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Contents

Dedication

Preface

Acknowledgments

PART I

Diagnostic Procedures

1 Philosophy and Approach to Diagnostic Parasitology

2 Collection, Preservation, and Shipment of Fecal Specimens

Safety

Fresh-specimen collection

Collection of the specimen

Number of specimens to be collected (standard recommendation)

Number of specimens to be collected (pros and cons of various options)

Collection times

Specimen type, specimen stability, and need for preservation

Preservation of specimens

Preservatives

Use of fixatives

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

3 Macroscopic and Microscopic Examination of Fecal Specimens

Macroscopic examination

Microscopic examination (ova and parasite examination)

Direct wet smear

Concentration (sedimentation and flotation)

Permanent stained smear

Specialized stains for coccidia (Cryptosporidium, Cystoisospora, and Cyclospora species) and the microsporidia

Modified Kinyoun’s acid-fast stain (cold method)

Modified Ziehl-Neelsen acid-fast stain (hot method)

Carbol fuchsin negative stain for Cryptosporidium (from W. L. Current)

Rapid safranin method for Cryptosporidium

Rapid safranin method for Cyclospora, using a microwave oven

Auramine O stain for coccidia (from Thomas Hänscheid)

Modified trichrome stain for the microsporidia (Weber—green)

Modified trichrome stain for the microsporidia (Ryan—blue)

Modified trichrome stain for the microsporidia (Kokoskin—hot method)

Acid-fast trichrome stain for Cryptosporidium and the microsporidia

4 Additional Techniques for Stool Examination

Culture of larval-stage nematodes

Harada-Mori filter paper strip culture

Filter paper/slant culture technique (petri dish)

Charcoal culture

Baermann technique

Agar plate culture for Strongyloides stercoralis

Egg studies

Estimation of worm burdens and Kato-Katz thick film

Hatching of schistosome eggs

Search for tapeworm scolex

India ink injection procedure for tapeworm proglottids

Qualitative test for fecal fat

Quantitation of reducing substances (Clinitest)

5 Examination of Other Specimens from the Intestinal Tract and the Urogenital System

Examination for pinworm

Cellulose tape preparations

Anal swabs

Sigmoidoscopy material

Direct saline mount

Permanent stained slide

Duodenal contents

Duodenal drainage

Duodenal capsule technique (Entero-Test)

Urogenital specimens

Trichomoniasis

Filariasis

Schistosomiasis

6 Sputum, Aspirates, and Biopsy Material

Expectorated sputum

Induced sputum

Aspirates

Lungs and liver

Lymph nodes, spleen, liver, bone marrow, spinal fluid, eyes, and nasopharynx

Cutaneous ulcer

Biopsy material

Skin

Lymph nodes

Muscle

Rectum and bladder

7 Procedures for Detecting Blood Parasites

Preparation of thick and thin blood films

Thick blood films

Thin blood films

Combination thick and thin blood films (on the same slide)

Combination thick and thin blood films (can be stained as either)

Buffy coat blood films

Staining blood films

Giemsa stain

Wright’s stain

General notes on staining procedures

Proper examination of thin and thick blood films

Thin blood films

Thick blood films

Determination of parasitemia

Diagnosis of malaria: review of alternatives to conventional microscopy

QBC microhematocrit centrifugation method

ParaSight F testy

NOW malaria test

Flow anti-pLDH Plasmodium monoclonal antibodies

Molecular testing

Automated blood cell analyzers

Diagnosis of leishmaniasis: review of alternatives to conventional microscopy

ICT for detection of anti-rK-39 antibodies

Concentration procedures

Cytocentrifugation technique

Knott concentration procedure

Membrane filtration technique

Gradient centrifugation technique

Triple-centrifugation method for trypanosomes

Special stain for microfilarial sheath

Delafield’s hematoxylin

8 Parasite Recovery: Culture Methods, Animal Inoculation, and Xenodiagnosis

Culture methods

Intestinal protozoa

Pathogenic free-living amebae

Blastocystis spp. (Blastocystis hominis)

Pathogenic flagellates

Flagellates of blood and tissue

Toxoplasma gondii

Plasmodium and Babesia spp.

Cryptosporidium spp.

Microsporidia

Animal inoculation

Leishmania spp

Trypanosoma spp.

Toxoplasma gondii

Xenodiagnosis

9 Fixation and Special Preparation of Fecal Parasite Specimens and Arthropods

Fixation of parasite specimens and arthropods

Protozoa

Solutions to induce relaxation in adult helminths

Nematodes

Trematodes

Cestodes

Helminth eggs and larvae

Arthropods

Mounting and staining of parasite specimens for examination

Nematodes

Trematodes

Cestodes

Mounting of arthropods for examination

Mites

Fleas and lice

Ticks

Miscellaneous arthropods

10 Artifacts That Can Be Confused with Parasitic Organisms

Protozoa

Amebae

Flagellates

Ciliates

Coccidia and microsporidia

Cryptosporidium spp. and Cyclospora cayetanensis

Cystoisospora belli

Microsporidia

Blood and body fluids

Malaria parasites and Babesia spp.

Leishmaniae and trypanosomes

Microfilariae

Body fluids: ciliated epithelial cells

Helminths

Adult worms and larvae

Eggs

Human cells

Polymorphonuclear leukocytes

Eosinophils

Macrophages

Lymphocytes

Red blood cells

Charcot-Leyden crystals

Nonhuman elements seen in feces (yeast cells)

Insect larvae

Spurious infections

Delusory parasitosis (delusional infestation)

11 Equipment, Supplies, Safety, and Quality System Recommendations for a Diagnostic Parasitology Laboratory: Factors Influencing Future Laboratory Practice

Equipment

Microscope

Centrifuge

Fume hood

Biological safety cabinet

Refrigerator-freezer

Supplies

Glassware

Miscellaneous supplies

ATCC quality control organisms

Safety: personnel and physical facilities

General precautions

Handwashing

Personal protective equipment (OSHA 2001 blood borne)

Handling specimens

Processing specimens

Spills

Disposal of contaminated materials

Standard precautions

Hepatitis exposure protocol

Dangerous properties of industrial materials

Current OSHA regulations for the use of formaldehyde

Latex allergy

Quality systems

Extent of services

Proficiency testing

In-house quality control

Patient outcome measures

Continuous quality improvement, total quality management, or 10-step and FOCUS-PDCA for performance improvement activities

CLIA ’88 inspection process

New quality guidelines

ISO guidelines

CLSI (NCCLS) model

Factors influencing future laboratory practice

Managed care

Financial considerations

Current regulations

Decentralized testing

Laboratory services

Technological trends

Clinical decision support

Personnel issues

Changing demographics

Emerging diseases

Bioterrorism

12 Medical Parasitology: Case Histories

Protozoal infections

Case 1

Case 2

Case 3

Case 4

Case 5

Case 6

Case 7

Helminth infections

Case 8

Case 9

Case 10

Case 11

Case 12

Case 13

Case 14

Blood parasite infections

Case 15

Case 16

Case 17

Case 18

Case 19

Case 20

Diagnostic methods and proficiency testing

Case 21

Case 22

Case 23

Case 24

Case 25

PART II

Clinically Important Human Parasites

13 Intestinal Nematodes

Ascaris lumbricoides

Enterobius vermicularis

Trichuris trichiura

Capillaria philippinensis

Hookworms (Ancylostoma duodenale, Necator americanus, and Ancylostoma ceylanicum)

Trichostrongylus spp.

Strongyloides spp.

14 Tissue Nematodes

Trichinella spp.

Baylisascaris procyonis

Lagochilascaris minor

Toxocara canis and T. cati (visceral larva migrans and ocular larva migrans)

Ancylostoma braziliense and A. caninum (cutaneous larva migrans)

Human eosinophilic enteritis

Dracunculus medinensis

Angiostrongylus (Parastrongylus) cantonensis (cerebral angiostrongyliasis)

Angiostrongylus (Parastrongylus) costaricensis (abdominal angiostrongyliasis)

Gnathostoma spinigerum

Gnathostoma doloresi, G. nipponicum, G. hispidum, and G. binucleatum

Anisakis simplex, A. physeteris, Pseudoterranova decipiens, Contracaecum osculatum, Hysterothylacium aduncum, and Porrocaecum reticulatum (larval nematodes acquired from saltwater fish)

Capillaria hepatica

Thelazia spp.

15 Filarial Nematodes

Basic life cycle

The endosymbiont

Human pathogens

Wuchereria bancrofti

Brugia malayi

Brugia timori

Zoonotic Brugia infections (American brugian filariasis)

Tropical pulmonary eosinophilia

Loa loa

Mansonella ozzardi

Mansonella perstans

Mansonella streptocerca

Onchocerca volvulus

Dirofilaria Dirofilaria and Dirofilaria Nochtiella spp.

16 Intestinal Cestodes

Diphyllobothrium latum

Taenia solium

Taenia saginata

Taenia asiatica (Asian Taenia or Taenia saginata asiatica)

Hymenolepis (Rodentolepis) nana

Hymenolepis diminuta

Dipylidium caninum

17 Tissue Cestodes: Larval Forms

Echinococcus granulosus (cystic hydatid disease)

Echinococcus multilocularis (alveolar disease, hydatid disease)

Echinococcus vogeli (polycystic hydatid disease) and Echinococcus oligarthrus (unicystic hydatid disease): neotropical echinococcosis

Taenia (Multiceps) spp. (Taenia multiceps, Taenia serialis) (coenurosis)

Spirometra mansonoides and Diphyllobothrium spp. (sparganosis)

18 Intestinal Trematodes

Fasciolopsis buski

Echinostoma ilocanum

Heterophyes heterophyes

Metagonimus yokogawai

Gastrodiscoides hominis

19 Liver and Lung Trematodes

Liver flukes

Clonorchis sinensis

Opisthorchis viverrini

Opisthorchis felineus

Fasciola hepatica

Fasciola gigantica

Less common liver flukes

Dicrocoelium dendriticum, Dicrocoelium hospes, and Eurytrema pancreaticum

Lung flukes

Paragonimus westermani

Paragonimus kellicotti

20 Blood Trematodes: Schistosomes

Schistosoma mansoni

Schistosoma japonicum

Schistosoma mekongi

Schistosoma malayensis

Schistosoma haematobium

Schistosoma intercalatum

21 Intestinal Protozoa: Amebae

Entamoeba histolytica

Entamoeba dispar

Entamoeba moshkovskii

Entamoeba bangladeshi

Entamoeba hartmanni

Entamoeba coli

Entamoeba polecki

Entamoeba gingivalis

Endolimax nana

Iodamoeba bütschlii

Blastocystis spp.

22 Intestinal Protozoa: Flagellates and Ciliates

Giardia lamblia (G. duodenalis, G. intestinalis)

Dientamoeba fragilis

Pentatrichomonas hominis (Trichomonas hominis)

Trichomonas tenax

Chilomastix mesnili

Enteromonas hominis

Retortamonas intestinalis

Balantidium coli

23 Intestinal Protozoa (Coccidia), Microsporidia, and Algae

Coccidia

Cryptosporidium spp.

Cyclospora cayetanensis

Cystoisospora (Isospora) belli

Sarcocystis spp.

Microsporidia

Algae (Prototheca)

24 Free-Living Amebae

Naegleria fowleri

Acanthamoeba spp.

Balamuthia mandrillaris

Sappinia (diploidea) pedata

25 Protozoa from Other Body Sites

Trichomonas vaginalis

Toxoplasma gondii

26 Malaria and Babesiosis

Malaria

Babesiosis

27 Leishmaniasis

Cutaneous leishmaniasis: general comments

Old World (Eastern) leishmaniasis: cutaneous leishmaniasis

New World (Western) leishmaniasis: cutaneous leishmaniasis

Visceral leishmaniasis: general comments

Old World (Eastern) leishmaniasis: visceral leishmaniasis

New World (Western) leishmaniasis: visceral leishmaniasis

28 Trypanosomiasis

African trypanosomiasis

Trypanosoma brucei gambiense

Trypanosoma brucei rhodesiense

American trypanosomiasis

Trypanosoma cruzi

Trypanosoma rangeli

29 Unusual Parasitic Infections

Aquatic protist

Rhinosporidium seeberi

Protozoa

Lophomonas blattarum

Dictyostelium polycephalum

Myxozoan parasites

Trypanosoma evansi, Trypanosoma lewisi

Nematodes (the roundworms)

Ancylostoma ceylanicum

Halicephalobus gingivalis

Oesophagostomum spp.

Eustrongylides spp.

Mermis nigrescens

Dioctophyma renale

Ternidens deminutus

Mammomonogamus laryngeus (Syngamus laryngeus)

Ascaris suum

Gongylonema pulchrum

Haycocknema perplexum

Cestodes

Diplogonoporus spp.

Bertiella studeri

Inermicapsifer madagascariensis

Raillietina celebensis

Mesocestoides spp.

Taenia crassiceps

Trematodes

Alaria americana

Plagiorchis spp.

Neodiplostomum seoulense

Spelotrema brevicaeca

Brachylaima sp.

Nanophyetus (Troglotrema) salmincola

Stellantchasmus falcatus

Phaneropsolus spinicirrus, Phaneropsolus bonnei, and Prosthodendrium molenkempi

Haplorchis taichui

Gymnophalloides seoi

Metorchis conjunctus (North American liver fluke)

Schistosoma mattheei

Philophthalmus lacrimosus

Achillurbainia spp.

Pentastomids

Armillifer spp., Linguatula serrata, and Sebekia spp.

Acanthocephalans

Macracanthorhynchus hirudinaceus and Moniliformis moniliformis

30 Parasitic Infections in the Compromised Host

Entamoeba histolytica

Free-living amebae

Blastocystis spp.

Giardia lamblia

Toxoplasma gondii

Cryptosporidium spp.

Cyclospora cayetanensis

Cystoisospora (Isospora) belli

Sarcocystis spp.

Microsporidia

Leishmania spp.

Strongyloides stercoralis

Plasmodium spp.

Babesia spp.

American trypanosomiasis

Crusted scabies

31 Health Care-Associated and Laboratory-Acquired Infections

Health care-associated (nosocomial) infections

Gastrointestinal infections

Cryptosporidium spp.

Giardia lamblia

Entamoeba histolytica

Microsporidia

Cystoisospora (Isospora) belli

Hymenolepis nana

Taenia solium

Blood and tissue infections

Plasmodium spp.

Babesia spp.

Trypanosoma brucei gambiense and T. brucei rhodesiense

Trypanosoma cruzi

Leishmania donovani

Toxoplasma gondii

Infections with ectoparasites

Pediculus spp. and Phthirus pubis

Sarcoptes scabiei

Myiasis

Infections in the pediatric patient

Cryptosporidium spp.

Giardia lamblia

Pediculus humanus capitis

Sarcoptes scabiei

Infections in the compromised patient

Laboratory infections

Intestinal protozoa

Free-living amebae

Plasmodium spp.

Trypanosoma brucei gambiense and T. brucei rhodesiense

Trypanosoma cruzi

Leishmania spp.

Toxoplasma gondii

Specimen handling

Summary

32 Immunology of Parasitic Infections

Amebiasis

Giardiasis

Toxoplasmosis

African trypanosomiasis

American trypanosomiasis

Malaria

Helminth infections

Summary

33 Antibody and Antigen Detection in Parasitic Infections

Protozoal infections

Amebiasis

Babesiosis

Chagas’ disease

Cryptosporidiosis

Cyclosporiasis

Giardiasis

Leishmaniasis

Malaria

Toxoplasmosis

Trichomoniasis

Helminth infections

Cysticercosis

Hydatid disease

Fascioliasis

Filariasis

Paragonimiasis

Schistosomiasis

Strongyloidiasis

Toxocariasis

Trichinosis

Intradermal tests

Casoni test

Montenegro test

34 Histologic Identification of Parasites

Protozoa

Amebae

Flagellates

Ciliates

Coccidia

Microsporidia

Helminths

Nematodes

Cestodes

Trematodes

Blood Parasites

Malaria

Leishmania

Trypanosomes

Filaria

35 Medically Important Arthropods

Arthropods and their relationship to disease

Biological vectors of microorganisms

Bites and envenomation

Tissue invasion

Entomophobia and delusional infestation (parasitosis)

Class Insecta (insects)

Order Diptera (flies, mosquitoes, and midges)

Myiasis

Order Hemiptera (true bugs)

Order Coleoptera (beetles)

Order Siphonaptera (fleas) (Ctenocephalides spp., Xenopsylla cheopis, Pulex irritans [human flea], Tunga penetrans, Nosopsyllus fasciatus, Echidnophaga gallinacea, and “sand fleas”)

Order Anoplura (sucking lice)

Order Mallophaga (biting and chewing lice)

Order Hymenoptera (bees, wasps, and ants)

Order Blattaria (cockroaches)

Class Arachnida (ticks, mites, spiders, and scorpions)

Subclass Acari (ticks, mites, and chiggers)

Subclass Araneae (spiders)

Subclass Scorpiones (scorpions)

Other arthropods

Class Chilopoda (centipedes)

Class Diplopoda (millipedes)

Class Crustacea (copepods, crabs, crayfish, etc.)

Control of arthropods of medical importance

Physical control

Biological control

Chemical control

36 Treatment of Parasitic Infections

Albendazole (Albenza) (Amedra)

Amphotericin B (AmBisome) (Gilead)

Amphotericin B (Fungizone) (X-Gen)

Artemether (Artenam) (Aarenco, Belgium)

Atovaquone (Mepron) (GlaxoSmithKline)

Atovaquone-proguanil (Malarone) (GlaxoSmithKline)

Benznidazole (Rochagan) (Roche, Brazil)

Bithionol (Bitin) (CDC)

Chloroquine phosphate (Aralen) (Sanofi, others)

Crotamiton (Eurax) (Ranbaxy)

Dapsone (Jacobus)

Diethylcarbamazine citrate USP (Hetrazan) (CDC)

Diloxanide furoate (Furamide, Entamide) (Boots, England)

Eflornithine (difluoromethylornithine, Ornidyl) (Sanofi) (CDC)

Fumagillin (Fumidil-B)

Furazolidone (Furoxone)

Iodoquinol/diiodohydroxyquin (Yodoxin) (Glenwood, others)

Ivermectin (Stromectol, Sklice) (Merck, Sanofi)

Lumefantrine/artemether (Coartem, Riamet) (Novartis)

Malathion (Ovide) (Taro Pharmaceuticals)

Mebendazole (generics)

Mefloquine hydrochloride (generics)

Melarsoprol (Mel-B) (CDC)

Metronidazole (Flagyl, IV Flagyl) (Searle/Pfizer, Baxter)

Miltefosine (Impavido, Miltex) (Paladin, Canada) (CDC)

Niclosamide (Yomesan, Niclocide) (Bayer, Germany)

Nifurtimox (Lampit) (Bayer HealthCare) (CDC)

Nitazoxanide (Alinia) (Romark)

Paromomycin (generics) (Sun Pharma)

Pentamidine isethionate (Pentam 300, Nebupent) (APP Pharmaceuticals)

Permethrin (Nix [Insight Pharmaceuticals], Elimite [Prestium Pharma])

Polyhexamethylene biguanide (Baquacil) (Zeneca)

Praziquantel (Biltricide) (Bayer)

Primaquine phosphate (Sanofi-Aventis)

Propamidine isethionate (Brolene) (Aventis, Canada)

Pyrantel pamoate (Pin-X, Reese’s Pinworm Medicine) (Quartz Specialty Pharmaceuticals, Reese)

Pyrethrin with piperonyl butoxide (Rid) (Bayer, others)

Pyrimethamine (Daraprim) (Amedra)

Quinidine gluconate (generics)

Quinine sulfate or quinine dihydrochloride (many manufacturers)

Spiramycin (Rovamycine) (Sanofi-Aventis)

Stibogluconate sodium (Pentostam, Solustibosan) (GlaxoSmithKline) (CDC)

Suramin sodium (Germanin) (Bayer, Germany) (CDC)

Thiabendazole (Mintezol) (Merck)

Tinidazole (Tindamax) (Mission Pharmaceuticals)

Triclabendazole (Egaten) (Novartis)

APPENDIXES

APPENDIX 1 Information Tables

A1.1 Classification of human parasites

A1.2 Distribution of selected parasitic infections in the Americas

A1.3 Distribution of selected parasitic infections in Europe

A1.4 Distribution of selected parasitic infections in Africa

A1.5 Distribution of selected parasitic infections in Asia

A1.6 Distribution of selected parasitic infections in Oceania

A1.7 Cosmopolitan distribution of common parasitic infections (North America, Mexico, Central America, South America, Europe, Africa, Asia, and Oceania)

A1.8 Body sites and specimen collection

A1.9 Body sites and possible parasites recovered (trophozoites, cysts, oocysts, spores, adults, larvae, eggs, amastigotes, and trypomastigotes)

A1.10 Body site, specimen and procedures, recommended methods, relevant parasites, and comments

A1.11 Examination of tissue and body fluids

A1.12 Key characteristics of protozoa of the intestinal tract and urogenital system

A1.13 Key characteristics of tissue protozoa

A1.14 Key characteristics of helminths

A1.15 Key characteristics of most common parasites found in blood

A1.16 Diagnostic laboratory report information that should be relayed to the physician

A1.17 Pros and cons of stool specimen collection and testing options

A1.18 Approaches to stool parasitology: test ordering

A1.19 Pros and cons of ova and parasite examination options

A1.20 Laboratory test reports: optional comments

A1.21 Estimated prevalence of parasitic diseases worldwide

APPENDIX 2 Flowcharts and Staining Tables for Diagnostic Procedures

Flowcharts

A2.1 Procedure for processing fresh stool for the ova and parasite examination

A2.2 Procedure for processing liquid specimens for the ova and parasite examination

A2.3 Procedure for processing preserved stool for the ova and parasite examination by using the traditional two-vial collection kit

A2.4 Procedure for processing sodium acetate-acetic acid-formalin (SAF)-preserved stool for the ova and parasite examination

A2.5 Use of various fixatives and their recommended stains: fecal specimens preserved using polyvinyl alcohol (PVA)

A2.6 Use of various fixatives and their recommended stains: fecal specimens preserved in the Universal Fixative, TOTAL-FIX

Tables

A2.1 Steps in the trichrome staining procedure (mercuric chloride-based PVA-preserved stool specimens)

A2.2 Steps in the trichrome staining procedure (non-mercuric chloride-based PVA-preserved stool specimens)

A2.3 Steps in the iron hematoxylin staining procedure (mercuric chloride-based PVA-preserved stool specimens) (Spencer-Monroe method)

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

A2.5 Steps in the iron hematoxylin staining procedure (incorporating the carbol fuchsin step)

A2.6 Steps in the trichrome staining procedure (Universal Fixative [no mercury, no formalin, no PVA])

A2.7 Oil-mounted permanent stained smears (no Permount is used)

A2.8 Tips on stool processing and staining

APPENDIX 3 Common Problems in Parasite Identification

Figures

A3.1–A3.26 Paired drawings of “look alikes”

A3.27 Relative sizes of helminth eggs

Tables

A3.1 Entamoeba spp. trophozoites versus macrophages

A3.2 Entamoeba spp. cysts versus polymorphonuclear leukocytes (PMNs)

A3.3 Entamoeba histocolytica versus Entamoeba coli precysts and cysts

A3.4 Endolimax nana versus Dientamoeba fragilis

A3.5 Adult nematodes and/or larvae found in stool specimens: size comparisons

APPENDIX 4 Quality Control Recording Sheets

A4.1 Diagnostic parasitology quality control (QC) (reagents)

A4.2 Diagnostic parasitology quality control (QC) (reagents)—example for multiple reagents

A4.3 Diagnostic parasitology quality control (QC) (culture)—example of a worksheet

A4.4 Equipment maintenance

APPENDIX 5 Commercial Supplies and Suppliers

Tables

A5.1 Sources of commercial reagents and supplies

A5.2 Addresses of suppliers listed in Table A5.1

A5.3 Sources of available reagents for immunodetection of parasitic organisms or antigens

A5.4 Addresses of suppliers listed in Table A5.3

A5.5 Commercial suppliers of diagnostic parasitology products

A5.6 Sources of additional teaching materials, including case histories

A5.7 Sources of parasitologic specimens

APPENDIX 6 Reference Sources

APPENDIX 7 “Late-Breaking” Published Information

APPENDIX 8 Molecular Panels for Parasitology

APPENDIX 9 Frequently Asked Questions about Diagnostic Parasitology

APPENDIX 10 Current Procedural Terminology (CPT) Codes in Parasitology

GLOSSARY

INDEX