and Muscles
Necrotizing Fasciitis
Progressive Bacterial Synergistic Gangrene
Myositis
Figure 76-3 Erysipelas caused by group A streptococci.
Figure 76-4 Cellulitis.
Wound Infections
Postoperative Infections
Box 76-2 Organisms Producing Myositis or Other Muscle Pathology
Bites
Figure 76-5 Human bite infection.
Figure 76-6 Animal bite infection caused by Pasteurella spp.
Box 76-3 Organisms Encountered in Postoperative Wound Infections
Burns
Special Circumstances Regarding Skin and Soft Tissue Infections
Infections Related to Vascular and Neurologic Problems
Sinus Tracts and Fistulas
Figure 76-7 Sacral decubitus ulcer.
Figure 76-8 Actinomycosis. A, Note “lumpy jaw.” B, Side view. Note sinuses in skin of face and neck.
Systemic Infections and Skin Manifestations
Laboratory Diagnostic Procedures
Infections of the Epidermis and Dermis
Erysipeloid
Superficial Mycoses and Erythrasma
Erysipelas and Cellulitis
Figure 76-9 Bullae on the arm of a patient with Vibrio vulnificus sepsis.
Box 76-4 Organisms Involved in Systemic Infection with Cutaneous Lesions
Vesicles and Bullae
Infections of the Subcutaneous Tissues
Infections of the Muscle Fascia and Muscles
Wound Infections
Postoperative
Bites
Burns
Procedure 76-1
Semiquantitative Bacteriologic Culture of Tissue
Principle
Method
Chapter Review
Case Study 76-1
Questions
Case Study 76-2
Questions
Case Study 76-3
Figure 76-10 Demonstration of the swarming film of growth of Clostridium septicum at 24 hours (A) and Clostridium sporogenes (B).
Questions
Bibliography
Chapter 77 Normally Sterile Body Fluids, Bone and Bone Marrow, and Solid Tissues
Objectives
Specimens From Sterile Body Sites
Fluids
Pleural Fluid
Peritoneal Fluid
Figure 77-1 The location of the pleural space in relation to the parietal and visceral pleura and the rest of the respiratory tract.
TABLE 77-1 Microbiology Laboratory Body Fluid Collection Sites
Primary Peritonitis.
Figure 77-2 The abdominal cavity. The retroperitoneal and preperitoneal spaces are considered as extraperitoneal (outside) spaces.
TABLE 77-2 Pleural Fluid Effusion Characteristics
Secondary Peritonitis.
Peritoneal Dialysis Fluid
Pericardial Fluid
Joint Fluid
Box 77-1 Common Etiologic Agents of Pericarditis and Myocarditis
Viruses
Bacteria (relatively uncommon)
Fungi (relatively uncommon)
Parasites (relatively uncommon)
Box 77-2 Most Frequently Encountered Etiologic Agents of Infectious Arthritis
Bacterial
Fungal
Viral
Bone
Bone Marrow Aspiration or Biopsy
Bone Biopsy
Solid Tissues
Box 77-3 Infectious Agents in Tissue Requiring Special Media
Laboratory Diagnostic Procedures
Specimen Collection and Transport
Fluids and Aspirates
Bone
Tissue
Figure 77-3 Mincing a piece of tissue for culture using a sterile forceps and scissors. Note: Perform this procedure in a biosafety cabinet.
Specimen Processing, Direct Examination, and Culture
Fluids and Aspirates
Bone
Solid Tissue
Case Study 77-1
Questions
Bibliography
Chapter Review
Part VIII Clinical Laboratory Managementy
Chapter 78 Quality in the Clinical Microbiology Laboratory
Objectives
Quality Program
Specimen Collection and Transport
Standard Operating Procedure Manual
Personnel
Reference Laboratories
Patient Reports
Proficiency Testing (PT)
Performance Checks
Instruments
Commercially Prepared Media Exempt From QC
User-Prepared and Nonexempt, Commercially Prepared Media
Antimicrobial Susceptibility Tests
Stains and Reagents
Antisera
Kits
Maintenance of QC Records
Maintenance of Reference QC Stocks
Bacteriology
Mycology
Mycobacteriology
Virology
Parasitology
QA Program
Q-Probes
In-House QA Audits
Conducting a QA Audit
Continuous Daily Monitoring
Chapter Review
Box 78-1 QA (Quality Assurance) Audit on STAT (Immediately; derived from Latin statim) Turnaround Times
Background
Study Design
Results
Analysis
Conclusions
Recommendations
Bibliography
Chapter 79 Infection Control
Objectives
Incidence of HAI
Types of HAI
Urinary Tract Infections
Lung Infections
Surgical Site Infections
Central Line-Associated Bloodstream Infection
Emergence of Antibiotic-Resistant Microorganisms
Hospital Infection Control Programs
Role of the Microbiology Laboratory
Characterizing Strains Involved in an Outbreak
Preventing HAI
TABLE 79-1 Examples of Notifiable Infectious Conditions in Texas*
Box 79-1 Infection Control Measures for Standard Precautions
Surveillance Methods
TABLE 79-2 Transmission-Based Precautions
Chapter Review
Bibliography
Chapter 80 Sentinel Laboratory Response to Bioterrorism
Objectives
General Considerations
Biocrime
Government Laws and Regulations
Box 80-1 List of Select Agents*
Viruses
Bacteria
Rickettsiae
Toxins
Biosecurity
Laboratory Response Network
Role of the Sentinel Laboratory
Figure 80-1 Laboratory Network for Biological Terrorism.
TABLE 80-1 Algorithm for Sentinel Laboratories for Likely Bioterrorism Agents*
Figure 80-2 Colony of Bacillus anthracis.
Figure 80-3 Gram stain of Bacillus anthracis.
Box 80-2 Sentinel Level Clinical Microbiology Laboratory Guidelines*
Chapter Review
Bibliography
Glossary
Glossary
Index
Index
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T
U
V
W
X
Y
Z
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