PHIDIAS: Pathogen-Host Interaction Data Integration and Analysis System

Table of Contents:

Taxonomy Information
Lifecycle Information
Genome Information
Biosafety Information
Culturing Information
Epidemiology Information
Diagnostic Tests Information
Infected Hosts Information
Phinet: Pathogen-Host Interaction Network
Lab Animal Pathobiology & Management
References
Data Provenance and Curators

Taxonomy Information

Species:
1. Cryptosporidium parvum (Website 1):
  1. GenBank Taxonomy No.: 5807
  2. Description: Cryptosporidium parvum is a protozoan parasite belonging to the phylum Apicomplexa, subclass Coccidia. C. parvum causes a self-limiting infection of the small intestine in immunocompetent humans or animals, but it also can be persistent and life threatening in immunocompromised individuals, particularly those with AIDS. Even though C. parvum was described in 1907, it was not recognized as a pathogen of mammals until 1971 when the infection was linked to calf diarrhea. Chronic cryptosporidiosis became recognized with the emergence of the human immunodeficiency virus (HIV) and AIDS. Human cryptosporidiosis is attributed to two major genotypes, of which type 1 is found exclusively in humans, while type 2 is zoonotic and found in other mammals, including humans(Sestak et al., 2002). Cryptosporidium parvum is zoonotic, apparently lacking host specificity among mammals(Fayer et al., 1997).

Lifecycle Information

Monoxenous life cycle
1. Stage Information:
  1. Oocyst:
    - Size: Oocysts recovered from different host species may vary in size and shape ranging from 4.5 to 7.9 um in length by 4.2 to 6.5 um in width and being ovoid to elliptical in shape with shape indices (length/width) ranging from 1.0 to 1.4.
    - Shape: Oocysts recovered from different host species may vary in size and shape ranging from 4.5 to 7.9 um in length by 4.2 to 6.5 um in width and being ovoid to elliptical in shape with shape indices (length/width) ranging from 1.0 to 1.4.
    - Picture(s):
      - Stained Oocysts (Website 99)
        
        Description: Oocysts of Cryptosporidium parvum stained by the modified acid-fast method. Against a blue-green background, the oocysts stand out in a bright red stain. Sporozoites are visible inside the two oocysts to the right. Copyright: CDC.
    - Description: The oocyst is the stage transmitted from an infected host to a susceptible host by the fecal-oral route. Routes of transmission can be (1) person-to-person through direct or indirect contact, possibly including sexual activities, (2) animal-to-animal, (3) animal-to-human, (4) water-borne through drinking water or recreational water, (5) food-borne, and (6) possibly airborne. To determine how many oocysts of C. parvum were required for seronegative healthy persons to become infected, 29 volunteers ingested a single dose of 30 to 1 million oocysts from a calf. After ingesting 30 oocysts, one of five persons became infected. After ingesting 1000 or more oocysts seven of seven became infected. The median infective dose (ID50) was calculated to be 132 oocysts. With further data the ID50 was recalculated to be 87 oocysts and different isolates of C. parvum were found to have highly different ID50 values(Fayer et al., 2000). The sporulated oocyst is the only exogenous stage. Consisting of four sporozoites within a tough two-layered wall, it is excreted from the body of an infected host in the feces. The endogenous phase begins after the oocyst is ingested by a suitable host(Fayer et al., 1997).
  2. Sporozoite:
    - Size: Free sporozoites were fusiform and measured 3.5 to 4.2 x 0.53 to 0.6 um.
    - Shape: Free sporozoites were fusiform and measured 3.5 to 4.2 x 0.53 to 0.6 um.
    - Description: Sporozoites and merozoites of Cryptosporidium spp. appear similar to those of other coccidia with organelles typical of the phylum, such as the pellicle, rhoptries, micronemes, electron-dense granules, nucleus, ribosomes, subpellicular microtubules, and apical rings. However, they lack other organelles such as typical polar rings, mitochondria, micropores, and the conoid. Posterior to the apical rings, sporozoites and merozoites have a cylindrical collar that appears to be the site of origin for the inner membrane complex and the subpellicular microtubules(Fayer et al., 1997). Unlike other coccidia, the sporozoites are free within the oocysts and not surrounded by sporocysts(Spano and Crisanti, 2000).
  3. Trophozoite:
    - Shape: Spherical
    - Description: Trophozoites contain a prominent nucleolus within a single nucleus surrounded by cytoplasm, and a well-developed attachment/feeder organelle. During nuclear division of schizogony, division spindles, nuclear plaques, and centrioles have been observed(Fayer et al., 1997). The trophozoite stage is intracellular beneath the host cell membrane but is extracytoplasmic(Marshall et al., 1997). Upon attachment to an enterocyte and discharge of secretory molecules from the zoite apical organelles, C. parvum does not simply induce the typical invagination of the host plasma membrane (described, for example, in Plasmodium, Toxoplasma and Eimeria), but additionally initiates a profound structural rearrangement of the enterocyte microvilli, which elongate and eventually fuse around the invading zoite. The parasite thereby establishes itself intracellularly within a parasitophorous vacuole that is delimited by a host-derived membrane, but which lies in a singular extra-cytoplasmic position, giving the impression of being attached to the apical surface of the enterocyte. As a consequence of this peripheral location with respect to the host cell cytoplasm, all C. parvum intracellular stages (trophozoites, type I and type II meronts, gametocytes, zygotes and immature oocysts) develop the so-called feeder organelle, another peculiarity of the Cryptosporidium genus. This unique organelle has a multilamellar structure and is situated at the base of the parasitophorous vacuole. It is believed to mediate the uptake of nutrients from the host cell(Spano and Crisanti, 2000).
  4. Type I Meront:
    - Description: Asexual multiplication, called schizogony or merogony, results when the trophozoite nucleus divides. C. parvum has two types of schizonts or meronts. For C. parvum, type I schizonts develop six or eight nuclei, and each is incorporated into a merozoite, a stage structurally similar to the sporozoite(Fayer et al., 1997). Type I meronts form 8 merozoites which are liberated from the parasitophorous vacuole when mature(O'Donoghue, 1995). Upon attachment to an enterocyte and discharge of secretory molecules from the zoite apical organelles, C. parvum does not simply induce the typical invagination of the host plasma membrane (described, for example, in Plasmodium, Toxoplasma and Eimeria), but additionally initiates a profound structural rearrangement of the enterocyte microvilli, which elongate and eventually fuse around the invading zoite. The parasite thereby establishes itself intracellularly within a parasitophorous vacuole that is delimited by a host-derived membrane, but which lies in a singular extra-cytoplasmic position, giving the impression of being attached to the apical surface of the enterocyte. As a consequence of this peripheral location with respect to the host cell cytoplasm, all C. parvum intracellular stages (trophozoites, type I and type II meronts, gametocytes, zygotes and immature oocysts) develop the so-called feeder organelle, another peculiarity of the Cryptosporidium genus. This unique organelle has a multilamellar structure and is situated at the base of the parasitophorous vacuole. It is believed to mediate the uptake of nutrients from the host cell(Spano and Crisanti, 2000).
  5. Merozoite from Type I Meront:
    - Description: Sporozoites and merozoites of Cryptosporidium spp. appear similar to those of other coccidia with organelles typical of the phylum, such as the pellicle, rhoptries, micronemes, electron-dense granules, nucleus, ribosomes, subpellicular microtubules, and apical rings. However, they lack other organelles such as typical polar rings, mitochondria, micropores, and the conoid. Posterior to the apical rings, sporozoites and merozoites have a cylindrical collar that appears to be the site of origin for the inner membrane complex and the subpellicular microtubules(Fayer et al., 1997). Each mature merozoite, theoretically, leaves the schizont to infect another host cell and develop into another type I or type II schizont which produces four merozoites. It is thought that only merozoites from type II schizonts initiate sexual multiplication (gametogony) upon infecting new host cells by differentiating into either a microgamont (male) or macrogamont (female) stage(Fayer et al., 1997). Type I meronts form 8 merozoites which are liberated from the parasitophorous vacuole when mature. The merozoites then invade other epithelial cells where they undergo another cycle of type I merogony or develop into type II meronts(O'Donoghue, 1995).
  6. Type II Meront:
    - Description: Each mature merozoite, theoretically, leaves the schizont to infect another host cell and develop into another type I or type II schizont which produces four merozoites. It is thought that only merozoites from type II schizonts initiate sexual multiplication (gametogony) upon infecting new host cells by differentiating into either a microgamont (male) or macrogamont (female) stage(Fayer et al., 1997). The type II meronts form 4 merozoites which do not undergo further merogony but produce sexual reproductive stages (called gamonts)(O'Donoghue, 1995). Upon attachment to an enterocyte and discharge of secretory molecules from the zoite apical organelles, C. parvum does not simply induce the typical invagination of the host plasma membrane (described, for example, in Plasmodium, Toxoplasma and Eimeria), but additionally initiates a profound structural rearrangement of the enterocyte microvilli, which elongate and eventually fuse around the invading zoite. The parasite thereby establishes itself intracellularly within a parasitophorous vacuole that is delimited by a host-derived membrane, but which lies in a singular extra-cytoplasmic position, giving the impression of being attached to the apical surface of the enterocyte. As a consequence of this peripheral location with respect to the host cell cytoplasm, all C. parvum intracellular stages (trophozoites, type I and type II meronts, gametocytes, zygotes and immature oocysts) develop the so-called feeder organelle, another peculiarity of the Cryptosporidium genus. This unique organelle has a multilamellar structure and is situated at the base of the parasitophorous vacuole. It is believed to mediate the uptake of nutrients from the host cell(Spano and Crisanti, 2000).
  7. Merozoite from Type II Meront:
    - Description: Sporozoites and merozoites of Cryptosporidium spp. appear similar to those of other coccidia with organelles typical of the phylum, such as the pellicle, rhoptries, micronemes, electron-dense granules, nucleus, ribosomes, subpellicular microtubules, and apical rings. However, they lack other organelles such as typical polar rings, mitochondria, micropores, and the conoid. Posterior to the apical rings, sporozoites and merozoites have a cylindrical collar that appears to be the site of origin for the inner membrane complex and the subpellicular microtubules(Fayer et al., 1997). The type II meronts form 4 merozoites which do not undergo further merogony but produce sexual reproductive stages (called gamonts)(O'Donoghue, 1995).
  8. Microgamont:
    - Description: Microgamonts have been found less frequently than other stages. Immature microgamonts resemble schizonts but contain small, compact nuclei. The single surface membrane later doubles at sites around the margin where microgametes form(Fayer et al., 1997). Microgamonts develop into microgametocytes which produce up to 16 non-flagellated microgametes(O'Donoghue, 1995). Upon attachment to an enterocyte and discharge of secretory molecules from the zoite apical organelles, C. parvum does not simply induce the typical invagination of the host plasma membrane (described, for example, in Plasmodium, Toxoplasma and Eimeria), but additionally initiates a profound structural rearrangement of the enterocyte microvilli, which elongate and eventually fuse around the invading zoite. The parasite thereby establishes itself intracellularly within a parasitophorous vacuole that is delimited by a host-derived membrane, but which lies in a singular extra-cytoplasmic position, giving the impression of being attached to the apical surface of the enterocyte. As a consequence of this peripheral location with respect to the host cell cytoplasm, all C. parvum intracellular stages (trophozoites, type I and type II meronts, gametocytes, zygotes and immature oocysts) develop the so-called feeder organelle, another peculiarity of the Cryptosporidium genus. This unique organelle has a multilamellar structure and is situated at the base of the parasitophorous vacuole. It is believed to mediate the uptake of nutrients from the host cell(Spano and Crisanti, 2000).
  9. Microgamete:
    - Size: Microgametes are rod shaped (1.4 x 0.5 um for C. parvum), with a flattened anterior end.
    - Shape: Microgametes are rod shaped (1.4 x 0.5 um for C. parvum), with a flattened anterior end.
    - Description: Microgametes are rod shaped (1.4 x 0.5 um for C. parvum), with a flattened anterior end and lack both flagellae and mitochondria typically observed in microgametes of other coccidia. Most of the microgamete consists of a condensed nucleus. A plasmalemma completely surrounds the body. Beneath it a single membrane extends approximately two thirds the body length. Originating at an anterior conical structure, eight microtubules extend posterior in close proximity to the surface of the nucleus. Three to five concentric lamellae extend outward at 90 degrees to the long axis at the posterior margin of the apical cap. Electron-dense granules of undetermined function are found in the cytoplasm at midbody(Fayer et al., 1997).
  10. Macrogamont:
    - Size: Macrogamonts of C. parvum are approximately 4 to 6 um.
    - Shape: Macrogamonts of C. parvum are spherical to ovoid.
    - Description: Macrogamonts of C. parvum are approximately 4 to 6 um and spherical to ovoid, have a large central nucleus with a prominent nucleolus, and contain lipid bodies, amylopectin granules, and unique wall-forming bodies in the cytoplasm(Fayer et al., 1997). Upon attachment to an enterocyte and discharge of secretory molecules from the zoite apical organelles, C. parvum does not simply induce the typical invagination of the host plasma membrane (described, for example, in Plasmodium, Toxoplasma and Eimeria), but additionally initiates a profound structural rearrangement of the enterocyte microvilli, which elongate and eventually fuse around the invading zoite. The parasite thereby establishes itself intracellularly within a parasitophorous vacuole that is delimited by a host-derived membrane, but which lies in a singular extra-cytoplasmic position, giving the impression of being attached to the apical surface of the enterocyte. As a consequence of this peripheral location with respect to the host cell cytoplasm, all C. parvum intracellular stages (trophozoites, type I and type II meronts, gametocytes, zygotes and immature oocysts) develop the so-called feeder organelle, another peculiarity of the Cryptosporidium genus. This unique organelle has a multilamellar structure and is situated at the base of the parasitophorous vacuole. It is believed to mediate the uptake of nutrients from the host cell(Spano and Crisanti, 2000).
  11. Zygote:
    - Description: The fertilized macrogamont, or zygote, develops into an oocyst with either a thin or a thick wall(Fayer et al., 1997). The resultant zygotes undergo further asexual development (sporogony) leading to the production of sporulated oocysts containing 4 sporozoites(O'Donoghue, 1995). Upon attachment to an enterocyte and discharge of secretory molecules from the zoite apical organelles, C. parvum does not simply induce the typical invagination of the host plasma membrane (described, for example, in Plasmodium, Toxoplasma and Eimeria), but additionally initiates a profound structural rearrangement of the enterocyte microvilli, which elongate and eventually fuse around the invading zoite. The parasite thereby establishes itself intracellularly within a parasitophorous vacuole that is delimited by a host-derived membrane, but which lies in a singular extra-cytoplasmic position, giving the impression of being attached to the apical surface of the enterocyte. As a consequence of this peripheral location with respect to the host cell cytoplasm, all C. parvum intracellular stages (trophozoites, type I and type II meronts, gametocytes, zygotes and immature oocysts) develop the so-called feeder organelle, another peculiarity of the Cryptosporidium genus. This unique organelle has a multilamellar structure and is situated at the base of the parasitophorous vacuole. It is believed to mediate the uptake of nutrients from the host cell(Spano and Crisanti, 2000).
  12. Thin-walled oocyst:
    - Description: The fertilized macrogamont, or zygote, develops into an oocyst with either a thin or a thick wall. Those that develop into thick-walled oocysts have type I and II wall-forming bodies similar to other coccidia. Those that develop into thin-walled oocysts lack the characteristic wall-forming bodies. Initially, two unit membranes form simultaneously external to the plasmalemma, while the sporont separates from the feeder/attachment organelle. Then, wall-forming body material is transported or exocytosed across the oocyst pellicle (i.e. plasmalemma and inner membrane), where it forms a thin, moderately coarse outer layer and a finely granular inner layer. Between these two layers of the oocyst wall is an electron-lucent zone that consists of the two oocyst membranes sandwiched between the outer and inner layers of the oocyst wall. The outer layer of the wall is continuous and of uniform thickness. The inner layer contains a suture at one pole which spans 1/3 to 1/2 the circumference of the oocyst(Fayer et al., 1997). Some reports suggest that oocysts with thin walls release sporozoites that autoinfect the host, whereas those with thicker walls leave the body to infect other hosts(Fayer et al., 1997). Approximately 20% of the zygotes develop into thin-walled oocysts, which represent auto-infective life cycle forms that can maintain the parasite in the host. This stage and the persistent meronts are believed to be responsible for the life-threatening disease in immunodeficient persons who do not have repeated exposure to environmentally resistant forms(Marshall et al., 1997).
  13. Thick-walled oocyst:
    - Size: Oocysts recovered from different host species may vary in size and shape ranging from 4.5 to 7.9 um in length by 4.2 to 6.5 um in width and being ovoid to elliptical in shape with shape indices (length/width) ranging from 1.0 to 1.4.
    - Shape: Oocysts recovered from different host species may vary in size and shape ranging from 4.5 to 7.9 um in length by 4.2 to 6.5 um in width and being ovoid to elliptical in shape with shape indices (length/width) ranging from 1.0 to 1.4.
    - Description: The fertilized macrogamont, or zygote, develops into an oocyst with either a thin or a thick wall. Those that develop into thick-walled oocysts have type I and II wall-forming bodies similar to other coccidia. Those that develop into thin-walled oocysts lack the characteristic wall-forming bodies. Initially, two unit membranes form simultaneously external to the plasmalemma, while the sporont separates from the feeder/attachment organelle. Then, wall-forming body material is transported or exocytosed across the oocyst pellicle (i.e. plasmalemma and inner membrane), where it forms a thin, moderately coarse outer layer and a finely granular inner layer. Between these two layers of the oocyst wall is an electron-lucent zone that consists of the two oocyst membranes sandwiched between the outer and inner layers of the oocyst wall. The outer layer of the wall is continuous and of uniform thickness. The inner layer contains a suture at one pole which spans 1/3 to 1/2 the circumference of the oocyst. Some reports suggest that oocysts with thin walls release sporozoites that autoinfect the host, whereas those with thicker walls leave the body to infect other hosts(Fayer et al., 1997).
2. Progression Information:
  1. oocyst:
    - From Stage: Oocyst
    - To Stage: Sporozoite
    - Description: The oocyst is the stage transmitted from an infected host to a susceptible host by the fecal-oral route. Routes of transmission can be (1) person-to-person through direct or indirect contact, possibly including sexual activities, (2) animal-to-animal, (3) animal-to-human, (4) water-borne through drinking water or recreational water, (5) food-borne, and (6) possibly airborne(Fayer et al., 2000). The sporulated oocyst is the only exogenous stage. Consisting of four sporozoites within a tough two-layered wall, it is excreted from the body of an infected host in the feces. The endogenous phase begins after the oocyst is ingested by a suitable host. The oocyst wall of Cryptosporidium spp., like that of other coccidia, has distinct inner and outer layers but is unique in having a suture at one end. The suture dissolves during excystation, opening the wall through which the sporozoites leave the oocyst. Sporozoites excyst from the oocyst and parasitize epithelial cells of the gastrointestinal or respiratory tract(Fayer et al., 1997).
  2. sporozoite:
    - From Stage: Sporozoite
    - To Stage: Trophozoite
    - Description: Sporozoites excyst from the oocyst and parasitize epithelial cells of the gastrointestinal or respiratory tract(Fayer et al., 1997). Invasion of a host cell by coccidian sporozoites is a dynamic event of considerable interest as the attachment and entry processes involve the sequential secretion of the contents of discrete compartments from within the sporozoite. The released materials are thought to participate in a number of ways, including the penetration event itself and the formation of the vacuolar membrane which initially surround the intracellular parasite. The machinery mediating this invasion process is collectively housed in the anterior region of the sporozoite and is known as the apical complex(Tetley et al., 1988). Each sporozoite differentiates into a spherical trophozoite(Fayer et al., 1997). Freed sporozoites attach to epithelial cells where they become enclosed within parasitophorous vacuoles and develop attachment organelles (stages generally referred to as trophozoites)(O'Donoghue, 1995).
  3. trophozoite:
    - From Stage: Trophozoite
    - To Stage: Type I Meront
    - Description: Asexual multiplication, called schizogony or merogony, results when the trophozoite nucleus divides (Fayer et al., 1997). C. parvum has two types of schizonts or meronts. For C. parvum, type I schizonts develop six or eight nuclei, and each is incorporated into a merozoite, a stage structurally similar to the sporozoite(Fayer et al., 1997).
  4. MerontI:
    - From Stage: Type I Meront
    - To Stage: Merozoite from Type I Meront
    - Description: Type I meronts form 8 merozoites which are liberated from the parasitophorous vacuole when mature. The merozoites then invade other epithelial cells where they undergo another cycle of type I merogony or develop into type II meronts(O'Donoghue, 1995). Each mature merozoite, theoretically, leaves the schizont to infect another host cell and develop into another type I or type II schizont which produces four merozoites. It is thought that only merozoites from type II schizonts initiate sexual multiplication (gametogony) upon infecting new host cells by differentiating into either a microgamont (male) or macrogamont (female) stage(Fayer et al., 1997).
  5. merozoite:
    - From Stage: Merozoite from Type I Meront
    - To Stage: Type II Meront
    - Description: Each mature merozoite, theoretically, leaves the schizont to infect another host cell and develop into another type I or type II schizont which produces four merozoites(Fayer et al., 1997). The type II meronts form 4 merozoites which do not undergo further merogony but produce sexual reproductive stages (called gamonts)(O'Donoghue, 1995).
  6. MerontII:
    - From Stage: Type II Meront
    - To Stage: Merozoite from Type II Meront
    - Description: It is thought that only merozoites from type II schizonts initiate sexual multiplication (gametogony) upon infecting new host cells by differentiating into either a microgamont (male) or a macrogamont (female) stage(Fayer et al., 1997).
  7. merozoite2:
    - From Stage: Merozoite from Type II Meront
    - To Stage: Macrogamont
    - Description: Each mature merozoite, theoretically, leaves the schizont to infect another host cell and develop into another type I or type II schizont which produces four merozoites. It is thought that only merozoites from type II schizonts initiate sexual multiplication (gametogony) upon infecting new host cells by differentiating into either a microgamont (male) or macrogamont (female) stage (Fayer et al., 1997). Macrogamonts of C. parvum are approximately 4 to 6 um and spherical to ovoid, have a large central nucleus with a prominent nucleolus, and contain lipid bodies, amylopectin granules, and unique wall-forming bodies in the cytoplasm(Fayer et al., 1997).
  8. merozoite2:
    - From Stage: Merozoite from Type II Meront
    - To Stage: Microgamont
    - Description: Each mature merozoite, theoretically, leaves the schizont to infect another host cell and develop into another type I or type II schizont which produces four merozoites. It is thought that only merozoites from type II schizonts initiate sexual multiplication (gametogony) upon infecting new host cells by differentiating into either a microgamont (male) or macrogamont (female) stage (Fayer et al., 1997). Microgamonts have been found less frequently than other stages. Immature microgamonts resemble schizonts but contain small, compact nuclei. The single surface membrane later doubles at sites around the margin where microgametes form(Fayer et al., 1997).
  9. microgamont:
    - From Stage: Microgamont
    - To Stage: Microgamete
    - Description: Each microgamont becomes multinucleate, and each nucleus is incorporated into a microgamete, a sperm cell equivalent(Fayer et al., 1997).
  10. microgamete macrogamont:
    - From Stage: Microgamete, Macrogamont
    - To Stage: Zygote
    - Description: Little of the fertilization process of a macrogamont by a microgamete has been recorded, suggesting that the process is rapid. Microgametes attach at their apical cap to the surface of host cells harboring macrogamonts. Only the microgamete nucleus and associated microtubules have been observed within macrogamonts. Fusion of nuclei has not been observed (Fayer et al.,1997). It is assumed that only fertilized macrogamonts develop into oocysts that sporulate in situ and contain four sporozoites (Fayer et al., 1997). The fertilized macrogamont, or zygote, develops into an oocyst with either a thin or a thick wall(Fayer et al., 1997).
  11. zygote:
    - From Stage: Zygote
    - To Stage: Thin-walled oocyst, Thick-walled oocyst
    - Description: The fertilized macrogamont, or zygote, develops into an oocyst with either a thin or a thick wall (Fayer et al., 1997). Some reports suggest that oocysts with thin walls release sporozoites that autoinfect the host, whereas those with thicker walls leave the body to infect other hosts(Fayer et al., 1997). Approximately 20% of the zygotes develop into thin-walled oocysts, which represent auto-infective life cycle forms that can maintain the parasite in the host. This stage and the persistent meronts are believed to be responsible for the life-threatening disease in immunodeficient persons who do not have repeated exposure to environmentally resistant forms(Fayer et al., 1997, Marshall et al., 1997).
3. Picture(s):
  - Illustration of lifecycle (Website 99)
    
    Description: Life cycle of Cryptosporidium parvum and C. hominis. (from: Juranek DD. Cryptosporidiosis. In: Strickland GT, Editor. Hunters Tropical Medicine, 8th edition.) Sporulated oocysts, containing 4 sporozoites, are excreted by the infected host through feces and possibly other routes such as respiratory secretions (1). Transmission of Cryptosporidium parvum and C. hominis occurs mainly through contact with contaminated water (e.g., drinking or recreational water). Occasionally food sources, such as chicken salad, may serve as vehicles for transmission. Many outbreaks in the United States have occurred in waterparks, community swimming pools, and day care centers. Zoonotic and anthroponotic transmission of C. parvum and anthroponotic transmission of C. hominis occur through exposure to infected animals or exposure to water contaminated by feces of infected animals (2). Following ingestion (and possibly inhalation) by a suitable host (3), excystation occurs. The sporozoites are released and parasitize epithelial cells (b,c) of the gastrointestinal tract or other tissues such as the respiratory tract. In these cells, the parasites undergo asexual multiplication (schizogony or merogony) (d, e, f) and then sexual multiplication (gametogony) producing microgamonts (male)(g) and macrogamonts (female)(h). Upon fertilization of the macrogamonts by the microgametes (i), oocysts (j, k) develop that sporulate in the infected host. Two different types of oocysts are produced, the thick-walled, which is commonly excreted from the host (j), and the thin-walled oocyst (k), which is primarily involved in autoinfection. Oocysts are infective upon excretion, thus permitting direct and immediate fecal-oral transmission. Copyright: CDC.
4. Description: C. parvum is an obligate intracellular parasite, transmitted as highly durable oocysts in feces. Ingested oocysts excyst in the ileum, releasing sporozoites which infect the intestinal epithelium. Subsequent development includes both a cyclic asexual reproduction and the production of gametes giving rise to further oocysts, which are either excreted or reinfect the host(Bankier et al., 2003). Cryptosporidium spp. have a monoxenous life cycles where all stages of development (asexual and sexual) occur within one host(O'Donoghue, 1995).

Genome Summary

Genome of Cryptosporidium parvum
1. Description: Electrophoretic analysis suggests that the C. parvum genome contains eight chromosomes of between 0.9 and 1.5 Mbp, giving a total genome size of 10.4 Mbp (Bankier et al., 2003)(Bankier et al., 2003). Three main sequencing projects are in progress, two in the US and one in the UK. An Expressed Sequence Tag (EST) project is being carried out at the University of California, with the objective of determining 300- to 600-bp-long single-pass sequences from about 1000 genes expressed in the sporozoite stage. The project involves sequencing the 5' ends of cDNA inserts randomly selected from three directionally cloned cDNA libraries, which were constructed in the phage vector Uni-ZAP XR using the mRNA of C. parvum sporozoites (Iowa isolate, genotype 2). Almost 600 ESTs have been analyzed so far, of which 68% represent unique sequences corresponding to about 265 kb of novel coding genetic information. Thirty-seven percent of the non redundant ESTs shared significant homology with GenBank sequences. A collaborative Gene Sequence Tag (GST) project is being conducted by researchers at the Universities of California and Minnesota. Unlike the EST approach, the GST project consists in the generation of short DNA sequences from random genomic inserts of the Iowa isolate, thus enabling identification of genes that are expressed in all developmental stages of the parasite, as well as genomic sequences devoid of coding function. Over 2000 GSTs have been characterized so far by sequencing about 500 bp from both ends of randomly sheared genomic DNA inserts derived from a pBlueScript II (SK-) plasmid library. Overall, the sequenced GSTs account for approximately 10% of the C. parvum genome, with about 20% of unique GSTs showing significant homology with sequences present in GenBank. A slightly different approach to the characterization of random C. parvum genomic sequences, a Sequence Tagged Sites project, has been undertaken by scientists at the MRC in Cambridge, UK Cryptosporidium parvum genomic DNA (Moredun isolate, genotype 2) was cut to completion with either AluI or RsaI and the fragments were cloned into the M13mp18 phage vector to obtain two distinct libraries. Randomly selected clones were analyzed by single-pass sequencing, yielding 161 Sequence Tagged Sites greater than 100 bp for a total of 43 kb of unique sequence information. The same group has recently commenced a new project aimed at determining the nucleotide sequence of the whole genome of the Iowa C. parvum isolate(Spano and Crisanti, 2000). Cryptosporidium parvum appears to contain neither mitochondria (although they have been observed in other Cryptosporidium spp.) nor the plastid commonly found in apicomplexan parasites(Piper et al., 1998).
2. Chromosome 6(Website 101)
  1. GenBank Accession Number: BX526834
  2. Size: 1164703 bp for the complete sequence of C. parvum chromosome 6(Website 101).
  3. Gene Count: A total of 474 protein-coding genes are predicted, giving a mean density of one per 2.46 kbp. Only 122 of the genes (25.7%) have predicted introns delimited by the usual eukaryotic GT...AG motifs at either end and these have an average of 2.7 exons per gene (Bankier et al., 2003). With a mean density of one predicted gene per 2.46 kb, chromosome 6 of C. parvum is particularly gene-dense. In comparison, the P. falciparum genome (approximately twice the size of that of C. parvum) contains one predicted gene per 4.3 kb and, among the fully sequenced eukaryotes, only S. cerevisiae and Encephalitozoon cuniculi have higher densities of predicted genes(Bankier et al., 2003).
  4. Description: The apicomplexan Cryptosporidium parvum is one of the most prevalent protozoan parasites of humans. We report the physical mapping of the genome of the Iowa isolate, sequencing and analysis of chromosome 6, and 0.9 Mbp of sequence sampled from the remainder of the genome. To construct a robust physical map, we devised a novel and general strategy, enabling accurate placement of clones regardless of clone artifacts. Analysis reveals a compact genome, unusually rich in membrane proteins. As in Plasmodium falciparum, the mean size of the predicted proteins is larger than that in other sequenced eukaryotes. We find several predicted proteins of interest as potential therapeutic targets, including one exhibiting similarity to the chloroquine resistance protein of Plasmodium. Coding sequence analysis argues against the conventional phylogenetic position of Cryptosporidium and supports an earlier suggestion that this genus arose from an early branching within the Apicomplexa. In agreement with this, we find no significant synteny and surprisingly little protein similarity with Plasmodium. Finally, we find two unusual and abundant repeats throughout the genome. Among sequenced genomes, one motif is abundant only in C. parvum, whereas the other is shared with (but has previously gone unnoticed in) all known genomes of the Coccidia and Haemosporida. These motifs appear to be unique in their structure, distribution and sequences(Bankier et al., 2003).

Biosafety Information

Biosafety information for Cryptosporidium parvum
1. Level: 2.
2. Precautions: Biosafety Level 2 practices and facilities are recommended for activities with infective stages of the parasites listed. Primary containment (e.g., biological safety cabinet) or personal protection (e.g., face shield) may be indicated when working with cultures of Naegleria fowleri, or Cryptosporidium(Website 104).

Culturing Information

Culture in Different Host Cells :
1. Description: The most successful host cells used to study C. parvum are epithelial-like. These include human colonic adenocarcinoma (Caco-2), human endometrial carcinoma (RL95-2), galactose-adapted human colonic carcinoma (HT29.74), human ileocaecal adenocarcinoma (HCT-8), and Madin-Darby canine kidney (MDCK). When development of C. parvum was compared in 11 different host cells, it was concluded that the yield of parasites in HCT-8 cells was superior to all other cells based on counts of live parasites grown in monolayers on coverslips. Both Caco-2 and MDCK cells are more delicate than HCT-8 cells, however, and host cells as well as parasites are easily disrupted when coverslips are inverted onto microscope slides, compromising observation and counting. MCDK cells were found superior to other cells in a similar study. Nevertheless, these studies demonstrate that one of the most important factors determining successful in vitro cultivation of C. parvum is the choice of host cell(Upton, 1997).
2. Medium: N-[2-hydroxyethyl]peperazine-N'[2-ethanesulfonic acid] (HEPES), the most commonly used buffer for in vitro cultivation of coccidia, has a pKa of 7.5 at 25 degrees celcius and useful buffering in a pH range of 6.8 to 8.2. When cultures of host cells are placed in a CO2 incubator, the pH falls well into the acidic range. Because C. parvum sporozoites are most infective in a pH range of 7.2 to 7.6, other types of biological buffers may prove more useful. Considering that Plasmodium spp gametocyte emergence and exflagellation can be induced by increasing the pH to 7.7 - 8.0 or by ion exchange mechanisms, the reason that so few C. parvum oocysts are generated in vitro may, in part, reflect microgamete inactivity at suboptimal pH(Upton, 1997). A medium was formulated that enhanced the numbers of C. parvum in vitro tenfold. This formula consisted of RPMI 1640 containing L-glutamate, supplemented with an additional 2mM L-glutamine, 15 mM HEPES buffer, 50 mM glucose, 35 ug/ml Asorbic acid, 4.0 ug/ml para-aminobenzoic acid, 2.0 ug/ml calcium pantothenate, and 1.0 ug/ml folic acid. After adjusting the pH to 7.4 and filter sterilizing the mixture, fetal bovine serum (FBS) was added to a concentration of 10%. Insulin and antibiotics were eliminated, however, 100 U/ml penicillin, 100 ug;/ml streptomycin, and 0.25 ug/ml amphotericin B can be used, if desired. Macrolides at high concentrations must be avoided as they inhibit C. parvum development in vitro(Upton, 1997).
3. Optimal pH: Both pH and extracellular ions significantly effect motility and penetration of host cells by Coccidian sporozoites. C. parvum sporozoites are most infective in a pH range of 7.2 to 7.6. Considering that Plasmodium spp gametocyte emergence and exflagellation can be induced by increasing the pH to 7.7 - 8.0 or by ion exchange mechanisms, the reason that so few C. parvum oocysts are generated in vitro may, in part, reflect microgamete inactivity at suboptimal pH(Upton, 1997).
4. Doubling Time: Parasite numbers reach a maximum approximately 48 to 72 h after inoculation. A general rule of thumb is that developing stage number will equal (or roughly double) the number of original oocysts or sporozoites in the inoculum(Arrowood, 2002).
5. Note: Numbers of parasites used to infect cultures will greatly influence the final number of developmental stages. Generally lower parasite to host cell ratios result in proportionally higher levels of infection. An oocyst-to-host cell ratio of about 1:1 or 1:2 is generally optimal for C. parvum. A higher ratio should be employed for short-term binding assays(Upton, 1997). Truly ideal and routine methods that support cryptosporidial development in vitro are not yet available. Methods that permit continuous development (as is routinely used with Toxoplasma spp.), efficient production of mature, infectious oocysts, and cryopreservation methods that would allow cloned stocks to be developed are missing. Consequently, primary isolates from clinical specimens are quite valuable for ongoing research studies(Arrowood, 2002).

Epidemiology Information:

Outbreak Locations:
1. The mean prevalence rate for Cryptosporidium infection is between 1 and 3% in Europe and North America but is considerably higher in underdeveloped continents, ranging from 5% in Asia to approximately 10% in Africa(Marshall et al., 1997). Cryptosporidiosis has now been reported from over 40 countries in six continents in both immunocompetent as well as immunocompromised patients around the world. In a review of over 130,000 presumably immunocompetent patients with diarrhea in 43 studies in developing areas (Asia, Africa and Latin America) and in 35 studies in industrialized countries (in Europe, North America and Australia), it was noted that 6.1% and 2.1% in developing and developed areas with diarrhea (vs. 1.5% and 0.15% in controls without diarrhea) had Cryptosporidium infections(Dillingham et al., 2002).
Transmission Information:
1. From: Mammal(Casemore et al., 1997, Dillingham et al., 2002). , To: Human(Fayer et al., 2000). , With Destination:Human(Fayer et al., 1997). --(at lifecycle progression level: oocyst)
  Mechanism: Cryptosporidium parvum has been identified in about 80 species of mammals, and cross-transmission has been demonstrated between a variety of host species. There is, therefore, a potentially large zoonotic reservoir for animals and humans(Casemore et al., 1997). While apparently not the case for genotype 1 C. parvum, genotype 2 C. parvum and other genotypes and Cryptosporidium species increasingly appear to have less stringent host species specificities. An impressive range of 152 different mammalian species have been reported to be infected with C. parvum or with a C. parvum-like organism(Dillingham et al., 2002). To initiate infection, oocysts must be ingested with food, water, or by close personal contact with infected people, animals or contaminated surfaces(Fayer et al., 2000).
2. From: Environment(Dillingham et al., 2002). , To: Human(O'Donoghue, 1995). , With Destination:Human(Fayer et al., 1997). --(at lifecycle progression level: oocyst)
  Mechanism: Probably most common is waterborne transmission, whether in fully chlorinated drinking water (that has been contaminated usually via contaminated surface water) or by sewage effluent, since sewage treatment often does not kill the parasite. There have been some 50 waterborne outbreaks reported from throughout the US, UK, Canada and New Zealand, and the documentation of widespread fecal contamination with oocysts in wastewater, activated sludge, ground and surface water, and treated drinking water. Although questions remain about viability, species and sources of oocysts found in tap water, numerous outbreaks (including the huge Milwaukee outbreak) amply document the importance of waterborne transmission of C. parvum infections in humans(Dillingham et al., 2002). All infections have presumably been acquired by the ingestion (or inhalation) of infective oocysts excreted by infected hosts(O'Donoghue, 1995). Sporozoites excyst from the oocyst and parasitize epithelial cells of the gastrointestinal or respiratory tract(Fayer et al., 1997).
3. From: Environment(Dillingham et al., 2002). , To: Human(Fayer et al., 2000). , With Destination:Human(Fayer et al., 1997). --(at lifecycle progression level: oocyst)
  Mechanism: 31 outbreaks affecting over 10,000 people have associated cryptosporidiosis with exposure to recreational water, often despite its full chlorination, and often related to frequent fecal accidents by diapered infants, toddlers, or incontinent individuals(Dillingham et al., 2002). All infections have presumably been acquired by the ingestion (or inhalation) of infective oocysts excreted by infected hosts(O'Donoghue, 1995). Sporozoites excyst from the oocyst and parasitize epithelial cells of the gastrointestinal or respiratory tract(Fayer et al., 1997).
4. From: Environment(Dillingham et al., 2002). , To: Human(Fayer et al., 2000). , With Destination:Human(Fayer et al., 1997). --(at lifecycle progression level: oocyst)
  Mechanism: Several documented food-associated outbreaks have implicated fresh pressed cider in Maine and New York, improperly pasteurized milk in the UK, chicken salad in Minnesota, uncooked green onions in Spokane, Washington, and an infected cook who cut fresh vegetables and fruit in a Washington, DC cafeteria(Dillingham et al., 2002). All infections have presumably been acquired by the ingestion (or inhalation) of infective oocysts excreted by infected hosts(O'Donoghue, 1995). Sporozoites excyst from the oocyst and parasitize epithelial cells of the gastrointestinal or respiratory tract(Fayer et al., 1997).
5. From: Human(Dillingham et al., 2002). , To: Human(Fayer et al., 2000). , With Destination:Human(Fayer et al., 1997). --(at lifecycle progression level: oocyst)
  Mechanism: Person-to-person transmission occurred in households in 5.4% (of household contacts who developed symptomatic disease in the Milwaukee outbreak) to 19% (of family members of infected children in Fortaleza, Brazil developing disease or seroconversion) (Dillingham et al., 2002). Association with anal sexual exposure also likely reflects person-to-person direct spread as well(Dillingham et al., 2002). All infections have presumably been acquired by the ingestion (or inhalation) of infective oocysts excreted by infected hosts(O'Donoghue, 1995). To initiate infection, oocysts must be ingested with food, water, or by close personal contact with infected people, animals or contaminated surfaces(Fayer et al., 2000).
6. From: Environment(Fayer et al., 2000). , To: Human(Fayer et al., 2000). , With Destination:Human(Fayer et al., 1997). --(at lifecycle progression level: oocyst)
  Mechanism: It was shown that C. parvum oocysts ingested by Canada geese (Branta canadensis) and Peking ducks (Anas platyrhynchos) passed through the gastrointestinal tract, were excreted in the feces for nearly 1 week, and were capable of infecting mice. Later, viable oocysts of C. parvum were recovered from feces of Canada geese in fields where they rested along their migration route (Fayer et al., 2000). What appeared to be oocysts of C. parvum were found in the intestinal tracts of cockroaches (Periplaneta americana) collected in the household where a child had cryptosporidiosis, suggesting that roaches had a role in disseminating the parasite. House flies, exposed under laboratory conditions to bovine feces containing oocysts of C. parvum and wild filth flies trapped in a barn where a calf had cryptosporidiosis, had oocysts both in their feces and on their external surfaces. Although most oocysts of C. parvum ingested by dung beetles were destroyed by digestion, some passed through the intestinal tract and appeared morphologically normal in beetle feces. Oocysts also were recovered from the external surfaces of beetles, suggesting they may be capable of disseminating oocysts in the environment. Six genera of rotifers (microscopic invertebrates found worldwide in lakes, ponds, puddles, moss, damp soil, or virtually anywhere water can accumulate) were observed ingesting oocysts of C. parvum; it was not determined whether oocysts were digested or rendered nonviable(Fayer et al., 2000). All infections have presumably been acquired by the ingestion (or inhalation) of infective oocysts excreted by infected hosts(O'Donoghue, 1995). Sporozoites excyst from the oocyst and parasitize epithelial cells of the gastrointestinal or respiratory tract(Fayer et al., 1997).
7. From: Environment(Fayer et al., 2000). , To: Human(Fayer et al., 2000). , With Destination:Human(Fayer et al., 1997). --(at lifecycle progression level: oocyst)
  Mechanism: Although there have been no proven cases of airborne transmission in humans the concept was theorized by investigators in 1987. There are, however, numerous reports of high rates of cough or other pulmonary symptoms in children and immune compromised persons with cryptosporidiosis. Although lethal respiratory cryptosporidiosis has been reported for persons with AIDS, malignant lymphoma, and bone marrow transplantation, the occurrence of respiratory cryptosporidiosis is rarely reported(Fayer et al., 2000). All infections have presumably been acquired by the ingestion (or inhalation) of infective oocysts excreted by infected hosts(O'Donoghue, 1995). Sporozoites excyst from the oocyst and parasitize epithelial cells of the gastrointestinal or respiratory tract(Fayer et al., 1997).
Environmental Reservoir:
1. Mammalian Reservoir:
  1. Description: To date, C. parvum has been reported in some 79 mammalian species, and potentially all pose a health risk either by direct contact or indirectly through fecal contamination of food or water consumed by people(Tzipori and Griffiths, 2002). An impressive range of 152 different mammalian species have been reported to be infected with C. parvum or with a C. parvum-like organism(Dillingham et al., 2002).
  2. Survival: Most oocysts are fully sporulated and infective when excreted from infected hosts and they are very resistant to a range of environmental conditions. Laboratory studies have shown that oocysts stored in aqueous solutions have remained viable for up to 3 months at ambient temperature (15 to 20 C) and for up to one year when refrigerated (4 to 6 C). Infectivity was lost after oocysts had been heated to 65 C for at least 30 min or when desiccated for at least 4 h. Snap freezing has been shown to kill oocysts whereas slow freezing was less effective and some oocysts have survived freezing at -22 C for up to 1 month(O'Donoghue, 1995).
Intentional Releases:
1. Intentional Release Information:
  1. Description: Deliberate sabotage of industrialized water supplies is possible, but there is no evidence it has ever occurred, despite countless threats to municipal water supplies (Khan et al., 2001). The potential for water to serve as a vehicle for an agent and to cause mass casualties in the modern era was verified by the largest documented waterborne disease outbreak in the United States since record-keeping began in 1920. An estimated 403,000 people developed cryptosporidiosis in Milwaukee in 1993, of whom 4,400 were hospitalized and at least 54 died, in association with water obtained from a municipal water plant. Although the treated water met all the state and federal quality standards that were then in effect, C. parvum oocysts were able to get through the treatment system in sufficient numbers to infect a large proportion of the population served. Information based on mathematical modeling suggested that some individuals might have become infected when exposed to only one oocyst(Khan et al., 2001).
  2. Emergency Contact: Since 1971, CDC, the U.S. Environmental Protection Agency (EPA), and the Council of State and Territorial Epidemiologists have maintained a collaborative surveillance system consisting of the collection and periodic reporting of data on the occurrences and causes of waterborne-disease outbreaks (WBDOs) (Levy et al., 1998). State, territorial, and local public health agencies have the primary responsibility for detecting and investigating WBDOs and voluntarily reporting them to CDC on a standard form (CDC form 52.12). CDC annually requests reports from state and territorial epidemiologists or from persons designated as the WBDO surveillance coordinators. When needed, additional information about water quality and treatment is obtained from the state's drinking water agency(Levy et al., 1998).
  3. Delivery Mechanism: The use of food-borne and waterborne agents would be less likely than airborne agents in a large-scale attack, because it is difficult to expose many people. Standard treatment of municipal water supplies would preclude survival of most biological agents (Moran, 2002). C. parvum can be spread by contamination of food or water and has been involved in outbreaks related to swimming pools. Because it is resistant to chlorine, C. parvum can survive in swimming pools and municipal water supplies(Moran, 2002).
  4. Containment: Standard body fluid precautions should prevent spread of these organisms. Patients should be instructed to be extra vigilant about handwashing after using the bathroom(Moran, 2002).

Diagnostic Tests Information

Organism Detection Test:
1. Differential Staining Methods :
  1. Description: Conventional detection methods include concentration and staining of fecal smears. Differential staining methods including safranin-methylene blue stain, Kinyoun, Ziehl-Neelsen and DMSO-carbol fuchsin stain oocysts red and counterstain the background. Differential staining, however, is time consuming and varies in sensitivity and specificity. Fluorochrome stains, although sensitive, are complex and oocyst-like structures in fecal debris often take up the stain. Negative staining techniques with nigrosin, light green, merbromide and malachite green stain background yeasts and bacteria but not oocysts. Many of these stains require an experienced microscopist, however, and are labor-intensive(Fayer et al., 2000). The "gold standard" and perhaps most widely used test for the detection of Cryptosporidium oocysts in stool remains the modified acid-fast or Kinyoun stain. The test should be specifically requested, because it will not be performed as part of a routine examination for ova and parasites. Interpretation of the stained smear requires experience, because other organisms in the stool may stain acid fast(Leav et al., 2003). Conventionally, diagnosis is made by concentration of stools followed by acid-fast staining (AF) or immunofluorescent staining. The threshold of detection in human stool specimens by these methods may require the presence of 50,000 (immunofluorescent staining) to 500,000 (AF) oocysts per g of stool(Balatbat et al., 1996).
2. Indirect Immunofluorescent Assays :
  1. Description: Immunologic techniques for the detection of cryptosporidia in stool specimens were introduced in 1985 and 1986. Indirect immunofluorescent assays were described for the detection of oocysts employing convalescent human serum and oocyst-immunized rabbit antiserum. Innumofluorescent assays employing oocyst-reactive monoclonal antibodies were also introduced. The immunofluorescent methodologies showed significantly increased sensitivities and specificities compared to conventional staining techniques and have found widespread application in research and clinical laboratories, as well as for monitoring oocyst presence in environmental samples. The assays generally work well with fresh or preserved stools (formalin, potassium dichromate), but some fixatives can cause problems (e.g. MIF)(Arrowood, 1997). Immunofluorescence assays demonstrating cryptosporidial life-cycle stages (e.g. oocysts) in infected tissues or biopsy specimens have been reported and can be performed using reagents available in commercial diagnostic kits. These immuno histological assays are primarily of research value, given the broad availability of stool-based diagnostic assays and the ready identification of Cryptosporidium in tissue sections (cryptosporidia are uniquely found on the lumenal surface of epithelial cells and are apparent in specimens stained with hematoxylin and eosin or other routine histology stains(Arrowood, 1997). Several immunofluorescent assays and EIA kits have become commercially available and show promising sensitivity and specificity. These tests use antibodies against Cryptosporidium antigens to detect the parasite in stool specimens. One of these kits, the ColorPAC Cryptosporidium/Giardia rapid assay (Becton-Dickinson), was recently recalled because of a cluster of false-positive results(Leav et al., 2003).
3. Direct Fluorescent Antibody :
  1. Description: Direct Fluorescent antibody. The most widely used antigen detection immunoassays for Giardia and Cryptosporidium are the direct fluorescent-antibody (DFA) tests, which detect intact organisms, and enzyme immunoassays (EIAs), which detect soluble stool antigen. DFA tests utilize fluorescein-labeled antibodies directed against cell wall antigens of Giardia cysts and Cryptosporidium oocysts and allow visualization of the intact parasites, providing a definitive diagnosis. The sensitivity and specificity of the most commonly used commercial DFA test, the MERIFLUOR DFA test, have been reported to be 96 to 100% and 99.8 to 100%, respectively, for both Giardia and Cryptosporidium. This test has a greater sensitivity than traditional examination of permanent smears for Giardia and a sensitivity equal to or greater than that of traditional examination of permanent smears prepared from concentrated stool specimens for Cryptosporidium(Johnston et al., 2003).
4. Oocyst concentration :
  1. Description: The flotation-concentration method by Sheather was found to provide the best results of all selected methods. The merthiolate iodine formaldehyde concentration (MIFC) method was the least specific one. The least suitable method concerning sensitivity and costs was the flotation method with caesium chloride (CsCl) with a specificity of 29%(Kvac et al., 2003). Only Sheather's method detected all samples as positive(Kvac et al., 2003).
Immunoassay Test:
1. Antigen-capture ELISA :
  1. Description: The diagnosis of the small (4- to 6-microns) Cryptosporidium oocysts is labor intensive and relies on stool concentration, with subsequent staining and microscopy. The primary purpose of this study was to evaluate the clinical utility of an antigen capture enzyme-linked immunosorbent assay (ELISA) in detecting Cryptosporidium oocysts in human stools. A total of 591 specimens (76 diarrheal, 515 control) obtained from 213 inhabitants of an urban slum in northeastern Brazil were examined by both ELISA and conventional microscopic examination (CME) of formalin-ethyl acetate- concentrated stool samples stained with modified acid-fast and auramine stains. Forty-eight diarrheal stools (63.2%) were positive for Cryptosporidium oocysts by CME, with 40 of these positive by ELISA. Thirty-five control stools (6.8%) had Cryptosporidium oocysts detected by CME, with 15 of these also positive by ELISA. All of the 480 nondiarrheal stools and all but one of the diarrheal stools negative by CME were negative by ELISA. The test had an overall sensitivity of 66.3% and a specificity of 99.8% (positive predictive value, 98.2%; negative predictive value, 94.8%). In the evaluation of human diarrheal stool samples, the test sensitivity increased to 83.3%, with a specificity of 96.4%, and, in analysis of samples from individual patients with diarrhea, the sensitivity was 87.9%, with a specificity of 100%. These results indicate that this stool ELISA is sensitive and specific for the detection of Cryptosporidium oocysts in human diarrheal stool specimens but has limited use in epidemiologic studies for the diagnosis of asymptomatic Cryptosporidium infection(Newman et al., 1993).
  2. False Positive: We evaluated a commercially produced enzyme-linked immunosorbent assay (ELISA; LMD Laboratories, Inc.) for the detection of Cryptosporidium spp. in 296 stool specimens submitted to the Mayo Clinic parasitology laboratory for routine examination. Eight ELISA false negatives and one false positive were observed(Rosenblatt and Sloan, 1993).
  3. False Negative: We evaluated a commercially produced enzyme-linked immunosorbent assay (ELISA; LMD Laboratories, Inc.) for the detection of Cryptosporidium spp. in 296 stool specimens submitted to the Mayo Clinic parasitology laboratory for routine examination. Eight ELISA false negatives and one false positive were observed(Rosenblatt and Sloan, 1993).
2. Enzyme immunoassays (EIAs) :
  1. Description: The most widely used antigen detection immunoassays for Giardia and Cryptosporidium are the direct fluorescent-antibody (DFA) tests, which detect intact organisms, and enzyme immunoassays (EIAs), which detect soluble stool antigen (Johnston et al., 2003). Commercially available EIAs use antibodies for the qualitative detection of Giardia- and Cryptosporidium-specific antigens in preserved stool specimens. The reported sensitivities of EIAs range from 94 to 97% and specificities range from 99 to 100%. Advantages of the EIA are as follows: (i) numerous samples can be screened at one time, and tests can be read objectively on a spectrophotometer instead of subjectively on a fluorescence microscope. However, problems with false-positive and false-negative test results have been reported(Johnston et al., 2003).
  2. False Positive: 62 false-positive results obtained with the Alexon ProSpecT Cryptosporidium enzyme immunoassay were deemed false-positive based on negative results obtained from extensive microscopic examinations(Doing et al., 1999).
3. Combination cassette format nonenzymatic rapid immunoassay for detection of Giardia and Cryptosporidium antigens :
  1. Time to Perform: minutes-to-1-hour
  2. Description: For Cryptosporidium, the detection system consisted of an immobilized monoclonal capture antibody and a colloidal-carbon-labeled monoclonal detector antibody, both directed against oocyst antigens. The assay procedure involved the addition of 2 drops of sample treatment buffer to a tube, the pipeting of 60 microliters (ul) of stool specimen diluted in fixative or transport medium into the tube, the addition of 2 drops of a Giardia capture antibody conjugate, and the addition of 2 drops of a colloidal-carbon-conjugated detection reagent for Giardia and Cryptosporidium. After the sample was mixed, it was immediately poured into the test device. Assay results were read after 10 min. Positive results were visualized as grey-black lines in the appropriate position in the results window. Samples showing discrepancies between microscopy and the rapid assay were analyzed using microplate EIAs for Giardia and Cryptosporidium(Chan et al., 2000).
  3. False Positive: The six Cryptosporidium false-positive samples came from two patients, indicating that the positive rapid assay results were reproducible across specimens from the same individuals. Testing of these samples using the Cryptosporidium microplate EIA showed them all to be Cryptosporidium negative, as did reexamination of the microscopy slides(Chan et al., 2000).
4. ImmunoCard STAT! Cryptosporidium/Giardia rapid assay (Meridian Bioscience, Inc.) :
  1. Time to Perform: minutes-to-1-hour
  2. Description: The ImmunoCard STAT! Cryptosporidium/Giardia rapid assay (Meridian Bioscience, Inc.) is a solid-phase qualitative immunochromatographic assay that detects and distinguishes between Giardia lamblia and Cryptosporidium parvum in aqueous extracts of human fecal specimens (fresh, frozen, unfixed, or fixed in 5 or 10% formalin or sodium acetate-acetic acid-formalin). By using specific antibodies, antigens specific for these organisms are isolated and immobilized on a substrate. After the addition of appropriate reagents, a positive test is detected visually by the presence of a gray-black color bar (regardless of the intensity) next to the organism name printed on the test device (Garcia et al., 2003). The assay can be performed in approximately 12 min on formalin-fixed (5 or 10% formalin or sodium acetate-acetic acid-formalin) or unfixed stool specimens(Garcia et al., 2003).
  3. False Negative: The one specimen false negative for C. parvum was confirmed to be positive by immunofluorescene(Garcia et al., 2003).
5. Immunochromatographic Dip-Strip Test for the Detection of Cryptosporidium Oocysts in Stool Specimens :
  1. Time to Perform: minutes-to-1-hour
  2. Description: In the Crypto-Strip immunochromatographic test, liquid sample migrates by capillary action up the test strip, first rehydrating a specific anti-Cryptosporidium, gold-conjugate, monoclonal mouse antibody and then reaching a nitrocellulose membrane. A first line in the membrane (detection line) presents anti-Cryptosporidium monoclonal antibodies. A second line (control line) displays anti-mouse-immunoglobulin antibodies. On reaching the second line, the remaining conjugate is blocked. Gold-conjugate antibody fixed on either line appears as a pink/purple color. In order to perform the test, approximately 50 mg of stool sample is added to a tube containing 0.5 ml of a dilution buffer provided with the kit. After mixing and waiting for 1-2 min, one test strip is dipped into the stool suspension for 5-10 min at room temperature before being read(Llorente et al., 2002).
  3. False Negative: Forty-nine of the 50 known positive samples tested positive with the Crypto-Strip test, whereas all 25 negative samples tested negative(Llorente et al., 2002).
6. EIA. ColorPAC Giardia/Cryptosporidium rapid assay and ProSpecT Giardia/Cryptosporidium microplate assay for detection of Giardia and Cryptosporidium in fecal specimens :
  1. Time to Perform: unknown
  2. Description: Detection of Giardia and Cryptosporidium in clinical stool specimens using the ColorPAC and ProSpecT enzyme immunoassays revealed 98.7% agreement for Giardia detection and 98.1% agreement for Cryptosporidium detection. Sensitivities were uniformly 100%. The specificities of the ColorPAC immunoassay for Giardia and Cryptosporidium detection were 100 and 99.5%, respectively, and those for the ProSpecT assay were 98.4 and 98.6%, respectively. The false-positive reactions with the ProSpecT assay occurred with specimens that were grossly bloody(Katanik et al., 2001).
  3. False Positive: There was one false positive for Cryptosporidium in the ColorPAC test, and 3 in the ProSpecT test(Katanik et al., 2001).
Nucleic Acid Detection Test:


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