..

Contents

Preface xv

Acknowledgments XIX

PART I

Clinically Important Human ParasitesI Philosophy and Approach to Diagnostic

Parasitology 3

2 Intestinal Protozoa: Amebae 6

Entamoeba histolytica 6Entamoeba dispar 19Entamoeba mosh kovskii 21Entamoeba hartmanni 21Entamoeba coli 22

Entamoeba polecki 23Entamoeba gingivalis 24Endolimax nana 25Iodamoeba bütschlii 27

Blastocystishominis 27

3 Intestinal Protozoa: Flagellates and Ciliates 33Giardialamblia 33

Dientamoeba fragilis 47Pentatrichomonas hominis (Trichomonashominis) 49Trichomonas tenax 50Chilomastix mesnili 50Enteromonas hominis 51Retortamonas intestinalis 51Balantidium coli 51

4 Intestinal Protozoa (Coccidia and Microsporidia)andAlgae 57Coccidia 57

Cryptosporidiumspp. 57

Cyclospora cayetanensis 73Isospora (Cystoisospora) belli 80Sarcocystis spp. 83

Microsporidia 87

Algae (Prototheca) 98

5 Free-LivingAmebae 102

Naegleria fowleri 104Acanthamoeba spp. 109Balamuthia mandrillaris 118

Sappinia diploidea 121

6 Protozoa from Other Body Sites

Trichomonas vaginalis 123Toxoplasma gondii 130

123

7 Malaria and Babesiosis 142

Malaria 142Babesiosis 180

8 leishmaniasis 190

Old World leishmaniasis: cutaneousleishmaniasis 191Old World leishmaniasis: visceralleishmaniasis 198New World leishmaniasis: cutaneousleishmaniasis 205New World leishmaniasis: visceralleishmaniasis 212

9 Trypanosomiasis 218

African trypanosomiasis 218Trypanosomabruceigambiense 218Trypanosomabrucei rhodesiense 228

American trypanosomiasis 232Trypanosomacruzi 232Trypanosomarangeli 243

vii

viii Contents

10 Intestinal Nematodes 249

Ascaris lumbricoides 250Enterobius vermicularis 258Trichuris trichiura 261

Capillaria philippinensis 264Hookworms (Ancylostoma duodenale and Necatoramericanus) 266Trichostrongylus spp. 270Strongyloides spp. 271

11 Tissue Nematodes 283

Trichinella spp. 283Baylisascaris procyonis 294Lagochilascaris minar 298Toxocara canis and T. cati (visceral larva migransand ocular larva migrans) 298Ancylostoma braziliense and A. caninum (cutaneouslarva migrans) 302Human eosinophilic enteritis 303Dracunculus medinensis 304

Angiostrongylus (Parastrongylus) cantonensis(cerebral angiostrongyliasis) 307Angiostrongylus (Parastrongylus) costaricensis(abdominal angiostrongyliasis) 309Gnathostoma spinigerum 310Gnathostoma doloresi, G. nipponicum,G. hispidum, and G. binucleatum 312Anisakis simplex, A. physeteris, Pseudoterranovadecipiens, Contracaecum osculatum,Hysterothylacium aduncum, and Porrocaecumreticulatum (larval nematodes acquired fromsaltwater fish) 312Capillaria hepatica 315Thelazia spp. 315

12 Filarial Nematodes 3 19

Wuchereria bancrofti 321Brugia malayi 332Brugia timori 333Zoonotic Brugia infections (American brugianfilariasis) 334Tropical pulmonary eosinophilia 335Loa loa 335Mansonella ozzardi 338

Mansonella perstans 339Mansonella streptocerca 340Onchocerca volvulus 341

Dirofilaria Dirofilaria and DirofilariaNochtiella spp. 348

13 Intestinal Cestodes 357

Diphyllobothrium latum 357Taenia solium 362

Taenia saginata 371Taenia saginata asiatica (Asian Taenia or Taeniaasiatica) 373Hymenolepis nana 374Hymenolepis diminuta 376Dipylidium caninum 377

14 Tissue Cestodes: larval Forms 381

Echinococcus granulosus (cystic disease, hydatiddisease) 381Echinococcus multilocularis (alveolar disease,hydatid disease) 393Echinococcus oligarthrus and Echinococcus vogeli(polycystic hydatid disease) 399Taenia (Multiceps) spp. (Taenia multiceps, Taeniaserialis) (coenurosis) 402Spirometra mansonoides and Diphyllobothrium spp.(sparganosis) 402NOTE: Taenia solium (cysticercosis) is discussed inchapter 13.

15 Intestinal Trematodes 411

Fasciolopsis buski 411Echinostoma ilocanum 416

Heterophyes heterophyes 417Metagonimus yokogawai 419Gastrodiscoides hominis 420

16 Liver and lung Trematodes 423

Liver flukes 423Clonorchis sinensis 423

Opisthorchis viverrini 429Opisthorchis felineus 431Fasciola hepatica 432Fasciola gigantica 435

Less common liver flukes 436

Dicrocoelium dendriticum, Dicrocoelium hospes, andEurytrema pancreaticum 436

Lung flukes 438Paragonimus spp. 438

17 Blood Trematodes: Schistosomes --445

Schistosoma mansoni 446Schistosoma japonicum 458Schistosoma mekongi 463Schistosoma haematobium 466Schistosoma intercalatum 472

,..

18 Unusual Parasitic Infections 478

Aquatic Protist 478Rhinosporidium seeberi 478

Protozoa 482

Myxozoan parasites 482Nematodes 483

Oesophagostomum spp. 483Eustrongylides spp. 485Mermis nigrescens 486Micronema deletrix 487

Dioctophyma renale 487Ternidens deminutus 488

Mammomonogamus laryngeus (Syngamuslaryngeus) 488Ascaris suum 489

Gongylonema pulchrum 489Haycocknema perplexum 490

Cestodes 490

Diplogonoporus spp. 490Bertiella studeri 491

Inermicapsifer madagascariensis 491Raillietina celebensis 491

Mesocestoides spp. 492Taenia crassiceps 492

Trematodes 493Alaria americana 493

Plagiorchis spp. 493Neodiplostomum seoulense 494Spelotrema brevicaeca 494Brachylaima sr. 494Troglotrema salmincola 494Stellantchasmus falca tus 494Phaneropsolus bonnei and Prosthodendriummolenkempi 495Phaneropsolus spinicirrus 495Haplorchis taichui 496Gymnophalloides seoi 496Metorchis conjunctus (North American liverfluke) 497Schistosoma mattheei 498

Philophthalmus lacrimosus 498Achillurbainia spp. 499

Pentastomids 499

Armillifer spp., Linguatula serrata, and Sebekiaspp. 499

Acanthocephalans 500Macracanthorhynchus hirudinaceus and Moniliformismoniliformis 500

19 Parasitic Infections in the CompromisedHost 506

Entamoeba histolytica 508Free-living amebae 518

Giardia lamblia 522

Toxoplasma gondii 523Cryptosporidium spp. 524Cyclospora cayetanensis 528Isospora (Cystoisospora) belli 530Sarcocystis spp. 531Microsporidia 532Leishmania spp. 537Strongyloides stercoralis 540Crusted (Norwegian) scabies 540

Contents ix

20 Nosocomial and Laboratory-AcquiredInfections 549

Nosocomial infections 549

Nosocomial gastrointestinal infections 550Cryptosporidium spp. 550Giardia lamblia 553

Entamoeba histolytica 554

Microsporidia 555Isospora (Cystoisospora) belli 555Hymenolepis nana 555Taenia solium 555

Nosocomial blood and tissue infections 556

Plasmodium spp. 556Babesia spp. 556Trypanosoma brucei gambiense and T. bruceirhodesiense 556

Trypanosoma cruzi 556Leishmania donovani 557

Toxoplasma gondii 557

Nosocomial infections with ectoparasites 557Pediculus spp. and Phthirus pubis 557Sarcoptes scabiei 557Myiasis 557

Nosocomial infections in the pediatric patient 558Cryptosporidium spp. 558Giardia lamblia 558

Pediculus humanus capitis 558Sarcoptes scabiei 558

Nosocomial infections in the compromisedpatient 558Laboratory infections 560

Intestinal protozoa 560Free-living amebae 560Plasmodium spp. 560Trypanosoma brucei gambiense and T. bruceirhodesiense 560

Trypanosoma cruzi 562Leishmania spp. 562Toxoplasma gondii 562Specimen handling 563

Summary 563

x Contents

21 Immunology of Parasitic Infections 567Amebiasis 570Giardiasis 575

Toxoplasmosis 577African trypanosomiasis 579American trypanosomiasis 580Malaria 582

Summary 586

22 Antibody and Antigen Detection in ParasiticInfections 592

Protozoal infections 594Amebiasis 594Babesiosis 597

Chagas' distase 597Cryptosporidiosis 598Cyclosporiasis 598Giardiasis 598Leishmaniasis 598Malaria 600

Toxoplasmosis 601Trichomoniasis 603

Helminth infections 604

Cysticercosis 604Hydatid distase 605Fascioliasis 605Filariasis 606

Paragonimiasis 607Schistosomiasis 608

Strongyloidiasis 609Toxocariasis 610Trichinellosis 610

Intradermal tests 611Casoni test 611

Montenegro test 611

23 Histologic Identification of Parasites 616

24 Medically Important Arthropods 670

Arthropods and their relationship to disease 670Biological vectors of microorganisms 670Bites and envenomation 671Tissue invasion 676

Entomophobia and delusory parasitosis 676

Class Insecta (insects) 678arder Diptera (flies, mosquitoes, and midges) 679Myiasis 683arder Hemiptera (true bugs) 690arder Coleoptera (beetles) 692arder Siphonaptera (fleas) (Ctenocephalides spp.,Xenopsylla cheopis, Pulex irritans [human flea],Tunga penetrans, Nosopsyllus fasciatus, Echidnophagagallinacea, and "salid fleas") 693

arder Anoplura (sucking lice) 694arder Mallophaga (biting and chewing lice) 696arder Hymenoptera (bees, wasps, and ants) 696arder Blattaria (cockroaches) 698

Class Arachnida (ticks, mires, spiders, andscorpions) 699

Subclass Acari (ticks, mires, and chiggers) 699Subclass Araneae (spiders) 708Subclass Scorpiones (scorpions) 710

Other arthropods 711Class Chilopoda (centipedes) 711Class Diplopoda (millipedes) 712Class Crustacea (copepods, crabs, crayfish, ete.) 712

Control of arthropods of medical importance 712Physical control 712Biological control 712Chemical control 715

25 Treatment of Parasitic Infections 718

PART 11

Diagnostic Procedures 759

26 Collection, Preservation, and Shipment of FecalSpecimens 761

Safety 761

Fresh-specimen collection 762Collection of the specimen 762Number of specimens to be collected (standardrecommendation) 762Number of specimens to be collected (pros and cons ofvarious options) 763Collection times 764

Specimen type, specimen stability, and need forpreservarían 764

Preservarían of specimens 768Preservatives 768

Formalin 768MIF 769SAF 771Schaudinn's fluid 772PVA 772Modified PVA 773

Single-vial collection systems (other than SAF) 773Use of fixatives 774

Quality control for stool fixatives 774Procedure notes for use of preservatives 775Procedure limitations for use Ff preservatives 775

Shipment of diagnostic specimens, biological products,etiologic agents, or infectious substances 775

27 Macroscopic and Microscopic Examination ofFecal Specimens 782Macroscopic examinarían 782

Microscopic examination (ova and parasiteexamination) 783

Direct wet smear 783

Concentration (sedimentation and flotation) 788

Formalin-ethyl acetate sedimentationconcentration 789Iodine-trichrome stain for sediment 792Zinc sulfate flotation concentration 794Commercial fecal concentration devices 796

Automated workstation for the microscopic analysisof fecal concentra tes 797

Permanent stained smear 797

Preparation of material for staining 798Trichrome stain 802

Iron hematoxylin stain 807Modified iron hematoxylin stain (incorporating thecarbol fuchsin step) 811Polychrome IV stain 812Chlorazol black E stain 813

Specialized stains for coccidia (Cryptosporidium,Isospora, and Cyclospora species) and themicrosporidia 813

Modified Kinyoun's acid-fast stain (cold method) 813Modified Ziehl-Neelsen acid-fast stain (hotmethod) 816Carbol fuchsin negative stain for Cryptosporidium (fromW. lo Current) 819

Rapid safranin method for Cryptosporidium 819Rapid safranin method for Cyclospora, using amicrowave oyen 819

Auramine O stain for coccidia (from ThomasHanscheid) 819Modified trichrome stain for the microsporidia(Weber-green) 821Modified trichrome stain for the microsporidia (Ryan-bluf) 823Modified trichrome stain for the microsporidia(Kokoskin-hot method) 825Acid-fast trichrome stain for Cryptosporidium and themicrosporidia 826

28 AdditionalTechniques for Stool Examination 83 I

Culture of larval-stage nematodes 831Harada-Morifi!terpapel strip culture 832Filterpaper/slantculture technique (petri dish) 833Charcoalculture 834Baermanntechnique 835Agarplaceculturefor Strongyloidesstercoralis 837

Eggstudies 840Estimationof worm burdens 840Hatchingof schistosomeeggs 842

Searchfor tapeworm scolex 844Qualitative test for fecal fat 845Quantitation of reducing substances (Clinitest) 847

Contents xi

29 Examination of Other Specimens from theIntestinal Tract and the Urogenital System 850

Examination for pinworm 850Cellulose tape preparations 851Anal swabs 851

Sigmoidoscopy material 852Direct saline mount 853Permanent stained slide 853

Duodenal contents 854

Duodenal drainage 854Duodenal capsule technique (Entero-Test) 854

Urogenital specimens 855Trichomoniasis 855Filariasis 856Schistosomiasis 856

30 Sputum, Aspirates, and Biopsy Material 859

Expectorated sputum 859

Induced sputum 861

Aspirates 868Lungs and liver 868Lymph nades, spleen, liver, baile marrow, spinal fluid,eres, and nasopharynx 869Cutaneous ulcer 871

Biopsy material 871Skin 876

Lymph nades 876Muscle 876Rectum and bladder 877

31 Procedures for Detecting Blood Parasites 881

Preparation of thick and thin blood films 881Thick blood films 882Thin blood films 884

Combination thick and thin blood films (on the sameslide) 884Combination thick and thin blood films (can be stainedas either) 884Buffy coat blood films 886

Staining blood films 887Giemsa sta in 888

Wright's stain 889General notes on staining procedures 891

Proper examination of thin and thick bloodfilms 891

Thin blood films 891Thick blood films 893

Determination of parasitemia 893

Diagnosis of malaria: review of alternatives toconventional microscopy 894

QBC microhematocrit centrifugation method 895ParaSight F test 896NOW malaria test 900

,

xii Contents

11

Flow anti-pLDH Plasmodium monoclonalantibodies 901PCR 902

Automated blood cell analyzers 903

Diagnosis of leishmaniasis: review of alterna ti ves toconventional microscopy 903

ICT for detection of anti-rK-39 antibodies 903

Concentration procedures 903Cytocentrifugation technique 903Knott concentration procedure 904Membrane filtration technique 904Gradient centrifugation technique 905Triple-centrifugation method for trypanosomes 905

Special stain for microfilarial sheath 905Delafield's hematoxylin 905

32 Parasite Recovery: Culture Methods,AnimalInoculation, and Xenodiagnosis 910

Culture methods 910

Intestinal protozoa 911Pathogenic free-living amebae 917Pathogenic fIagellates 922Flagellates of blood and tissue 926Toxoplasma gondii 931Plasmodium and Babesia spp. 931Cryptosporidium spp. 932Microsporidia 932

Animal inoculation 932

Leishmania spp. 932Trypanosoma spp. 933Toxoplasma gondii 933

Xenodiagnosis 933

33 Fixation and Special Preparation of FecalParasite Specimens and Arthropods 936

Fixation of parasite specimens and arthropods 936Protozoa 937Solutions to induce relaxation in adult helminths 938Nematodes 939Trematodes 940Cestodes 940

Helminth eggs and larvae 940Arthropods 940

Mounting and staining of parasite specimens forexamination 941

Nematodes 941Trematodes 942Cesto des 943

Mounting of arthropods for examination 944Mites 944Fleas and lice 944Ticks 945

Miscellaneous arthropods 945

34 ArtifactsThat Can Be Confused with ParasiticOrganisms 947Protozoa 947

Amebae 947

Flagellates 949Ciliates 949

Coccidia and microsporidia 949Cryptosporidium spp. and Cyclosporacayetanensis 949Isospora belli 950Microsporidia 951

Blood and body fluids 951Malaria parasites and Babesia spp. 951Leishmaniae and trypanosomes 951Microfilariae 951

Body fIuids: ciliated epithelial cells 953Helminths 953

Adult worms and larvae 953

Eggs 954Human cells 954

Polymorphonuclear leukocytes 956Eosinophils 956Macrophages 956Lymphocytes 957Red blood cells 957

Charcot-Leyden crystals 957

Nonhuman elements seen in feces (yeast cells) 957Insect larvae 958

Spurious infections 958

Delusory parasitosis 959

35 Equipment, Supplies, Safety, and Quality SystemRecommendations for a Diagnostic ParasitologyLaboratory: Factors Influencing FutureLaboratory Practice 96 I

Equipment 961Microscope 961Centrifuge 965Fume hood 965

Biological safety cabinet 965Refrigerator-freezer 966

Supplies 966Glassware 966

Miscellaneous supplies 967ATCC qua lity control organisms 967

Safety: personnel and physical facilities 967General precautions 967Handwashing 968Personal protective equipment (OSHA 2001 bloodborne) 969Handling specimens 970Processing specimens 970Spills 972

T

Disposal of contaminated materials 973Standard precautions 974Hepatitis exposure protocol 976Dangerous properties of industrial materials 976Currellt OSHA regulations for the use offormaldehyde 977Latex allergy 978

Quality systems 979Extent of services 979

Proficiencytesting 979In-house quality control 982Patient outcome measures 986

Continuous quality improvement, total qualitymanagement, or lO-step and FOCUS-PDCA forperformance improvement activities 987CLlA '88 inspection process 989

New qua lity quidelines 989ISO guidelines 989CLSI (NCCLS) model 990

Faetors influeneing future laboratory praetiee 990Managed care 992Financial considerations 993

Decentralized testing 993Laboratory services 993Technological trends 994Clinical decision support 994Personnel issues 995

Changing demographics 995Emerging diseases 995

36 MedicalParasitology: Case Histories 998Protozoal infeetions 998Helminth infeetions 1007

Bloodparasite infeetions 1016Diagnostic methods 1024

APPENDIXES 1027

APPENDIX 1InformationTables 1028

A1.1 Classification of bullan parasites 1028A1.2 Distribution of selected parasitie infeetions in the

Amerieas 1031

AI.3 Distribution of selected parasitie infections inEurope 1031

A1.4 Distribution of selected parasitic infections inAfrica 1032

AI.5 Distribution of selected para sitie infeetions inAsia 1033

A1.6 Distribution of seleeted parasitie infeetions inOeeania 1033

AI.7 Cosmopolitan distribution of eommon parasitieinfeetions (North Ameriea, Mexieo, Central

Contents xiii

Ameriea, South America, Europe, Afriea, Asia,and Oeeania) 1034

A 1.8 Body sites and specimen collection 1035A 1.9 Body sites and possible parasites recovered

(trophozoites, eysts, ooeysts, spores, adults,larvae, eggs, amastigotes, andtrypomastigotes) 1036

A 1.1OBody site, specimen and procedures,reeommended methods, relevant parasites, andeomments 1037

A 1.11 Examination of tissue and body fluids 1042Al .12 Key eharacteristics of protozoa of the intestinal

traet and urogenital system 1044A 1.13 Key characteristics of tissue protozoa 1047Al. 14 Key characteristics of helminths 1049A 1.15 Key characteristies of parasites found in

blood 1052

A 1.16 Diagnostic laboratory report information thatshould be relayed to the physician 1054

A 1.17 Pros and eons of stool speeimen eollection andtesting options 1055

A 1.18 Approaches to stool parasitology: testordering 1057

A 1.19 Pros and eons of ova and parasite examinationoptions 1058

A 1.20 Laboratory test reports: optionalcomments 1060

A 1.21 Estimated prevalenee of parasitic distasesworldwide 1061

APPENDIX 2Flowcharts and Staining Tables for DiagnosticProcedures 1062

Flowcharts

A2.1 Proeedure for processing fresh stool for the ovaand parasite examination 1062

A2.2 Procedure for processing liquid speeimens for theova and parasite examination 1063

A2.3 Procedure for processing preserved stool forthe ova and parasite examination by using thetraditional two-vial colleetion kit 1064

A2.4 Procedure for processing sodium acetate-aceticacid-formalin (SAF)-preserved stool for the ovaand parasite examination 1065

A2.5 Use of various fixatives and their recommendedstains 1066

Tables

A2.1 Steps in the trichrome staining procedure(mereurie ehloride-based PVA-preserved stoolspecimens) 1067

A2.2 Steps in the trichrome staining proeedure(non-mercurie ehloride-based PVA-preserved stoolspecimens) 1068

xiv Contents

A2.J Steps in the iron hematoxylin staining procedure(mercuric chloride-based PVA-preserved stoolspecimens) (Spencer-Monroe method) 1069

A2.4 Steps in the iron hematoxylin staining procedure(mercuric chloride-based PVA-preserved stoolspecimens) (Tompkins-Miller method) 1070

A2.S Steps in the iron hematoxylin staining procedure(incorporating the carbol fuchsin step) 1071

A2.6 Oil-mounted permanent stained smears (noPermount is used) 1072

A2.7 Tips on stool processing and staining 1073

APPENDIX3Common Problems in Parasite Identification

FiguresAJ.I-AJ.2S Paired drawings of "look alikes"AJ.26 Relative sizes of helminth eggs 1080TableAJ.I

1074

1074

Adult nematodes and/or larva e found inÚool

specimens: size comparisons 1081

APPENDIX4Quality Control Recording Sheets 1082A4.1 Diagnostic parasitology quality control (QC)

(reagents) 1083A4.2 Diagnostic parasitology quality control (QC)

(reagents)-example for multiple reagents 1084A4.J Diagnostic parasitology quality control (QC)

(culture)-example of a worksheet 1085A4.4 Equipment maintenance 1086

APPENDIX5Commercial Supplies and Suppliers 1089Tables

AS.I Sources of commercial reagents andsupplies 1090

AS.2 Addresses of suppliers listed in Table A5.1 1092AS.J Sources of available reagents for immunodetection

of parasitic organisms or antigens 1094AS.4 Addresses of suppliers listed in Table A5.3 1095AS.S Commercial suppliers of diagnostic parasitology

products 1096AS.6 Sources of parasitologic specimens 1099AS.7 Sources of Kodachrome study slides (35 mili,

2 x 2) for rental 1100AS.8 Sources of additional teaching materials, including

case histories 1100

~

~.

I

APPENDIX 6Reference Sources 1101

APPENDIX 7Color Plates of Diagnostic Stages of HumanParasites I I04

APPENDIX 8

"late-Breaking" Published Information 1120

GLOSSARY 1151

INDEX 1165