Practical Guide to Diagnostic Parasitology

Lynne Shore Garcia is the director of LSG & Associates, a firm providing training, teaching, and consultation services for diagnostic medical parasitology and health care administration. A former manager of the UCLA Clinical Microbiology Laboratory, she is a sought-after speaker (nationally and internationally) and author of hundreds of articles, book chapters, and books including two ASM Press books, Clinical Laboratory Management, Second Edition and Diagnostic Medical Parasitology, Sixth Edition.

Garcia 3e TOC draft from mss

Preface 000

Acknowledgments 000

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 Hanscheid)

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 butschlii

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