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Home , Ciliate, Coccidia, Cyclospora cayetanensis, Eimeria, Isospora, Retortamonas

MORPHOLOGY OF DIAGNOSTIC STAGES OF INTESTINAL PARASITES OF HUMANS*

M. M. Brooke, Sc.D. D. M. Melvin, Ph.D.

Division of Laboratory Training and Consultation Laboratory Program Office

Second Edition* 1984 Reprinted 1989

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES Public Health Service Centers For Disease Control Atlanta, 30333

HHS Publication No. (CDC) 89-8116

*Updated from the original printed version in 2001.

ii PREFACE

Although the morphology of the diagnostic stages of the intestinal parasites found in humans is described in many textbooks and manuals, it is not generally readily available in the average laboratory. The characteristics commonly used to distinguish species are tabulated and illustrated in this publication. Thus, the technologist performing parasitology examinations in public health and medical laboratories can quickly refer to them. The booklet is designed to assist the microscopist in identifying the organisms found, but it is not a substitute for the practical training in laboratory procedures and differentiation of species under the supervision of persons experienced in diagnostic parasitology.

In this second edition, several species of , , and schistosomes have been added. Because of the increased number of drawings, those of the flagellates and of the and coccidia have been separated into two figures.

We thank Mrs. Margery Borom and Miss Jean Ryan, formerly of the Centers for Disease Control (CDC), for preparing the original drawings of the organisms and Mr. Ed Biel, Chief, Publications, Graphics Section, CDC, for the revisions and additions.

M. M. Brooke, Sc.D.

Associate Director

Division of Laboratory Training and Consultation

Dorothy M. Melvin, Ph.D.

Chief, Parasitology Training Section

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CONTENTS INTRODUCTION…………………………………………………………..…. vii …………………………………...………………………………. 1 Amebae……………...…………………………………………………..…... 1 Flagellates……………………………………………………………..…….. 2 Ciliate………………………………………...………………………………. 3 Coccidia………………………………………...……………………………. 3 ………….………………………...….…………………..……... 4 HELMINTHS………………………………...….…………………….……... 5 ……………………………………….………………...... …….. 5 Cestodes……………………………………………….………….…………. 6 Trematodes...... …...... 6 TABLES 1. Characteristics of Intestinal Amebae Visible in Different Types of Fecal Preparations.…………………………….………………… 2 2. Characteristics of Intestinal Flagellates, Ciliate, and Coccidia Visible in Different Types of Fecal Preparations.………………………… 4 3. Differential Morphology of Protozoa Found in Stool Specimens of Humans: Amebae 3 - Trophozoites………………..……………………8 4. Differential Morphology of Protozoa Found in Stool Specimens of Humans: Amebae - Cysts………………………………………………..9 5. Differential Morphology of Protozoa Found in Stool Specimens of Humans: Flagellates - Trophozoites……………………..…………….10 6. Differential Morphology of Protozoa Found in Stool Specimens of Humans: Flagellates - Cysts…………………………………….…..….11 7. Differential Morphology of Protozoa Found in Stool Specimens of Humans: Ciliate, Coccidia, and Blastocystis………………………….12 8. Differential Morphology of Diagnostic Stages of Helminths Found in Humans: Eggs…………………………………………………….13 9. Differential Morphology of Diagnostic Stages of Helminths Found in Humans: Larvae………………………………………………… 18 10. Differential Morphology of Diagnostic Stages of Helminths Found in Humans: Tapeworm Gravid Proglottids………………………. 19 11. Differential Morphology of Diagnostic Stages of Helminths Found in Humans: Tapeworm Scoleces……………………..………….. 20 FIGURES 1. Protozoa Found in Stool Specimens of Humans-Amebae……..……..21 2. Protozoa Found in Stool Specimens of Humans-Flagellates….….…..22 3. Protozoa Found in Stool Specimens of Humans-Ciliate, Coccidia, Blastocystis………………… ………………………...…..……..23 4. and Cestode Eggs Found in Stool Specimens of Humans…………………………………..….……………..……………..24 5. Trematode Eggs Found in Stool Specimens of Humans……………...25 6. Relative Sizes of Helminth Eggs……………………………...………….26 7. Hookworm and Strongyloides Larvae…………….………..……………27 8. Gravid Proglottids and Scoleces of Cestode Parasites of Humans…………………………….……………….….…..…28 REFERENCES……………………………….……..………………………..…..29

v INTRODUCTION The diagnostic stages of intestinal parasites are differentiated on the basis of specific morphologic features that can be seen microscopically. The characteristics that are visible in different types of preparations are listed in tables 1 and 2; the characteristics commonly used to distinguish species are presented in tables 3-11 for the microscopist's ready reference. The descriptions, however, do not include all the morphologic characteristics of the various stages, and supplemental references may be needed in some cases. Although is a (Camp et al., 1974; Honigberg, 1974), morphologically, it resembles the amebae. Therefore, in this manual it is included with the amebae to facilitate species identification. Several parasite species were added to this second edition. Drawings of the trophozoites and cysts of Enteromonas hominis and Retortamonas intestinalis were included with those of the other flagellates. Information on the coccidia** was updated, and both descriptions and diagrams of the diagnostic stages of and were included. Blastocystis hominis, now identified as a protozoan, was also included in the tables and figures. Because of the increased number of drawings, diagrams of the flagellates and the ciliate and coccidia were separated into two figures. Also in this edition, two additional species of trematodes, Schistosoma mekongi and Schistosoma intercalatum, were described in the table of differential morphology of helminth eggs. Drawings of these eggs, however, were not included in the figures. Some of the parasites listed occur only infrequently or accidentally in humans; but, since their diagnostic stages may be confused with those of the more common organisms, they were included here. For example, Entamoeba polecki is rarely found in human feces; however, it closely resembles Entamoeba histolytica and Entamoeba coli, and microscopists unaware of its existence may mistake it for these species. Similarly, several helminth species of lower that occasionally parasitize humans are included. The diagnostic stages may be misidentified by inexperienced microscopists. For example, Trichostrongylus (Nematode) eggs may be confused with hookworm eggs, and Hymenolepis diminuta (Cestode) eggs may be confused with Hymenolepis nana eggs. The protozoa found in the mouth, Entamoeba gingivalis and tenax, were omitted from this manual, since the diagnostic stages of these parasites are not found in feces. Trichomonas vaginalis was not included because it is a parasite of the urogenital system rather than of the intestinal tract. Technical methods for examining specimens are presented in the manual "Laboratory Procedures for the Diagnosis of Intestinal Parasites" (Melvin and Brooke, 1982). The morphology of the intestinal parasites is described in many parasitology textbooks. Some of those which emphasize morphology of the diagnostic stages and have good illustrations are listed in the references.

**At the time of this publication the coccidian parasite cayetanensis had not been classified.

vii PROTOZOA The intestinal protozoa of humans belong to four groups: amebae, flagellates, , and coccidia. In addition to these, Blastocystis hominis now has been identified as a protozoan (Zierdt and coworkers, 1967,1973,1976, 1983). With the exception of the coccidia and lamblia (flagellate) which inhabit the small intestine, the protozoa live in the caecum and colon. The coccidia differ from the other protozoan species in being obligatory parasites. Oocysts or sporocysts, passed in the feces, are the diagnostic stages. Organisms belonging to the three other groups have two stages, trophozoites and cysts, in their cycle, except Dientamoeba fragilis and Trichomonas* hominis (flagellates), which have only a trophozoite stage. Both trophozoites and cysts are passed in feces and are diagnostic stages. The features listed in tables 3-7 are those commonly seen in fecal preparations and used for identifications. Features or structures that are difficult to see or are demonstrated only by special techniques are omitted. Not all of the characteristics listed, however, can be seen in a single type of preparation. The fecal preparations used to demonstrate protozoa are either wet mounts or permanently stained smears. Wet mounts may be unstained (saline or formalin) or stained (iodine, buffered methylene blue, Quensel's stain, merthiolate-iodine- formaldehyde [MIF], or other temporary stains). Iodine is primarily a cyst stain, and iodine preparations are usually routinely prepared in examining fecal specimens for protozoa. Buffered methylene blue and Quensel's stains are used to stain amebae trophozoites. Others, such as MIF, will stain both stages, but they may be less satisfactory for trophozoites than for cysts. Fecal smears may be permanently stained with hematoxylin, trichrome, chlorazol black, or other suitable stains. Scaled drawings of the intestinal protozoa are presented in figures 1-3. AMEBAE Characteristics used to distinguish species of intestinal amebae are as follows: Trophozoites Motility- progressive or nonprogressive. Appearance-finely granular, coarsely granular, or vacuolated. Inclusions-erythrocytes, , molds. Nucleus Number present. Peripheral - present or absent. If present, the distribution along the inner surface of the nuclear membrane and the size of the granules are important. Karyosome- location and size. Size Sizes overlap, but they can be used as a secondary distinguishing feature. Size is the chief criterion for distinguishing Entamoeba histolytica from Entamoeba hartmanni. Cysts Nucleus Number present. Peripheral chromatin-present or absent. If present, the distribution of the granules along the inner surface of the nuclear membrane is important. Karyosome- location and size.

*Since initial publication of this manual, the name Trichomonas has been replaced by Pentatrichomonas. (Kirby, 1945) (Honigberg, 1978)

1 Cytoplasm Chromatoid bodies-present or absent. If present, the shape is important. Glycogen- appearance. Size Sizes overlap, but they can be used as a secondary distinguishing feature. Size is the major criterion for distinguishing Entamoeba histolytica from Entamoeba hartmanni. Shape Shapes vary, but they may be useful as a secondary distinguishing feature. As stated above, both unstained and stained preparations are necessary to demonstrate all of the features. Table 1 indicates the structures or characteristics that can be seen in different types of preparations. Species identification of trophozoites can rarely be made from single features, such as nucleus or cytoplasm, or from a single organism. Several features and several organisms should be examined. Cysts are less variable and can usually be more easily identified than trophozoites. Tables 3 and 4 list the common morphologic characteristics of the amebae. Drawings of the trophozoite and cyst stages are presented in figure 1. TABLE 1 CHARACTERISTICS OF INTESTINAL AMEBAE VISIBLE IN DIFFERENT TYPES OF FECAL PREPARATIONS Characteristic Unstained Temporary Stains Permanent Stains Saline Formalin Iodine Buffered (Cysts) Methylene Blue (Trophs)1 Trophozoites Motility + - - - Cytoplasm Appearance + + + + Inclusions (rbc, bacteria) + + + + Nucleus - +2 + + Cysts Nuclei - + + + Chromatoid bodies + + +3 + Glycogen - - + - ( present)

1 Quensel’s stain may be substituted for buffered methylene blue. 2 Nuclei of trophozoites are visible in formalin-fixed material but are usually not sufficiently distinctive for species identification. 3 Chromatoid bodies are more easily seen in unstained wet mounts than in iodine preparations. FLAGELLATES

Characteristics used to distinguish flagellate species are as follows: Trophozoites Motility Shape Number of nuclei. The appearance of the nucleus is not commonly used to distinguish species. Other features such as undulating membrane, sucking disk, and prominent . Number and location of flagella. Although the number and location of flagella vary with the species, they are often difficult to see and count. Therefore, the number and location of flagella are not practical diagnostic features for routine identification.

2 Cysts Shape Size Number of nuclei Fibrils As with amebae, not all of the characteristics of flagellates can be seen in a single type of preparation, and both unstained and stained preparations may be necessary. Nuclei are not visible in unstained saline mounts, but they can be seen in formalin-fixed material. Most of the other features can be seen in either saline or formalin unstained mounts. Cyst structures are usually easily seen in iodine preparations. Quensel's and buffered methylene blue solutions, however, do not stain the trophozoites. If fresh feces are available, trophozoites can be readily distinguished by their characteristic motion in saline mounts. Methocel solutions can be used to slow the motion, particularly of Trichomonas, for better observation of flagella and undulating membranes. Neutral red dye added to the solution will stain the trophozoites to some extent. Table 2 indicates the structures or characteristics that can be seen in different types of preparations.

Tables 5 and 6 list the diagnostic characteristics of the intestinal flagellates. Drawings are presented in figure 2. CILIATE Only one ciliate species, coli (figure 3), parasitizes humans. It is also the largest protozoan parasite of humans, and the trophozoites and cysts are easily detected in saline mounts of fresh feces. The size, shape, and motility of the trophozoites are readily recognized by even beginning parasitology students. Although the cysts are less easily identified, they, too, present few real diagnostic problems. In formalin-fixed feces, the trophozoites may be somewhat distorted, but they are usually recognizable. Formalin does not significantly alter the appearance of the cyst.

Good stained preparations of B. coli are difficult to obtain. Most of the structural details, however, can be seen in unstained mounts, and neither temporary nor permanent stains are needed. The organism is so characteristic in appearance that identification is usually made without difficulty. The common diagnostic characteristics are listed in table 7 and drawings of the trophozoite and cyst are presented in figure 3. COCCIDIA† The coccidian species of humans belong to the genera , Sarcocystis, and Cryptosporidium. The diagnostic stages are often difficult to locate in feces, and they are easily overlooked because of their almost transparent appearance. Correct lighting and careful focusing are essential for detection. In Isospora belli infections, immature oocysts are generally passed in feces, although fully mature oocysts may be present in some cases. In Sarcocystis hominis and Sarcocystis suihominis infections, mature oocysts or free single sporocysts are usually passed in feces. The oocysts and sporocysts of S. hominis and S. suihominis are more or less identical and reliable species identification cannot be made from these stages. When found, they are reported as " Sarcocystis species." Species differentiation is based on the intermediate host involved ( for S. hominis and swine for S. suihominis) and differences in the sarcocyst stage in the tissues of these hosts. In Cryptosporidium infections, either immature oocysts or mature oocysts containing four naked sporozoites (no sporocysts are present) are passed.

The usual permanently stained fecal smears are of little or no value in demonstrating coccidia. Cryptosporidium oocysts, however, can be identified in acid-fast stains and

† Despite the first human cases of being diagnosed in 1977 and 1978, the coccidian parasite was not classified until 1994. Please see the Cyclosporiasis chapter in this CD ROM.

3 stained preparations are routinely examined in diagnosing . Giemsa stains are also used but acid-fast stains are preferred by most workers.

Unstained wet mounts are generally used to detect and identify oocysts of Isospora and Sarcocystis, and may be of value for Cryptosporidium. Iodine and MIF stains, also may be helpful in some cases; iodine is particularly useful for distinguishing Cryptosporidium oocysts, which do not stain, from yeasts, which stain brown or yellow. The diagnostic characteristics of coccidia are listed in table 7; oocysts and sporocysts are presented in figure 3.

The immature oocysts of Isospora species frequently cannot be readily distinguished from immature oocysts of species, which parasitize lower animals. Occasionally, Eimeria may be accidentally ingested with tissue and passed out in human feces. Therefore, if the genus of coccidia is in doubt, it is preferable to identify the organisms as "coccidia " and attempt to obtain mature oocysts for positive identification. This can be done by mixing the feces containing oocysts with 2% potassium dichromate solution and letting them stand at room temperature for 48 hours or longer. Mature oocysts of Eimeria contain 4 sporocysts with 2 sporozoites each; those of Isospora contain 2 sporocysts with 4 sporozoites each.

BLASTOCYSTIS

Blastocystis hominis, long considered to be a yeast, has now been identified as a protozoan (Zierdt et al., 1967; Zierdt, 1973) and is currently classified in the subphylum Sporozoa (Zierdt and Tan, 1976; Zierdt, 1983). There is evidence to suggest that it may be pathogenic in some cases. Several forms, including round "granular forms" and "ameba forms," have been described from feces and cultures (Zierdt and Tan, 1976; Zierdt, 1973; Zierdt et al., 1967) but the stage most often recognized in stool specimens is the spherical "vacuolated form " (McClure et al., 1980). This form is described in table 7 and depicted in figure 3. TABLE 2 CHARACTERISTICS OF INTESTINAL FLAGELLATES, CILIATE, AND COCCIDIA VISIBLE IN DIFFERENT TYPES OF FECAL PREPARATIONS Characteristic Unstained Temporary Stains Permanent Stains Saline Formalin Iodine Neutral Red 1 (Cysts) (Trophs) Flagellates Trophozoites Motility + - + - Shape + + + + (may be distorted) Nucleus - + + + Flagella " - + " Other features 2 + + + + Cysts Shape + + + + Nuclei - + + + Fibrils " + + + Ciliate () Trophozoites Motility + - + - + + + + Cilia + + + + Cysts Macronucleus + + " + Coccidia Oocysts/Sporocysts + + + "3 1 Neutral red dye in methocel solutions. 2 The undulating membrane of Trichomonas and the spiral groove of may not be visible in all cases. 3 Cryptosporidium oocysts can be demonstrated in acid-fast stains.

4 HELMINTHS The common helminth parasites of humans belong to three groups: nematodes (roundworms), cestodes (tapeworms), and trematodes (flukes). The diagnostic stages of helminths are more easily identified by most technologists than the diagnostic stages of protozoa. Their distinguishing features are less variable, and being larger, they are more readily detected. Although the laboratory occasionally receives adult helminths, especially Ascaris and Enterobius, diagnosis is usually made from eggs, or in some cases, larvae, or body segments (proglottids). Differential characteristics of eggs are listed in table 8; characteristics of larvae are listed in table 9; and proglottids and scoleces are described in tables 10 and 11, respectively. Diagrams of eggs are presented in figures 4, 5, and 6; larvae are shown in figure 7; proglottids and scoleces are depicted in figure 8. Characteristics used to identify species of eggs are as follows: Size Shape Stage of development when passed in the feces Thickness of the eggshell Color Presence of structures such as mammillated coat, operculum, spine, or plugs Helminth eggs and larvae are best seen in unstained wet mounts. Neither temporary nor permanent stains are needed. Proglottids and scoleces usually require special clearing or staining procedures. NEMATODES The common diagnostic stage of the nematodes is the egg, except for Strongyloides stercoralis in which the first stage, or rhabditiform, larva is the usual diagnostic stage. Occasionally, hookworm larvae may also be present in feces and must be distinguished from those of Strongyloides. The rhabditiform larvae of hookworm and Strongyloides can be differentiated by the length of the buccal cavity and by the appearance of the genital primordium. In rare cases, filariform (third stage) larvae may be found in feces or they can be obtained by culturing specimens containing eggs or rhabditiform larvae. Filariform larvae differ from rhabditiform larvae in size and in the appearance of the esophagus. The third stage larva is about twice as long as the first stage larva and lacks the bulb configuration of the esophagus that is characteristic of the rhabditiform larva. The filariform larvae of Strongyloides are distinguished from those of hookworm by the appearance of the tip of the tail and the length of the esophagus. Characteristics of the eggs are listed in table 8; those of the larvae are described in table 9. Drawings of the nematode eggs are presented in figures 4 and 6; the larvae are presented in figure 7. Eggs of the nematode species are shed more or less continuously and may be present in every fecal specimen from an infected individual. Strongyloides larvae, however, are passed irregularly and will not be found in every specimen.

5 CESTODES In certain of the cestodes (Hymenolepis nana, Hymenolepis diminuta, and Diphyllobothrium latum), the egg is the usual diagnostic stage. In other species (Taenia and Dipylidium caninum), eggs may be present in feces, but the proglottid is the usual diagnostic stage. Except in the case of D. latum, the cestode eggs are embryonated when they leave the worm's body. The embryo is a six-hooked, spherical structure called an oncosphere. Diphyllobothrium latum eggs are unsegmented when passed and become embryonated outside of the host. They may resemble the eggs of hookworm, infertile Ascaris, or Paragonimus westermani and must be distinguished from these species. Occasionally, portions of the strobila (chain of proglottids) are passed, and the species can be identified by the characteristic appearance of the proglottids. The eggs of Taenia saginata and Taenia solium are identical, and species identification cannot be made from this stage. When found, they should be reported as "Taenia species." The common diagnostic stage, however, is the gravid proglottid rather than the egg. Proglottids, either singly or in short chains, become detached from the strobila and are passed with the feces. Taenia saginata proglottids occasionally migrate out of the anus independent of defecation. Specific identification is based on the number of lateral uterine branches in the gravid proglottid. These can be seen in uncleared segments, but they are more distinct in cleared proglottids. A hand lens or dissecting can be used to observe the structural details. The primary branches from one side of the central uterine stem are counted to distinguish between T. saginata and T. solium. Less frequently, and usually after treatment, scoleces may be obtained for species identification. Dipylidium caninum, a cestode parasite of dogs, , and occasionally humans, is also more frequently diagnosed by finding proglottids rather than eggs in the specimen. The eggs, when found, are usually contained in packets or capsules of 5 to 15 or more eggs. In rare cases, the scolex may be obtained for diagnosis. The eggs of the intestinal cestode species are described in table 8; the gravid proglottids are described in table 10; the scoleces are described in table 11. Drawings of the eggs are presented in figures 4 and 6, and drawings of gravid proglottids and scoleces are presented in figure 8. TREMATODES

The diagnostic stage of the trematode species parasitic in humans is the egg. The characteristics of the eggs of human species are described in table 8; drawings are presented in figures 5 and 6.

The eggs of Clonorchis, Opisthorchis, Heterophyes; and Metagonimus closely resemble each other, and for this reason, they are difficult to separate. The characteristics described in table 8 are not always apparent. The eggs of Fasciola hepatica and Fasciolopsis buski are also very similar, and it may be difficult to differentiate these species. Descriptions of the eggs of two additional species of schistosomes, Schistosoma mekongi, found in the Mekong River area of Southeast Asia, and Schistosoma intercalatum, found in Africa, are included in table 8. Both of these species inhabit the mesenteric venules, and eggs are passed in feces. The eggs of S. mekongi closely resemble those of Schistosoma japonicum except S. mekongi eggs are slightly smaller (see description in table 8). The eggs of S. intercalatum resemble those of S. haematobium except for minor differences in shape and size, as described in table 8. The S. intercalatum egg is longer and thinner than the S. haematobium egg, and the tip of the terminal spine of S. intercalatum may be bent.

6 Because the eggs of these newer species so closely resemble those of species already included in the drawings and because of space limitations, diagrams of S. mekongi and S. intercalatum are not included in figures 5 and 6.

The eggs of two of the human trematode parasites are ordinarily passed in urine (Schistosoma haematobium) or sputum (Paragonimus westermani)1. Eggs of both, however, are occasionally found in feces.

Since trematode eggs are usually found at irregular intervals in feces, several specimens may need to be examined to demonstrate their presence.

1Several species of Paragonimus have been identified as parasites of humans, but only P. westermani is included in this manual. The eggs of all species are similar but vary slightly in size and shape.

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1 Commensal form- usually found in asymptomatic or chronic cases; may contain bacteria. (Since original publication of this manual, more recent evidence supports differentiation of pathogenic E. histolytica from nonpathogenic E. dispar as two distinct species.) (Garcia et al, 1997) (Diamond et al, 1993)

2 Invasive form- usually found in acute cases; often contain red blood cells. 3 Flagellate- included with amebae for diagnostic purposes.

8 9

*

*Since initial publication of this manual, the genus name Trichomonas has been replaced by Pentatrichomonas. (Kirby, 1945) (Honigberg, 1978).

10 11 12

13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 REFERENCES TEXTBOOKS AND MANUALS Ash, L. R. and Orihel, T. C. 1997. Atlas of Human Parasitology. American Society of Clinical Pathologists, Chicago, . Beaver, P. C., Jung, R. C., and Cupp, E. W. 1984. Clinical Parasitology. Lea and Febiger, Philadelphia. Belding, D. L. 1965. Textbook of Parasitology. 3rd ed. Appleton-Century-Crofts, New York. Brooke, M. M., Melvin, D. M., and Healy, G. R. 1983. Common Intestinal Protozoa of Humans, Life Cycle Charts. Government Printing Office, Supt. of Documents, Washington, D.C. 20402. (GPO Stock #017-023-00164- 7) (NTIS #PB 80-186984)* Brown, H. W. and Neva, F. A. 1983. Basic Clinical Parasitology. 5th ed. Appleton- Century-Crofts, New York.

Burrows, R. B. 1965. Microscopical Diagnosis of the Parasites of Man. Yale University Press, New Haven, , and London. Centers for Disease Control. 1976. Amebiasis: Laboratory Diagnosis -Part II: Identification of Intestinal Amebae. HEW Pub. no. CDC- 77-8327, Part II. Government Printing Office, Washington, D.C. 20402 (GPO Stock #017-023- 00111-6) (NTIS #PB 80-145600)* Faust, E. C., Beaver, P. C. and Jung, R. C. 1975. Animal Agents and Vectors of Human Disease. 4th ed. Lea and Febiger, Philadelphia. Jeffrey, H.C. and Leach, R. M. 1966. Atlas of Medical Helminthology and Protozoology. Williams and Wilkins Co., Baltimore. Markell, E. K. and Voge, M. 1976. Diagnostic Medical Parasitology. 4th ed. W. B. Saunders, Philadelphia. Melvin, D. M., Brooke, M. M., and Sadun, E. H. 1959. Common Intestinal Helminths of Man, Life Cycle Charts, PHS Publication No.1235 (1964). Government Printing Office, Supt. of Documents, Washington, D.C. 20402. (NTIS #PB-80-186976)* Melvin, D.M. and Brooke, M.M., 1982. Laboratory Procedures for the Diagnosis of Intestinal Parasites. 3rd ed. HHS Pub. No. (CDC) 82-8282. Government Printing Office, Washington, D.C 20402. (GPO Stock #017-023-001-41-8)(NTIS #PB 83-124- 818)* Spencer, F. M., and Monroe, L. S. 1961. The Color Atlas of Intestinal Parasites. Charles C. Thomas Co., Springfield, Illinois. (Revised 1975.) RESEARCH PAPERS Camp, R. R., Mattern, C. F. T., and Honigberg, B. M. 1974. Study of Dientamoeba fragilis Jepps and Dobell. I. Electronmicroscopic observations of the binucleated stages. J. Protozool. 21(1): 69-79.

*NTIS- National Technical Information Service, 5285 Port Royal Road, Springfield, Virginia 22161

29 Harinasuta, C. and Kruatrachue, M. 1962. The first recognized endemic area of bilharziasis in Thailand. Ann. Trop. Med. Parasitol. 56:314-322. Heydorn, A. O., Gestrich, R., Mehlhorn, H., and Rommel, M. 1975. Proposal for a new nomenclature of the Sarcosporidia. Z. Parasitenk. 48:73-82. Honigberg, B. M. 1974. Study of Dientamoeba fragilis Jepps and Dobell. II. Taxonomic position and revision of the genus.J. Protozool. 21(1). 79-81. McClure, H. M., Strobert, E. A., and Healy, G. R. 1980. Blastocystis hominis in a pig-tailed macaque: a potential enteric for nonhuman primates. Lab. Anim. Sci. 30 (5) :890-894. Rommel, M. and Heydorn, A. O. 1972. Beiträge zum Lebenszykles der Sarkosporidien. III. Isospora hominis (Railliet und Lucet, 1891) Wenyon, 1923, eine Dauerform der Sarkosporidien des Rindes und des Schweins. Berl. u. München Tiertärztl. Wochenschr. 85(18):143-145. Taylor, R. G. and Moose, J. W. 1971. The egg from a human case of schistosomiasis in Laos. J. Parasitol. 57(1):78-80. Zierdt, C. H. 1973. Studies of Blastocystis hominis. J. Protozool. 20(1) :114-121. Zierdt, C. H. 1983. Blastocystis hominis, a protozoan parasite and intestinal pathogen of human beings. Clin. Microbiol. Newsletter 5(9): 57-59. Zierdt, C. H., Rude, W. S., and Bull, B. S. 1967. Protozoan characteristics of Blastocystis hominis. Am. J. Clin. Pathol. 48(5):495-501. Zierdt, C. H. and Tan, H. 1976. Endosymbiosis in Blastocystis hominis. Exp. Parasitol. 39:422-430. ADDITIONAL REFERENCES Diamond, L.S. and Clark, C.G. 1993. A redescription of Entamoeba histolytica Schaudinn, 1903 (amended Walker, 1911) separating it from Entamoeba dispar. Brumpt, J. Eukaryot. Microbiol. 40: 340-344. Garcia, L.S. and Bruckner, D.A. 1997. Diagnostic Medical Parasitology. 3rd ed. ASM Press, Washington, D.C. Honigberg, B.M. Trichomonads of importance in human medicine, pages 275-454, In: Parasitic Protozoa, Vol. II Intestinal flagellates, Histomonads, , Opalinids, and Ciliates, (ed) J.P. Kreier, Academic Press, 1978. Kirby, H. The structure of the common intestinal trichomonad of man. J. Parasitol. 31: 163-175, 1945.

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