Practical Guide to Diagnostic Parasitology

Preface
Acknowledgments

SECTION 1 Philosophy and Approach to Diagnostic Parasitology

• Neglected Tropical Diseases
• Why Perform Diagnostic Parasitology Testing?
• Travel
• Population Movements
• Control Issues
• Global Climate Change
• Epidemiologic Considerations
• Compromised Patients; Potential Sex Bias Regarding Infection Susceptibility, Aging
• Approach to Therapy
• Who Should Perform Diagnostic Parasitology Testing?
• Laboratory Personnel
• Nonlaboratory Personnel
• Where Should Diagnostic Parasitology Testing Be Performed?
• Inpatient Setting
• Outpatient or Referral Setting
• Decentralized Testing
• Physician Office Laboratories
• Over-the-Counter (Home Care) Testing
• Field Sites
• What Factors Should Precipitate Testing?
• Travel and Residence History
• Immune Status of the Patient
• Clinical Symptoms
• Documented Previous Infection
• Contact with Infected Individuals
• Potential Outbreak Testing
• Occupational Testing
• Therapeutic Failure
• What Testing Should Be Performed?
• Routine Tests
• Special Testing, Reference Laboratories
• Specialized Referral Test Options: DPDx and Other Sites
• Other (Nonmicrobiological) Testing
• What Factors Should Be Considered When Developing Test Menus?
• Physical Plant
• Client Base
• Customer Requirements and Perceived Levels of Service
• Personnel Availability and Level of Expertise
• Equipment
• Budget
• Risk Management Issues Associated with Stat Testing
• Primary Amebic Meningoencephalitis (PAM)
• Granulomatous Amebic Encephalitis and Amebic Keratitis
• Request for Blood Films
• Automated Instrumentation
• Patient Information
• Conventional Microscopy 

SECTION 2 Parasite Classification and Relevant Body Sites

• Protozoa (Intestinal)
• Amebae, Stramenopiles
• Flagellates
• Ciliates
• Apicomplexa (Including Coccodia)
• Microsporidia (Now Classified with the Fungi)
• Protozoa (Other Body Sites)
• Amebae
• Flagellates
• Apicomplexa (Including Coccidia)
• Microsporidia (Now Classified with the Fungi)
• Protozoa (Blood and Tissue)
• Apicomplexa (Including Sporozoa)
• Flagellates
• Leishmaniae
• Trypanosomes
• Nematodes (Intestinal)
• Nematodes (Tissue)
• Nematodes (Blood and Tissue)
• Cestodes (Intestinal)
• Cestodes (Tissue)
• Trematodes (Intestinal)
• Trematodes (Liver and Lungs)
• Trematodes (Blood)
• Pentastomids
• Acanthocephala
• Table 2.1 Classification of human parasites
• Table 2.2 Cosmopolitan distribution of common parasitic infections (North America, Mexico, Central America, South America, Europe, Africa, Asia, and Oceania)
• Table 2.3 Body sites and possible parasites recovered (trophozoites, cysts, oocysts, spores, adults, larvae, eggs, amastigotes, and trypomastigotes) 

SECTION 3 Collection Options

• Safety
• Collection of Fresh Stool Specimens
• Collection Method
• Number of Specimens To Be Collected
• Standard Approach
• Different Approaches
• Collection Times
• Posttherapy Collection
• Specimen Type, Stability, and Need for Preservation
• Preservation of Stool Specimens
• Overview of Preservatives
• Formalin
• Sodium Acetate-Acetic Acid-Formalin (SAF)
• Schaudinn’s Fluid
• Schaudinn’s Fluid containing PVA (mercury base)
• Schaudinn’s Fluid containing PVA (copper base, zinc base)
• Single-Vial Collection Systems (Other Than SAF)
• Universal Fixative (TOTAL-FIX)
• Quality Control for Preservatives
• Procedure Notes for Use of Preservatives (Stool Fixative Collection Vials)
• Procedure Limitations for Use of Preservatives (Stool Fixative Collection Vials)
• Collection of Blood
• Collection and Processing
• Stat Test Requests and Risk Management Issues
• Collection of Specimens from Other Body Sites
• Table 3.1 Fecal specimens for parasites: options for collection and processinga
• Table 3.2 Approaches to stool parasitology: test ordering
• Table 3.3 Preservatives and procedures commonly used in diagnostic parasitology (stool specimens)
• Table 3.4 Advantages of thin and thick blood films
• Table 3.5 Advantages and disadvantages of buffy coat films
• Table 3.6 Potential problems of using EDTA anticoagulant for the preparation of thin and thick blood films
• Table 3.7 Body sites and possible parasites recovered (trophozoites, cysts, oocysts, spores, adults, larvae, eggs, amastigotes, and trypomastigotes) 

SECTION 4 Specimen Test Options: Routine Diagnostic Methods and Body Sites

• Ova and Parasite Examination of Stool Specimens
• Other Diagnostic Methods for Stool Specimens
• Culture of Larval-Stage Nematodes
• Estimation of Worm Burdens through Egg Counts
• Hatching Test for Schistosome Eggs
• Screening Stool Samples for Recovery of a Tapeworm Scolex
• Testing of Other Intestinal Tract Specimens
• Examination for Pinworm
• Sigmoidoscopy Material
• Duodenal Drainage Material
• Duodenal Capsule Technique (Entero-Test)
• Urogenital Tract Specimens
• Sputum
• Aspirates
• Biopsy Specimens
• Blood
• Thin Blood Films
• Thick Blood Films
• Blood Staining Methods
• Buffy Coat Films
• QBC Microhematocrit Centrifugation Method
• Knott Concentration
• Membrane Filtration Technique
• Culture Methods
• Animal Inoculation and Xenodiagnosis
• Antibody and Antigen Detection
• Antibody Detection
• Antigen Detection, Nucleic Acid-Based Tests, and Molecular Panels
• Intradermal Tests
• Table 4.1 Body site, procedures and specimens, recommended methods and relevant parasites, and comments
• Table 4.2 Serologic, antigen, and probe tests used in the diagnosis of parasitic infections 

SECTION 5 Specific Test Procedures and Algorithms

• Microscopy
• CALIBRATION OF THE MICROSCOPE
• Ova and Parasite Examination
• DIRECT WET FECAL SMEAR
• Concentration (Sedimentation and Flotation)
• SEDIMENTATION CONCENTRATION (Formalin-Ethyl Acetate)
• SEDIMENTATION CONCENTRATION USING THE UNIVERSAL FIXATIVE (TOTAL-FIX)
• FLOTATION CONCENTRATION (Zinc Sulfate)
• Permanent Stained Smear
• PREPARATION OF MATERIAL FOR STAINING
• Fresh Material
• Preserved Material Containing PVA
• SAF-Preserved Material
• Universal Fixative (Total-Fix) Preserved Material
• Alternative Method for Smear Preparation Directly from Vial
• (Total-Fix)
• Stains Used in the Permanent Stained Smear
• TRICHROME STAIN (Wheatley’s Method)
• IRON HEMATOXYLIN STAIN (Spencer-Monroe Method)
• IRON HEMATOXYLIN STAIN (Tompkins-Miller Method)
• MODIFIED IRON HEMATOXYLIN STAIN (Incorporating the Carbol Fuchsin Step)
• POLYCHROME IV STAIN
• CHLORAZOL BLACK E STAIN
• Specialized Stains for Coccidia and Microsporidia
• KINYOUN’S ACID-FAST STAIN (Cold Method)
• MODIFIED ZIEHL-NEELSEN ACID-FAST STAIN (Hot Method)
• CARBOL FUCHSIN NEGATIVE STAIN FOR CRYPTOSPORIDIUM (W. L. Current)
• RAPID SAFRANIN METHOD FOR CRYPTOSPORIDIUM (D. Baxby)
• RAPID SAFRANIN METHOD FOR CYCLOSPORA, USING A MICROWAVE OVEN (Govinda Visvesvara)
• AURAMINE O STAIN FOR COCCIDIA (Thomas Hänscheid)
• MODIFIED TRICHROME STAIN FOR MICROSPORIDIA (Weber, Green Counterstain)
• MODIFIED TRICHROME STAIN FOR MICROSPORIDIA (Ryan, Blue Counterstain)
• MODIFIED TRICHROME STAIN FOR MICROSPORIDIA (Evelyn Kokoskin, Hot Method)
• Fecal Immunoassays for Intestinal Protozoa
• Entamoeba histolytica
• Cryptosporidium spp.
• Giardia lamblia
• Kits under Development
• Comments on the Performance of Fecal Immunoassays
• Enzyme Immunoassays (Antigen Detection, No Centrifugation Recommended)
• Fluorescence (Visual Identification of the Organisms, Centrifugation Recommended)
• Lateral-Flow Cartridges (Antigen Detection, No Centrifugation Recommended)
• Larval Nematode Culture
• HARADA-MORI FILTER PAPER STRIP CULTURE
• BAERMANN CONCENTRATION
• AGAR PLATE CULTURE FOR STRONGYLOIDES STERCORALIS
• Other Methods for Gastrointestinal Tract Specimens
• EXAMINATION FOR PINWORM (Cellulose Tape Preparations)
• SIGMOIDOSCOPY SPECIMENS (Direct Wet Smear)
• SIGMOIDOSCOPY SPECIMENS (Permanent Stained Smear)
• DUODENAL ASPIRATES
• Methods for Urogenital Tract Specimens
• RECEIPT OF DRY SMEARS
• DIRECT SALINE MOUNT
• PERMANENT STAINED SMEAR
• URINE CONCENTRATION (Centrifugation)
• URINE CONCENTRATION (Nuclepore Membrane Filter)
• Preparation of Blood Films
• THIN BLOOD FILMS
• THICK BLOOD FILMS
• COMBINATION THICK-THIN BLOOD FILMS
• RISK MANAGEMENT ISSUES ASSOCIATED WITH BLOOD FILMS
• USE OF A REFERENCE LABORATORY FOR PARASITE BLOOD DIAGNOSTIC TESTING
• BLOOD FILM REPORTING WITH ADDITIONAL REPORT COMMENTS
• BUFFY COAT BLOOD FILMS
• Blood Stains
• STAIN OPTIONS
• GIEMSA STAIN
• Blood Concentration
• BUFFY COAT CONCENTRATION
• KNOTT CONCENTRATION
• MEMBRANE FILTRATION CONCENTRATION
• Algorithm 5.1 Procedure for processing fresh stool for the O&P examination
• Algorithm 5.2 Procedure for processing liquid specimens for the O&P examination
• Algorithm 5.3 Procedure for processing preserved stool for the O&P examination—two-vial collection kit
• Algorithm 5.4 Procedure for processing SAF-preserved stool for the O&P examination
• Algorithm 5.5 Procedure for the use of Total-Fix (Universal Fixative, single vial system) (Alternate Method for Smear Preparation Directly from Vial)
• Algorithm 5.6 Use of various fixatives and their recommended stains
• Algorithm 5.7 Ordering algorithm for laboratory examination for intestinal parasites
• Algorithm 5.8 Procedure for processing blood specimens for examination
• Table 5.1 Body site, specimen, and recommended stain(s)
• Table 5.2 Approaches to stool parasitology: test ordering
• Table 5.3 Laboratory test reports: optional comments
• Table 5.4 Parasitemia determined from conventional light microscopy: clinical correlation 

Section 6 Commonly Asked Questions about Diagnostic Parasitology

• Stool Parasitology
• Specimen Collection
• Intestinal Tract
• Fixatives
• Specimen Processing
• O&P Exam
• Diagnostic Methods
• Direct Wet Examinations
• Concentrations
• Permanent Stains
• Stool Immunoassay Options
• Molecular Test Panels (FDA Approved)
• APTIMA Trichomonas vaginalis Assay
• Affirm VPIII Microbial Identification Test
• Cepheid Xpert TV Assay for Trichomonas vaginalis from men and women
• BD MAX Enteric Parasite Panel
• BioFire FilmArray Gastrointestinal panel
• Luminex (VERIGENE II GI Flex Assay, includes Parasites)
• Other Pending Molecular Tests
• Organism Identification
• Protozoa
• Helminths
• Reporting
• Organism Identification
• Quantitation
• Proficiency Testing
• Wet Preparations
• Permanent Stained Smears
• Tissues or Fluids
• Blood
• Specimen Collection
• Specimen Processing
• Diagnostic Methods
• Organism Identification
• Reporting
• Proficiency Testing
• General Questions 

SECTION 7 Parasite Identification

• Protozoa
• Amebae (Intestinal)
• Entamoeba histolytica
• Entamoeba histolytica/Entamoeba dispar
• Comments on Entamoeba moshkovskii and Entamoeba bangladeshi
• Entamoeba bangladeshi
• Entamoeba hartmanni
• Entamoeba coli
• Entamoeba gingivalis
• Entamoeba polecki
• Endolimax nana
• Iodamoeba bütschlii
• Blastocystis spp. (formerly Blastocystis hominis)
• Flagellates (Intestinal)
• Giardia lamblia (G. duodenalis, G. intestinalis)
• Dientamoeba fragilis
• Chilomastix mesnili
• Pentatrichomonas hominis
• Enteromonas hominis, Retortamonas intestinalis
• Ciliates (Intestinal)
• Balantidium coli
• Apicomplexa (Intestinal)
• Cryptosporidium spp.
• Coccidia (Intestinal)
• Cyclospora cayetanensis
• Cystoisospora (Isospora) belli
• Microsporidia (Intestinal)
• Enterocytozoon bieneusi
• Encephalitozoon intestinalis, Encephalitozoon spp.
• Sporozoa (Blood and Tissue)
• Plasmodium vivax
• Plasmodium falciparum
• Plasmodium malariae
• Plasmodium ovale wallickeri, Plasmodium ovale curtisi
• Plasmodium knowlesi
• Malaria: Key Diagnostic Points
• Babesia spp. (B. microti, B. duncani, B. divergens, B. venatorum)
• Toxoplasma gondii
• Flagellates (Blood and Tissue)
• Leishmania spp.
• Trypanosoma brucei gambiense (West), T. brucei rhodesiense (East)
• Trypanosoma cruzi
• Amebae (Other Body Sites)
• Naegleria fowleri
• Acanthamoeba spp., Balamuthia mandrillaris, Sappinia diploidea
• Flagellates (Other Body Sites)
• Trichomonas vaginalis
• NEMATODES
• Intestinal
• Ascaris lumbricoides
• Trichuris trichiura;General Comments on Capillaria philippinensis
• Necator americanus, Ancylostoma duodenale, A. ceylanicum (Hookworms); General comments on Trichostrongylus spp.
• Strongyloides stercoralis
• Enterobius vermicularis
• Tissue
• Ancylostoma braziliense, Ancylostoma caninum, Uncinaria stenocephala (Dog and Cat Hookworms)
• Toxocara canis, Toxocara cati (Dog and Cat Ascarid Worms)
• Dracunculus medinensis
• Trichinella spiralis
• Blood and Tissue
• Filarial Worms
• CESTODES
• Intestinal
• Taenia saginata
• Taenia solium
• Diphyllobothrium latum
• Hymenolepis (Rodentolepis) nana
• Hymenolepis diminuta
• Dipylidium caninum
• Tissue
• Echinococcus granulosus, E. multilocularis, E. vogeli, E. oligarthrus
• TREMATODES
• Intestinal
• Fasciolopsis buski
• Liver and Lungs
• Paragonimus westermani, Paragonimus mexicanus, Paragonimus kellicotti
• Fasciola hepatica
• Clonorchis sinensis (Chinese liver fluke) (Opisthorchis sinensis)
• Blood
• Schistosoma spp. (S. mansoni, S. haematobium, S. japonicum, S. mekongi, S. malayensis, S. intercalatum) 

SECTION 8 Common Problems in Parasite Identification

• Table 8.1 Entamoeba spp., trophozoites versus macrophages
• Table 8.2 Entamoeba spp., cysts versus polymorphonuclear leukocytes
• Table 8.3 Entamoeba histolytica versus Entamoeba coli precysts and cysts
• Table 8.4 Endolimax nana versus Dientamoeba fragilis 

SECTION 9 Identification Aids

• Diagnostic Considerations
• Table 9.1 Rapid diagnostic procedures
• Table 9.2 Diagnostic characteristics for organisms in wet mounts (direct or concentration sediment)
• Table 9.3 Diagnostic characteristics for organisms in permanent stained smears
• Key 9.1 Identification of intestinal amebae (permanent stained smear)
• Key 9.2 Identification of intestinal flagellates
• Key 9.3 Identification of helminth eggs
• Key 9.4 Identification of microfilariae
• Protozoa
• Table 9.4 Intestinal protozoa: trophozoites of common amebae
• Table 9.5 Intestinal protozoa: cysts of common amebae
• Table 9.6 Intestinal protozoa: trophozoites of less common amebae
• Table 9.7 Intestinal protozoa: cysts of less common amebae
• Table 9.8 Morphologic criteria used to identifyBlastocystisspp.
• Table 9.9 Intestinal protozoa: trophozoites of flagellates
• Table 9.10 Intestinal protozoa: cysts of flagellates
• Table 9.11 Intestinal protozoa: ciliates
• Table 9.12 Apicomplexa
• Table 9.13 Microsporidia (related to the fungi), general information
• Table 9.14 Microsporidia: recommended diagnostic techniques
• Table 9.15 Comparison of Naegleria fowleri, Acanthamoeba spp., Balamuthia mandrillaris, and Sappinia diploidea
• Table 9.16 Characteristics of Trichomonas vaginalis
• Table 9.17 Key characteristics of intestinal tract/urogenital system protozoa
• Helminths
• Table 9.18 Normal life spans of the most common intestinal nematodes
• Table 9.19 Characteristics of the most common intestinal nematodes
• Table 9.20 Tissue nematodes
• Table 9.21 Trichinella spiralis: life cycle stages and clinical conditions
• Table 9.22 Characteristics of human microfilariae
• Table 9.23 Characteristics of cestode parasites (intestinal)
• Table 9.24 Tissue cestodes
• Table 9.25 Characteristics of intestinal trematodes
• Table 9.26 Characteristics of liver and lung trematodes
• Table 9.27 Human paragonimiasis
• Table 9.28 Characteristics of blood trematodes
• Table 9.29 Key characteristics of helminths
• Blood Parasites
• Table 9.30 Malaria characteristics with fresh blood or blood collected using EDTA with no extended lag time
• Table 9.31 Potential problems using EDTA anticoagulant for the preparation of thin and thick blood films
• Table 9.32 Plasmodia in Giemsa-stained thin blood smears
• Table 9.33 Relevant issues for handling requests for blood parasite infections
• Table 9.34 Features of human leishmanial infectionsa
• Table 9.35 Characteristics of American trypanosomiasis
• Table 9.36 Characteristics of East and West African trypanosomiasis
• Table 9.37 Key characteristics of blood parasites 

Index

In the 21st century the field of diagnostic medical parasitology continues to see dramatic changes, including newly recognized pathogens and the changing endemicity and classification of familiar organisms; neglected tropical diseases and the impact of global climate change; and new methodologies and risk management issues. This classic clinical laboratory parasitology reference, now in its third edition, has been extensively revised and updated in a new full-color format. Still organized to provide maximum help to the user, particularly from the bench perspective, every section has been expanded with new images and discussion.

Specimen collection, preservation, and testing options are thoroughly discussed, from the routine ova and parasite examination to blood films, fecal immunoassays, and the newer molecular test panels. Specific test procedures, laboratory methods and reagents, and algorithms are provided. The ever-helpful “FAQ” section of commonly asked questions now offers expanded information on stool specimen fixatives and testing, thorough coverage of new techniques, and advice on reporting and commenting on results.

The heart of the Guide, covering identification of individual pathogens, has been expanded with more discussion and comparison of organisms and dozens of new color images. An entirely new section has been added that uses extensive figures and new tables to illustrate common problems with differentiating organisms from one another and from possible microscopic artifacts. The final section has been reorganized to include identification keys and dozens of tables summarizing organism characteristics to assist the bench microbiologist with routine diagnostic testing methods.