Morphology of Diagnostic Stages of Intestinal Parasites of

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Morphology of Diagnostic Stages of Intestinal Parasites of

Transcript Of Morphology of Diagnostic Stages of Intestinal Parasites of

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, Georgia 30333
HHS Publication No. (CDC) 89-8116
*Updated from the original printed version in 2001.
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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 flagellates, coccidia, and schistosomes have been added. Because of the increased number of drawings, those of the flagellates and of the ciliate 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 PROTOZOA…………………………………...………………………………. 1
Amebae……………...…………………………………………………..…... 1 Flagellates……………………………………………………………..…….. 2 Ciliate………………………………………...………………………………. 3 Coccidia………………………………………...……………………………. 3 Blastocystis………….………………………...….…………………..……... 4 HELMINTHS………………………………...….…………………….……... 5 Nematodes……………………………………….………………........…….. 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. Nematode 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
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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 Dientamoeba fragilis is a flagellate (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 Sarcocystis and Cryptosporidium 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 animals 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 Trichomonas 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 Cyclospora cayetanensis had not been classified.
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PROTOZOA
The intestinal protozoa of humans belong to four groups: amebae, flagellates, ciliates, 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 Giardia 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 tissue 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 life 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-iodineformaldehyde [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. Cytoplasm Appearance-finely granular, coarsely granular, or vacuolated. Inclusions-erythrocytes, bacteria, molds. Nucleus Number present. Peripheral chromatin- 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 genus name Trichomonas has been replaced by Pentatrichomonas. (Kirby, 1945) (Honigberg, 1978)
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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

-

-

+

-

(vacuole 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 cytostome. 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.

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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, Balantidium 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 Isospora, 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 (cattle 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 cyclosporiasis being diagnosed in 1977 and 1978, the coccidian parasite Cyclospora cayetanensis was not classified until 1994. Please see the Cyclosporiasis chapter in this CD ROM.
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stained preparations are routinely examined in diagnosing cryptosporidiosis. 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 Eimeria species, which parasitize lower animals. Occasionally, Eimeria may be accidentally ingested with animal 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 Saline Formalin

Temporary Stains

Iodine

Neutral Red 1

Permanent Stains

(Cysts)

(Trophs)

Flagellates

Trophozoites

Motility

+

-

+

-

Shape

+

+

+

+ (may be distorted)

Nucleus Flagella Other features 2

-

+

"

-

+

+

+

+

+

"

+

+

Cysts

Shape

+

+

+

+

Nuclei

-

+

+

+

Fibrils

"

+

+

+

Ciliate (Balantidium coli)

Trophozoites

Motility

+

-

+

-

Macronucleus

+

+

+

+

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 Chilomastix may not be visible in all cases. 3 Cryptosporidium oocysts can be demonstrated in acid-fast stains.

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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.
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StagesHumansMorphologyCoccidiaCharacteristics