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.
Table of Contents
Preface xix
About the Author xxiii
Section 1 Philosophy and Approach to Diagnostic Parasitology 1
Neglected Tropical Diseases 2
Why Perform Diagnostic Parasitology Testing? 3
Travel 3
Population Movements 3
Control Issues 4
Climate Change 4
Epidemiologic Considerations 4
Compromised Patients; Potential Sex Bias Regarding Infection Susceptibility; Aging 4
Approach to Therapy 5
Who Should Perform Diagnostic Parasitology Testing? 5
Laboratory Personnel 5
Nonlaboratory Personnel 5
Where Should Diagnostic Parasitology Testing Be Performed? 6
Inpatient Setting 6
Outpatient or Referral Setting 6
Decentralized Testing 6
Physician Office Laboratories 6
Over-the-Counter (Home Care) Testing 7
Field Sites 7
What Factors Should Precipitate Testing? 7
Travel and Residence History 7
Immune Status of the Patient 7
Clinical Symptoms 7
Documented Previous Infection 7
Contact with Infected Individuals 8
Potential Outbreak Testing 8
Occupational Testing 8
Therapeutic Failure 8
What Testing Should Be Performed? 8
Routine Tests 8
Special Testing and Reference Laboratories 9
Specialized Referral Test Options - DPDx and Other Sites 9
Other (Nonmicrobiological) Testing 10
What Factors Should Be Considered in Development of Test Menus? 10
Physical Plant 10
Client Base 10
Customer Requirements and Perceived Levels of Service 10
Personnel Availability and Level of Expertise 10
Equipment 10
Budget 11
Risk Management Issues Associated with Stat Testing 11
Primary Amebic Meningoencephalitis 11
Granulomatous Amebic Encephalitis and Amebic Keratitis 14
Request for Blood Films 15
Automated Instrumentation 15
Patient Information 16
Conventional Microscopy 16
Table 1.1 Common features of the neglected tropical diseases 18
Section 2 Parasite Classification and Relevant Body Sites 19
Protozoa (Intestinal) 20
Amebae, Stramenopiles 20
Flagellates 22
Ciliates 22
Apicomplexa (Including Coccidia) 23
Microsporidia (Now Classified with the Fungi) 23
Protozoa (Other Body Sites) 24
Amebae 24
Flagellates 24
Apicomplexa (Including Coccidia) 24
Microsporidia (Now Classified with the Fungi) 25
Protozoa (Blood and Tissue) 26
Apicomplexa (Including Sporozoa) 26
Flagellates 26
Nematodes (Intestinal) 29
Nematodes (Tissue) 29
Nematodes (Blood and Tissue) 30
Cestodes (Intestinal) 31
Cestodes (Tissue) 32
Trematodes (Intestinal) 33
Trematodes (Liver and Lungs) 33
Trematodes (Blood) 35
Pentastomids 35
Acanthocephala 36
Table 2.1 Classification of human parasites 37
Table 2.2 Cosmopolitan distribution of common parasitic infections 39
Table 2.3 Body sites and possible parasites recovered 40
Section 3 Collection Options 43
Safety 44
Collection of Fresh Stool Specimens 44
Collection Method 44
Number of Specimens To Be Collected 45
Collection Times 47
Posttherapy Collection 47
Specimen Type, Stability, and Need for Preservation 47
Preservation of Stool Specimens 48
Overview of Preservatives 48
Formalin 49
Sodium Acetate-Acetic Acid-Formalin (SAF) 50
Schaudinn’s Fluid 51
Schaudinn’s Fluid containing PVA (Mercury Base) 52
Schaudinn’s Fluid containing PVA (Copper Base, Zinc Base) 52
Single-Vial Collection Systems (Other than SAF) 53
Universal Fixative (Total-Fix) 54
Quality Control for Preservatives 54
Procedure Notes for Use of Preservatives (Stool Fixative Collection Vials) 55
Procedure Limitations for Use of Preservatives (Stool Fixative Collection Vials) 55
Collection of Blood 56
Collection and Processing 56
Stat Test Requests and Risk Management Issues 57
Collection of Specimens from Other Body Sites 57
Table 3.1 Fecal specimens for parasites: options for collection and processing 58
Table 3.2 Approaches to stool parasitology: test ordering 61
Table 3.3 Preservatives and procedures commonly used in diagnostic parasitology (stool specimens) 62
Table 3.4 Advantages of thin and thick blood films 64
Table 3.5 Advantages and disadvantages of buffy coat films 64
Table 3.6 Potential problems of using EDTA anticoagulant for the preparation of thin and thick blood films 65
Table 3.7 Body sites and possible parasites recovered (trophozoites, cysts, oocysts, spores, adults, larvae, eggs, amastigotes, and trypomastigotes) 66
Section 4 Specimen Test Options: Routine Diagnostic Methods and Body Sites 69
Ova and Parasite Examination of Stool Specimens 70
Other Diagnostic Methods for Stool Specimens 72
Culture of Larval-Stage Nematodes 72
Estimation of Worm Burdens through Egg Counts 73
Hatching Test for Schistosome Eggs 73
Screening Stool Samples for Recovery of a Tapeworm Scolex 74
Testing of Other Intestinal Tract Specimens 74
Examination for Pinworm 74
Sigmoidoscopy Material 75
Duodenal Drainage Material 76
Duodenal Capsule Technique (Entero-Test) 76
Urogenital Tract Specimens 77
Sputum 77
Aspirates 78
Biopsy Specimens 78
Blood 79
Thin Blood Films 79
Thick Blood Films 80
Blood Staining Methods 80
Buffy Coat Films 80
QBC Microhematocrit Centrifugation Method 80
Knott Concentration 81
Membrane Filtration Technique 81
Culture Methods 81
Animal Inoculation and Xenodiagnosis 82
Antibody and Antigen Detection 82
Antibody Detection 82
Antigen Detection, Nucleic Acid-Based Tests, and Molecular Panels 84
Intradermal Tests 84
UV Autofluorescence 84
Table 4.1 Body sites, procedures and specimens, recommended methods and relevant parasites, and comments 86
Table 4.2 Serologic, antigen, and probe tests used in the diagnosis of parasitic infections 94
Section 5 Specific Test Procedures and Algorithms 95
Microscopy 96
CALIBRATION OF THE MICROSCOPE 96
Ova and Parasite Examination 99
DIRECT WET FECAL SMEAR 99
CONCENTRATION (Sedimentation and Flotation) 104
SEDIMENTATION CONCENTRATION (Formalin-Ethyl Acetate) 105
SEDIMENTATION CONCENTRATION USING THE UNIVERSAL FIXATIVE (Total-Fix) 109
FLOTATION CONCENTRATION (Zinc Sulfate) 110
PERMANENT STAINED SMEAR 114
Stains Used in the Permanent Stained Smear 116
TRICHROME STAIN (Wheatley’s Method) 116
IRON HEMATOXYLIN STAIN (Spencer-Monroe Method) 122
IRON HEMATOXYLIN STAIN (Tompkins-Miller Method) 127
MODIFIED IRON HEMATOXYLIN STAIN (Incorporating the Carbol Fuchsin Step) 128
CHLORAZOL BLACK E STAIN 131
Specialized Stains for Coccidia and Microsporidia 132
KINYOUN’S ACID-FAST STAIN (Cold Method) 132
MODIFIED ZIEHL-NEELSEN ACID-FAST STAIN (Hot Method) 136
CARBOL FUCHSIN NEGATIVE STAIN FOR CRYPTOSPORIDIUM (W. L. Current) 139
RAPID SAFRANIN METHOD FOR CRYPTOSPORIDIUM (D. Baxby) 139
RAPID SAFRANIN METHOD FOR CYCLOSPORA, USING A MICROWAVE OVEN (Govinda Visvesvara) 139
AURAMINE O STAIN FOR APLICOMPLEXA (INCLUDING COCCIDIA) (Thomas Hänscheid) 140
MODIFIED TRICHROME STAIN FOR MICROSPORIDIA (Weber, Green Counterstain) 144
MODIFIED TRICHROME STAIN FOR MICROSPORIDIA (Ryan, Blue Counterstain) 146
MODIFIED TRICHROME STAIN FOR MICROSPORIDIA (Evelyn Kokoskin, Hot Method) 149
Fecal Immunoassays for Intestinal Protozoa 150
ENTAMOEBA HISTOLYTICA 150
CRYPTOSPORIDIUM SPP. 151
GIARDIA LAMBLIA 151
KITS UNDER DEVELOPMENT 151
COMMENTS ON THE PERFORMANCE OF FECAL IMMUNOASSAYS 151
ENZYME IMMUNOASSAYS (Antigen Detection, No Centrifugation Recommended) 152
FLUORESCENCE (Visual Identification of the Organisms, Centrifugation Recommended) 152
LATERAL-FLOW CARTRIDGES (Antigen Detection, No Centrifugation Recommended) 153
Larval Nematode Culture 153
HARADA-MORI FILTER PAPER STRIP CULTURE 153
BAERMANN CONCENTRATION 155
AGAR PLATE CULTURE FOR STRONGYLOIDES STERCORALIS 158
Other Methods for Gastrointestinal Tract Specimens 161
EXAMINATION FOR PINWORM (Cellulose Tape Preparations) 161
SIGMOIDOSCOPY SPECIMENS (Direct Wet Smear) 163
SIGMOIDOSCOPY SPECIMENS (Permanent Stained Smear) 166
DUODENAL ASPIRATES 168
Methods for Urogenital Tract Specimens 171
RECEIPT OF DRY SMEARS 171
DIRECT SALINE MOUNT 171
PERMANENT STAINED SMEAR 174
URINE CONCENTRATION (Centrifugation) 176
URINE CONCENTRATION (Nuclepore Membrane Filter) 179
Preparation of Blood Films 181
THIN BLOOD FILMS 182
THICK BLOOD FILMS 184
COMBINATION THICK-THIN BLOOD FILMS 186
RISK MANAGEMENT ISSUES ASSOCIATED WITH BLOOD FILMS 187
USE OF A REFERENCE LABORATORY FOR PARASITE BLOOD DIAGNOSTIC TESTING 188
BLOOD FILM REPORTING WITH ADDITIONAL REPORT COMMENTS 189
BUFFY COAT BLOOD FILMS 189
Blood Stains 190
STAIN OPTIONS 190
GIEMSA STAIN 190
Blood Concentration 194
BUFFY COAT CONCENTRATION 194
KNOTT CONCENTRATION 195
MEMBRANE FILTRATION CONCENTRATION 197
Algorithm 5.1 Procedure for processing fresh stool for the O&P examination 201
Algorithm 5.2 Procedure for processing liquid specimens for the O&P examination 202
Algorithm 5.3 Procedure for processing preserved stool for the O&P examination - two-vial collection kit 203
Algorithm 5.4 Procedure for processing SAF-preserved stool for the O&P examination 204
Algorithm 5.5 Procedure for the use of Total-Fix (universal fixative, single-vial system) (this fixative contains no mercury, no PVA, and no formalin) 205
ALTERNATE METHOD FOR SMEAR PREPARATION DIRECTLY FROM VIAL 206
Algorithm 5.6 Use of various fixatives and their recommended stains 207
Algorithm 5.7 Ordering algorithm for laboratory examination for intestinal parasites 208
Algorithm 5.8 Procedure for processing blood specimens for examination 209
Table 5.1 Body sites, specimens, and recommended stains 210
Table 5.2 Approaches to stool parasitology: test ordering 214
Table 5.3 Laboratory test reports: notes and optional comments 215
Table 5.4 Parasitemia determined from conventional light microscopy: clinical correlation 216
Section 6 Commonly Asked Questions about Diagnostic Parasitology 217
Stool Parasitology 218
Specimen Collection 218
Specimen Processing 221
Diagnostic Methods 222
Stool Immunoassay Options 227
MOLECULAR TEST PANELS (FDA CLEARED) 229
A. APTIMA Trichomonas vaginalis Assay 230
B. Affirm VPIII Microbial Identification Test 231
C. Cepheid Xpert TV Assay for Trichomonas vaginalis from Men and Women 232
D. BD MAX Enteric Parasite Panel 233
E. BioFire FilmArray Gastrointestinal Panel 234
F. Luminex (Verigene II GI Flex Assay; Includes Parasites) 236
G. Other Pending Molecular Tests 238
Organism Identification 238
Reporting 242
Proficiency Testing 245
Tissues or Fluids 245
Blood 246
Specimen Collection 246
Specimen Processing 248
Diagnostic Methods 249
Organism Identification 254
Reporting 256
Proficiency Testing 257
General Questions 258
Section 7 Parasite Identification 265
Protozoa
Amebae (Intestinal) 266
Entamoeba histolytica 266
Entamoeba histolytica/Entamoeba dispar 270
Comments on Entamoeba moshkovskii and Entamoeba bangladeshi 273
Entamoeba bangladeshi 274
Entamoeba hartmanni 275
Entamoeba coli 278
Entamoeba gingivalis 281
Entamoeba polecki 283
Endolimax nana 284
Iodamoeba bütschlii 287
Blastocystis spp. (formerly Blastocystis hominis) 290
Flagellates (Intestinal) Giardia lamblia (G. duodenalis, G. intestinalis) 295
Dientamoeba fragilis 298
Chilomastix mesnili 302
Pentatrichomonas hominis 304
Enteromonas hominis, Retortamonas intestinalis 307
Ciliates (Intestinal) 310
Balantidium coli 310
Apicomplexa (Intestinal) 315
Cryptosporidium spp. 315
Coccidia (Intestinal) 319
Cyclospora cayetanensis 319
Cystoisospora (formerly Isospora) belli 323
Microsporidia (Intestinal) 326
Enterocytozoon bieneusi 326
Encephalitozoon intestinalis, Encephalitozoon spp. 329
Sporozoa (Blood and Tissue) 334
Plasmodium vivax 334
Plasmodium falciparum 339
Plasmodium malariae 343
Plasmodium ovale wallickeri, Plasmodium ovale curtisi 346
Plasmodium knowlesi 350
Malaria 353
Babesia spp. (Babesia microti, B. duncani, B. divergens, B. venatorum) 355
Toxoplasma gondii 359
Flagellates (Blood and Tissue) 368
Leishmania spp. 368
Trypanosoma brucei gambiense (West), T. brucei rhodesiense (East) 372
Trypanosoma cruzi 376
Amebae (Other Body Sites) 381
Naegleria fowleri 381
Acanthamoeba spp., Balamuthia mandrillaris, Sappinia diploidea 385
Flagellates (Other Body Sites) 389
Trichomonas vaginalis 389
NEMATODES 392
Intestinal 392
Ascaris lumbricoides 392
Trichuris trichiura, Capillaria philippinensis 395
Necator americanus, Ancylostoma duodenale, Ancylostoma ceylanicum (Hookworms); Trichostrongylus spp. 398
Strongyloides stercoralis 401
Enterobius vermicularis 404
Tissue 407
Ancylostoma braziliense, Ancylostoma caninum, Uncinaria stenocephala (Dog and Cat Hookworms) 407
Toxocara canis, Toxocara cati (Dog and Cat Ascarid Worms) 410
Dracunculus medinensis 413
Trichinella spiralis 416
Blood and Tissue 419
Filarial Worms 419
CESTODES 425
Intestinal 425
Taenia saginata 425
Taenia solium 428
Diphyllobothrium latum 432
Hymenolepis (Rodentolepis) nana 435
Hymenolepis diminuta 438
Dipylidium caninum 440
Tissue 443
Echinococcus granulosus, Echinococcus multilocularis, Echinococcus vogeli,
Echinococcus oligarthrus 443
TREMATODES 448
Intestinal 448
Fasciolopsis buski 448
Liver and Lungs 451
Paragonimus westermani, Paragonimus mexicanus, Paragonimus kellicotti 451
Fasciola hepatica 454
Clonorchis (Opisthorchis) sinensis (Chinese liver fluke) 457
Blood 460
Schistosoma spp. (Schistosoma mansoni, S. haematobium, S. japonicum,
S. mekongi, S. malayensis, S. intercalatum) 460
Section 8 Common Problems in Parasite Identification 465
Table 8.1 Entamoeba sp. trophozoites versus macrophages 486
Table 8.2 Entamoeba sp. cysts versus PMNs 486
Table 8.3 Entamoeba histolytica versus Entamoeba coli precysts and cysts 487
Table 8.4 Endolimax nana versus Dientamoeba fragilis 488
Section 9 Identification Aids 489
DIAGNOSTIC CONSIDERATIONS 490
Table 9.1 Rapid diagnostic procedures 490
Table 9.2 Diagnostic characteristics for organisms in wet mounts (direct or concentration sediment) 491
Table 9.3 Diagnostic characteristics for organisms in permanent stained smears (e.g., Wheatley’s trichrome, iron-hematoxylin) 491
Identification Key 9.1 Identification of intestinal amebae (permanent stained smear) 491
Identification Key 9.2 Identification of intestinal flagellates 492
Identification Key 9.3 Identification of helminth eggs 493
Identification Key 9.4 Identification of microfilariae 494
PROTOZOA 495
Table 9.4 Intestinal protozoa: trophozoites of common amebae 495
Table 9.5 Intestinal protozoa: cysts of common amebae 497
Table 9.6 Intestinal protozoa: trophozoites of less common amebae 499
Table 9.7 Intestinal protozoa: cysts of less common amebae 499
Table 9.8 Morphologic criteria used to identify Blastocystis spp. 500
Table 9.9 Intestinal protozoa: trophozoites of flagellates 501
Table 9.10 Intestinal protozoa: cysts of flagellates 502
Table 9.11 Intestinal protozoa: ciliate (Balantidium coli) 503
Table 9.12 Apicomplexa 503
Table 9.13 Microsporidia (related to the Fungi): general information 504
Table 9.14 Microsporidia: recommended diagnostic techniques 505
Table 9.15 Comparison of Naegleria fowleri, Acanthamoeba spp., Balamuthia mandrillaris, and Sappinia diploidea 506
Table 9.16 Characteristics of Trichomonas vaginalis 507
Table 9.17 Key characteristics of intestinal tract and urogenital system protozoa 508
HELMINTHS 512
Table 9.18 Normal life spans of the most common intestinal nematodes 512
Table 9.19 Characteristics of the most common intestinal nematodes 513
Table 9.20 Tissue nematodes 515
Table 9.21 Trichinella spiralis: life cycle stages and clinical conditions 517
Table 9.22 Characteristics of human microfilariae 518
Table 9.23 Characteristics of cestode parasites (intestinal) 519
Table 9.24 Tissue cestodes 521
Table 9.25 Characteristics of intestinal trematodes 523
Table 9.26 Characteristics of liver and lung trematodes 524
Table 9.27 Human paragonimiasis 526
Table 9.28 Characteristics of blood trematodes 527
Table 9.29 Key characteristics of helminths 528
BLOOD PARASITES 532
Table 9.30 Malaria characteristics with fresh blood or EDTA-blood 532
Table 9.31 Potential problems with using EDTA anticoagulant for the preparation of thin and thick blood films 533
Table 9.32 Plasmodia in Giemsa-stained thin blood smears 534
Table 9.33 Relevant issues for handling requests for identification of infectious blood parasites 537
Table 9.34 Features of human leishmanial infections 540
Table 9.35 Characteristics of American trypanosomiasis 541
Table 9.36 Characteristics of East and West African trypanosomiasis 541
Table 9.37 Key characteristics of blood parasites 541
Index 547
