Coccidian parasites- Cryptosporidiosis

Basic discussion on Coccidian parasites with a focus on Cryptosporidiosis -morphology, life cycle, pathogenesis, clinical manifestations, and laboratory diagnosis and management.

1. COCCIDIAN PARASITES  Cryptosporidium spp. Dr. Suprakash Das Assit . Prof.

2. Introduction Coccidia are small protozoans (one-celled organisms) which are members of the class Sporozoa Parasitic group that typically requires alternation of sexual and asexual reproduction in the life cycle. The major phyla of protozoa include  Sarcomastigophora (includes the amebae and flagellates), the Apicomplexa (malaria parasites, coccidia, and Babesia) , the Microspora (the microsporidia), and the Ciliophora ( Balantidium coli ) Several protozoan genera are included in the phylum Apicomplexa and are referred to as coccidia .

3. Introduction All organisms included in the Apicomplexa are  Unicellular with an apical complex typically composed of polar rings, rhoptries, micronemes, and A conoid; subpellicular microtubules and Micropores are common. These structures can be seen in electron microscopy studies and are used to help classify the various coccidia . Intestinal Coccidia -> Genera that develop in the gastrointestinal tract of vertebrates throughout their entire life cycle include  Eimeria, Isospora , Cyclospora, Cryptosporidium.

4. Introduction Tissue coccidia  Those that are capable of or, require extraintestinal development are referred to as cyst-forming coccidia and include  Besnoitia , Caryospora , Frenkelia , Hammondia , Neospora , Sarcocystis , Toxoplasma.

5. Coccidia of Medical Importance The coccidian genera that cause disease in humans include Cryptosporidium, Cyclospora , Isospora , Sarcocystis , and Toxoplasma

6. Species Shape and size Other features Cryptosporidium spp. Oocyst  Generally round, 4–6 μm , Each mature oocyst containing sporozoites Oocyst usual diagnostic stage in stool; various other stages in life cycle can be seen in biopsy specimens taken from  gastrointestinal tract (brush border of epithelial cells, intestinal tract) and possibly other tissues (respiratory tract, biliary tract)

7. Species Shape and size Other features Cyclospora cayetanensis Organisms generally round, 8–10 μm ; They mimic Cryptosporidium spp. (acid fast) but are larger In wet smears they look like nonrefractile spheres; They will also autofluoresce with epifluorescence; Are acid-fast variable from no color to light pink to deep red; those that do not stain may appear wrinkled; in a trichrome-stained stool smear, They will appear as clear, round, somewhat wrinkled objects; they cause diarrhea in both immunocompetent and immunosuppressed patients

8. Species Shape and size Other features Isospora belli Ellipsoidal oocyst; Usual range, 20–30 μm long and 10–19 μm wide; Sporocysts rarely seen broken out of oocysts but measure 9–11 μm Mature oocyst contains 2 sporocysts with 4 sporozoites each; usual diagnostic stage in feces is immature oocyst containing spherical mass of protoplasm (diarrhea, intestinal tract) Sarcocystis hominis , S. suihominis , S. bovihominis Oocyst thin-walled and contains 2 mature sporocysts, each containing 4 sporozoites; thin oocyst wall frequently ruptures; ovoid sporocysts each measure 9–16 μm long and 7.5–12 μm wide Thin-walled oocyst or ovoid sporocysts occur in stool (intestinal tract)

9. Species Shape and size Other features S. “ lindemanni ” Shapes and sizes of skeletal and cardiac muscle sarcocysts vary considerably Sarcocysts contain several hundred to several thousand trophozoites , each of which measures 4–9 μm in width and 12–16 μm in length; the sarcocysts may also be divided into compartments by septa, not seen in Toxoplasma cysts (tissue/ muscle)

10. Cryptosporidium spp. Cryptosporidium is an intestinal coccidian parasite that cause infection of small intestine and is an important cause of Diarrhoea . In 1907 , Tyzzer first described this parasite in the peptic glands of a laboratory mouse. Suggested the name, Cryptosporidium. Subsequently, it has been found in chickens, turkeys, mice, rats, guinea pigs, horses, pigs, calves, sheep, rhesusmonkeys , dogs, cats, and humans .

11. Ernest Tyzzer

12. Cryptosporidium spp. Recently, molecular techniques like Multilocus analysis is used for classification among different species of Cryptosporidium. There are currently 11 valid species of cryptosporidium . Recent information also supports previous suggestions that cryptosporidiosis is a zoonosis , is not host specific, and is transmitted via the fecal-oral route . Previously C. parvum and was thought to be the primary Cryptosporidium species infecting humans is now classified as two separate species  C. parvum (mammals , including humans) and C. hominis ( primarily humans )

13. Cryptosporidium spp. HABITAT It is found attached to the surface epithelial cells of villi or crypts of the small intestine. MORPHOLOGY The parasite shows 6 distinct morphological forms 1] Oocyst 4] Meront 2] Sporozoite 5] Microgamont 3] Trophozoite 6] Macrogamont .

14. Diagnostic forms in Human Oocyst - It is the diagnostic form excreted in human faeces . Cryptosporidium oocyst is the smallest coccidian oocyst known to cause infection in humans. It is colorless, spherical to oval in shape, 4.5-6 µm in diameter. It doesn’t stain with Iodine and is Acid fast . Each oocyst contains up to 4 slender and fusiform sporozoites . These 4 sporozoites remain parallel to each other and released only after partial digestion of oocyst .

15. Diagnostic forms in Human It contains 1-8 small granules. Micropile polar granules , which are always present in coccidian oocysts, are characteristically absent in Cryptosporidian Oocyst . The oocysts are excreted in small numbers in feaces . Its number has no relationship with the severity of illness.

16. Diagnostic forms in Human The oocysts which sporulate inside the host are of 2 types-1] Thick walled 2] Thin-walled The Thick walled oocysts are infectious to other susceptible human host. the Thin walled oocysts always cause Autoinfection in the same host only.

17. Life Cycle Humans acquire infection by the ingestion of food or drink that is contaminated with faeces , containing sporulated thick walled oocyst of Cryptosporidium . On ingestion, the sporozoites are realesed from the oocysts in the small intestine and invade the enterocytes which they parasitise . Cryptosdporidium completes its life cycle through  Stages of asexual generation ( Schizogony ) and Sexual generation ( Gametogony ) in a single host . All these stages are truly intracellular .

18. Life Cycle ASEXUAL GENERATION (SCHIZOGONY) Inside the enterocytes, the sporozoites subsequently differentiate  intracellular Trophozoites  transitional form of the parasite. Trophozoites are round or oval, 2-2.5 µm in diameter. Each trophozoite consists of a large nucleus. These trophozoites multiply asexually by nuclear division to produce  2 types of Meronts; Type-1 & Type-2.

19. Life Cycle (SCHIZOGONY) These Meronts are  Crescent shaped, 1- 1.5 µm in diameter, Showing rounded anterior and posterior ends. These type-2 meronts in turn produce 4 merozoites each, which are k/a- Type-2 Merozoites .

20. Cryptosporidium Oocyst Cryptosporidium parvum oocysts stained with modified acid-fast . Against a blue-green background, the oocysts stand out in a bright red stain.

21. Cryptococcus Sporozoite Gliding Motility Leads to Active Cellular Invasion by Cryptosporidium parvum Sporozoites

22. TROPHOZOITE of C. parvum Trophozoite of Cryptosporidium parvum located on the surface of intestinal epithelial cell.

23. Mature Type-1 Meront It contains 6 or 8 Merozoites and in the pic 8 Merozoites .

24. Type I and Type II meronts Electron micrograph of developing Type I and Type II meronts after 2 h in culture. (a) Type II meront , showing three merozoites budding from the residual body with rhoptries (arrowheads). Inset: Para-crystalline substructure inside of the rhoptry's bulb region. Bar = 1 m.  

25. Type I and Type II meronts (b, c)  More than five early budding merozoites at the periphery are visible within the Type I meront, with several rhoptries (arrowheads) and amylopectin granules (Am). (d, e)  Longitudinal sections of a Type II meront (8 h), showing merozoites with 2–8 rhoptries (arrowheads) and rod-like micronemes. (f) Three separated meronts inside of an oocyst. Note the separation of the outer layer of the oocyst wall (arrow).

26. Life Cycle Sexual Generation ( Gemetogony ) Some of the Type-2 meronts invade new host cells and initiate sexual reproduction ( Gametogony ). Inside the host cell, they differentiate into either Male ( Microgametocyte ) or Female ( Macrogamont ) forms. Each microgametocyte produce 16 sperm-like Microgametes , which fertilise the Macrogamonts resulting in the formation of Oocysts (Zygote)

27. Life Cycle Four sporozoites are formed in each sporolating oocyst in situ. The sporulating oocysts are of 2 types- The Thin walled oocyst released the sporozoites inside the lumen of the intestine-> Autoinfection in the same host and repetition of the cycle of Schizogony and Gametogony . The Thick walled oocysts are excreted in the faeces and are infective .

28. Oocyst of Cryptosporidium parvum ( Ziehl-Neelsen stain, 1000x)

29. Microgametocyte About sixteen microgamets could be seen when microgametocyte is fully matured.

30. Macrogamont at Different Maturation

31. LIFE CYCLE

32. Life Cycle After excysting from oocysts in the lumen of the intestine (a), sporozoites (b) penetrate host cells and develop into trophozoites (c) within parasitophorous vacuoles confined to the microvillous region of the mucosal epithelium. Trophozoites undergo asexual division (merogony) (d and e) to form merozoites. After being released from type I meronts, the invasive merozoites enter adjacent host cells to form additional type I meronts or to form type II meronts (f). Type II meronts do not recycle but enter host cells to form the sexual stages, microgamonts (g) and macrogamonts (h).

33. Life Cycle Most of the zygotes ( i ) formed after the fertilization of the microgamont by the microgametes (released from the microgamont ) develop into environmentally resistant, thick-walled oocysts (j) that undergo sporogony to form sporulated oocysts (k) containing four sporozoites. Sporulated oocysts released in feces are the environmentally resistant life cycle forms that transmit the infection from one host to another. A smaller percentage of zygotes (approximately 20%) do not form a thick, two-layered oocyst wall; they have only a unit membrane surrounding the four sporozoites. These thin-walled oocysts (l) represent autoinfective life cycle forms that can maintain the parasite in the host without repeated oral exposure to the thick-walled oocysts present in the environment.

34. Virulence Factors Membrane aminopeptidase Cysteine proteas A heamolysin Lectin & Thromboplastin related adhesion molecule.

35. Pathologenesis of Cryptosporidium Diarrhoea The infection begins when the ingested Oocysts release Sporozoites , which subsequently attach and invade the intestinal epithelial cell. The parasite has a predilection for the Jejunum and terminal Ieilium . In patients with AIDS, other sites of the G.I Tract like Stomach, Duodenum, Colon & Biliary tract may be involved.

36. Pathologenesis of Cryptosporidium Diarrhoea The Cholera-like voluminous watery diarrhoea is the key feature of Cryptosporidiosis in the patients with AIDS. Diarrhoea is typically non-inflammatory and is Profuse. The exact mechanism of diarrhoea is not known but may be due to Secretory and Malabsorptive process.

37. Pathologenesis of Cryptosporidium Diarrhoea After the invasion of the enterocytes, the parasite does elicit a local inflammatory response  increased production of PGs and several cytokines  INF- γ These cytokines activate Phagocytes and attract new leucocytes  release soluble factors  (↑) intestinal secretion of Chloride & water & inhibition of absorption of nutrients  OSMOTIC DIARRHOEA . Bacterial fermentation of sugars and fatty acids of the unabsorbed nutrients present in the intestinal lumen causes offensive and foul-smelling stool, which is characteristic of Cryptosporidium Diarrhoea .

38. Pathological Findings Doesn’t invade beyond mucosal layer of intestine-> attach to the Brush Border of the Intestinal Surface. They appear as small basophilic round structures, staining readily with Geimsa and H&E stains. Pathological Changes  Blunting and loss of villi, Lengthening of crypts, and Infiltration of lamnia propria by Lymphocytes, Polymorphonuclear cells and Plasma cells are seen as pathological changes.

39. Clinical Manifestations The clinical manifestations of Cryptosporidium infection vary depending upon the immune status of the host and results in 2 distinct clinical entities- 1] A self-limiting diarrhoeal illness in Immunocompetent persons, mostly Children 2] Severe prolonged life-threatening diarrhoea in patients with AIDS.

40. Clinical Manifestations Immunocompetent Individuals Clinical symptoms  Nausea, Low-grade fever, Abdominal cramps, Anorexia, 5 to 10 watery, frothy bowel movements per day , which may be followed by constipation . Some patients may present with diarrhea as described above, and others may have relatively few symptoms, particularly later in the course of the infection. In patients with the typical watery diarrhea, the stool specimen contains very little fecal material but consists mainly of water and mucus flecks .

41. Clinical Manifestations Occasionally, these patients require fluid replacement and the diarrhea persists for more than 2 weeks. This is particularly true of infants, in whom excessive fluid loss may last for over 3 weeks. In general, when CD4 cells are present at levels greater than 200/ μl , infections are acute and resolve in approximately 2 weeks; however, When the CD4 cell count drops below 200/ μl , the infection may be chronic and may not resolve.

42. Clinical Manifestations Cryptosporidiosis may present as an acute relapse of inflammatory bowel disease and responds to standard therapy. Antibiotics confer no benefit. It appears that immunosuppressive therapy does not predispose to chronic or severe illness in these patients. Cryptosporidiosis may present with acute findings initially mimicking Crohn’s disease .

43. Clinical Manifestations Failure to thrive has also been attributed to chronic cryptosporidiosis in infants. Since diarrheal illness is a major cause of morbidity and mortality in young children living in developing countries, it is likely that cryptosporidiosis plays a major role in the overall health status of these children. Apparently, cryptosporidiosis has a lasting adverse effect on linear growth (height) , especially when acquired during infancy and when children are stunted before they become infected. It has also been suggested that Cryptosporidium may be implicated in the respiratory disease that often accompanies diarrheal illness in malnourished children .

44. Clinical Manifestations Immunocompromised Individuals (AIDS) Intestinal disease ( severely immunocompromised patients)  Cannot overcome the infection, Illness becomes progressively worse with time, May be a major factor leading to death . The length and severity of illness may also depend on the ability to reverse the immuno-suppression. Extraintestinal infections Respiratory problems, Cholecystitis, Hepatitis, Pancreatitis

45. Clinical Manifestations Gastric cryptosporidiosis - A close correlation between the intensity of the infection and the degree of histologic alterations was observed. These findings suggest that in patients with AIDS, cryptosporidiosis, and severe immunodepression , upper endoscopy with random gastric biopsies should be performed, even in the absence of endoscopically identified lesions . The diagnosis of gastric cryptosporidiosis relies on histologic findings- Cryptosporidium organisms are found mainly in areas showing reactive hyperplasia .

46. Clinical Manifestations When reviewing data from AIDS patients with four clinical syndromes, i.e., chronic diarrhea (36%), choleralike disease (33%), transient diarrhea (15%), and relapsing illness (15%) There is no statistically significant correlation between the histologic intensity of infection and the clinical severity of illness . However, infected patients had a much shorter survival time than did Cryptosporidium negative AIDS patients .

47. Clinical Manifestations BILIARY TRACT DISEASE Sclerosing cholangitis is well known as a complication of AIDS. Direct cytopathic effects are noted in infected monolayers ( human biliary epithelial cell line )  widespread apoptosis beginning within hours after exposure to the organism. It appears that more severely immunocompromised individuals are more likely to exhibit biliary tract disease.

48. Clinical Manifestations PANCREATITIS In AIDS patients with pancreatitis  these patients exhibit hyperplastic squamous metaplasia . The patients usually present with  Abdominal pain resistant to analgesics , Elevated serum amylase levels , and Abnormalities on sonography and computed tomography.

49. Clinical Manifestations RESPIRATORY TRACT DISEASE Because many patients with respiratory cryptosporidiosis also have other pathogens present, it is difficult to determine the significance of this complication in AIDS patients. Certainly for patients with respiratory symptoms, it is important to examine sputum, as well as stool specimens to confirm the diagnosis.

50. Clinical Manifestations Primary immunodeficiency diseases They can be categorized as follows: 1]Combined immunodeficiencies (which impact both T and B lymphocytes) 2]Antibody deficiencies 3]Complement deficiencies, and 4]Defects in phagocytes (decreased number and function). The most serious immunodeficiency in terms of risk is severe combined immunodeficiency syndrome. These patients are at risk for disseminated disease; often the prognosis is poor.

51. Clinical Manifestations MALIGNANT DISEASE . Although cryptosporidiosis can be seen in patients with malignant disease, infection with Cryptosporidium spp. does not appear to pose a special risk. Exceptions to this general statement seem to involve leukemia and other hematologic malignancies . Certainly there is more interest in patients who are candidates for bone marrow transplantation , particularly when assessing possible risks related to cryptosporidiosis

52. Clinical Manifestations SOLID-ORGAN TRANSPLANT Cryptosporidiosis has been found in both liver and kidney transplant recipients, as well as following small bowel transplantation . However, in most cases the cryptosporidiosis was not unusually severe and did not involve dissemination to extraintestinal sites .

53. Epidemiology & Prevention Cryptosporidium infection in the immunocompetent hosts has been described in more than 26 countries. Geographical distribution- WORLD- Serological and stool examination studies has shown high rates of Cryptosporidium infection in Latin America, Africa, Middle-east, & South Asia . Children are most commonly effected in developing countries. In persons with AIDS in developing countries , the rate of infection is 12%-48%. India- Infection has been identified in Vallore , Chandigarh, Bengaluru , Madurai, Punducherry and some other places.

54. Epidemiology & Prevention Humans are the key reservoir of infection. There are 3 modes of Infection - 1] Human-human transmission- Human faeces containing thick walled oocysts are the major source of infection for human-human faeco -oral transmission manily through drinking water . 2] Animal-human ( Zoonoses ) transmission- the infection is transmitted from the livestock such as Cattle, cat or dogs either directly by ingestion of oocyst in their faeces or indirectly by close contact .

55. Epidemiology & Prevention 3] Auto Infection - this is caused by sporozoites released by thin walled oocyst inside the lumen of intestine- responsible for persistence of infection in the infected host. Children( specially Urban) between 1-5 years are at greater risk. More common in warm, rainy and humid months of the year. Oocysts are extremely resistant to free Chlorine in portable water.

56. Risk factors for acquisition of cryptosporidiosis Risk factor Examples Deficient immunity AIDS, other acquired or congenital immunodepression syndormes (e.g., boys with mutation of the CD154 gene, congenital X-linked immunodeficiency with hyper- IgM ), immunosuppression , Malnutrition. Zoonotic contact Leisure activities such as camping, backpacking, farm visits. Occupational exposure Veterinary, agricultural, nursing or medical, laboratory, day care centers. Poor sanitary conditions Drinking water and food, inadequate sewage or waste disposal, inadequate fly control. Exposure to untreated water Leisure activities Inadequately treated water supply Inadequate treatment or breakdown in treatment process Consumption of raw foods Unpasteurized milk, raw meat

57. Risk factor Examples Travel Travel from developed to underdeveloped countries and from urban to rural areas Age (infants, young children) Weaning, teething, pica, finger sucking, wearing diapers; poor infection control. Contact with case of diarrhea Day care, household, parents

58. Biological factors which impact the epidemiology of Cryptosporidium. Small (4–6 μm ), environmentally resistant, and fully sporulated /infectious oocysts when passed. Large livestock animal and human reservoir host populations. Ubiquitous, able to cross-infect multiple host species (not species specific). Low infective dose (10–100 oocysts ). Large multiplication capability (10 10 ) in a single host. Resistance to disinfectants. Lack of effective therapy; resistance to available drugs.

59. Factors related to potential outbreaks of cryptosporidiosis FACTORS EXAMPLES Factors unrelated to water Sufficient number of parasites (viability, infectivity, virulence) Sufficient number of susceptible individuals for ingestion of oocysts leading to primary infection. Sufficient number of susceptible individuals for whom contact with primary case leads to secondary transmission. Adequate epidemiologic and laboratory surveillance and reporting. Water-related factors Abnormal weather conditions (heavy rainfall) ; Increased load for water treatment facilities High turbidity of surface water. Breakdown in integrity of groundwater aquifers (prolonged rain after drought) Suboptimal water treatment functions Problems with finished-water distribution

60. DIAGNOSIS Previously, most human cases were diagnosed after examination of small or large bowel biopsy material, often under both light and electron microscopy. However, because biopsies may miss the infected area of the mucosa, cases have recently been diagnosed by recovering oocysts from fecal material by a flotation method . Fresh fecal specimens can be concentrated by using Sheather’s sugar solution , and coverslip preparations can be examined by both phase-contrast and bright-field microscopy .

61. DIAGNOSIS Although examination of flotation material by phase-contrast microscopy has proven to be an excellent procedure, many laboratories have neither access to such equipment nor experience with phase-contrast microscopy. Also, since the organism is considered to be infectious to laboratory personnel, fixed specimens can be processed by using several acid-fast stains , many of which are very satisfactory in demonstrating the organisms in stool material. It is important to remember that the number of oocysts is directly correlated with the consistency of the stool; the more diarrheic the stool, the more oocysts are present .

62. DIAGNOSIS For the diagnosis of cryptosporidiosis, stool and other body fluid specimens should be submitted as fresh material or in- 5 or 10% formalin, sodium acetate-acetic acid-formalin , or one of the newer single-vial stool collection systems ( polyvinyl alcohol prepared with zinc sulfate-based Schaudinn’s fixative ). Fixed specimens are recommended because of potential biohazard considerations. Either fresh or preserved specimens can be examined using the routine stool formalin-ethyl acetate concentration and one of the modified acid-fast stains or the newer immunoassay kit reagents .

63. DIAGNOSIS Staining techniques for Cryptosporidium Oocysts Kinyoun’s modified acid-fast technique (Cold method) Modified Kohn’s stain Modified Koster stain Flurescent Stain - Auramine O Auramine-rhodamine Auramine-carbol fuchsin Acridin Orange 4,6- DAPI

64. DIAGNOSIS It is recommended that the stool specimen be centrifuged at 500 g for 10 min prior to use of any of the stains or fluorescent-antibody (FA) immunoassay reagents. centrifugation under these conditions ensures maximum recovery of the oocysts . Uncentrifuged fresh, frozen, or fixed fecal material can be used for the antigen( Cryptosporidium copro -Ag ) detection immunoassays . Multiple stool specimens may have to be examined to diagnose the infection; this is particularly true when dealing with formed stool specimens.

65. DIAGNOSIS Respiratory cryptosporidiosis has been found in AIDS patients. Sputum specimens from immunodeficient patients with undiagnosed respiratory illness should be submitted in 10% formalin and examined for Cryptosporidium oocysts by the same techniques as used for stool samples . Infection of the gallbladder and biliary tree should result in oocysts being passed in the stool . For Histopathological duagnosis biopsy specimen is taken from jejunum/ rectum and processed immediately.

66. DIAGNOSIS The use of immunoassays has proven to be very helpful in providing a more sensitive method of detecting organisms in stool specimens. A direct FA procedure with excellent specificity and sensitivity has been developed and results in a significantly increased detection rate over conventional staining methods. Enzyme immunoassay (EIA) also provides excellent specificity and sensitivity for laboratories using this approach. Some of these reagents, particularly the combination direct-FA product used to identify both Giardia cysts and Cryptosporidium oocysts , are being widely used in water testing and outbreak situations .

67. DIAGNOSIS Flow cytometry methods for the quantitation of Cryptosporidium oocysts in stool specimens have been developed as an alternative approach. Studies indicate that the results are approximately 10 times more sensitive than those of conventional immunofluorescence assays. PCR technology also offers alternatives to conventional diagnosis of Cryptosporidium in both clinical and environmental samples. Compared with microscopic examination by conventional acid-fast staining procedures, PCR is more sensitive and easier to interpret but requires more “hands-on” time and expertise, as well as being more expensive. An important advantage of PCR is the ability to directly differentiate between different Cryptosporidium genotypes , which is important in outbreak situations.

68. Cryptosporidiosis of Colon.- Endoscopic View

69. Intestinal coccidia : recommended diagnostic procedures

70. Organism Specimen Diagnostic procedure Cryptosporidium spp. Stool Modified acid-fast stains, Fluorescent stains, immunoassays Sputum Immunoassays Modified acid-fast stains Scraping Modified acid-fast stains or routine histology Biopsy specimen Routine histology Cyclospora cayetanensis Stool Modified acid-fast stains, Autofluorescence Biopsy specimen Routine histology

71. Organism Specimen Diagnostic procedure Isospora belli Stool Concentration sedimentation and modified acid-fast stains Biopsy specimen Routine histology Sarcocystis spp. Stool Concentration sedimentation and special stains Biopsy Routine histology

72. kit name Manufacturer and/or distributor Type of test Comments ProSpecT Cryptosporidium microplate assay Remel (www.remel.com) EIA Can be used with fresh, frozen, or formalin-preserved stool Crypto Cel Cellabs DFA ProSpecT Giardia / Cryptosporidium microplate assay Remel EIA Can be used with fresh, frozen, or formalin-preserved stool Commercially available kits for immunodetection of Cryptosporidium spp.

73. kit name Manufacturer and/or distributor Type of test Comments ColorPAC Giardia / Cryptosporidium rapid assay Becton-Dickinson (www.bd.com) Cartridge device, lateral flow Can be used with fresh, frozen, Can be used with fresh, frozen, or formalin-preserved stool or formalin-preserved stool Xpect Giardia /Cryptosporidium Remel Cartridge device, lateral flow Can be used with fresh, frozen, or formalin-preserved stool

74. Treatment Cryptosporidiosis tends to be self-limiting in patients who have an intact immune system. For patients who are receiving immunosuppressive agents, one method of therapy would be to discontinue such a regimen. Other approaches with specific therapeutic drugs have been tried, but to date the results are still somewhat controversial. One approach that has had a dramatic impact on cryptosporidiosis in AIDS patients is highly active antiretroviral therapy leading to an increased CD4 count. Resolution of the diarrhea resulting from cryptosporidiosis is apparently related to the enhanced CD4 count rather than to any change in the viral load or any therapeutic impact of the drugs themselves. Thus, it appears that cellular immunity is critical in clearing Cryptosporidium infection in these patients.

75. Chemotherapeutic agent Comments Macrolides - Spiramycin (oral) Absorption seems to be decreased in the presence of food. Decrease in diarrhea and oocyst excretion was seen in immunocompetent infants randomized to receive spiramycin (100 mg/kg/day) or placebo. A positive clinical response, as well as organism eradication, has also been reported in a group of AIDS patients. Spiramycin (intravenous) Data revealed a statistically significant drop in oocyst count compared to placebo; however, administration of intravenous spiramycin was associated with paresthesias , taste perversion, nausea, and vomiting . At doses of 75 mg/kg/day there were cases of severe colitis due to intestinal injury. Azithromycin There may be a therapeutic effect on biliary tree infection, particularly in patients receiving intravenous azithromycin . Intravenous azithromycin at doses up to 2 g daily for 2 weeks is well tolerated

76. Paromomycin It is an oligosaccharide aminoglycoside related to kanamycin ; it achieves high concentrations in the colon but is poorly absorbed. Studies indicate that the drug effect is probably static rather than cidal for Cryptosporidium. Although decreased bowel movements and oocyst shedding have been reported, a small number of patients developed biliary tract disease and some of these required cholecystectomy . Despite conflicting results, paromomycin currently is used as the first-line drug in treating cryptosporidiosis. Nitazoxanide This is a nitrothiazole benzamide with a wide spectrum of activity against coccidia , amebae , nematodes, cestodes , and trematodes . Preliminary data appear promising, with suggested activity against cryptosporidial infection in AIDS patients. The agent appears to be well tolerated.

77. Physical disinfection of Cryptosporidium oocysts Agent Conditions Heat 50–55°C, 5 min 45°C, 20 min 60°C, 6 min 64.2°C, 5 min 72.4°C, 1 min Freezing −196°C, 10 min −70°C, 1 h −20°C, 1 day, 3 days UV light 15,000 mW /s, 2.5 h 80 mW /s cm−2 120 mW /s cm−2 8,748 mW /s cm−2 Drying Air dried, 2 h Air dried, 4 h Air dried in feces, 1–4 days

78. Prevention of cryptosporidiosis Preventive measure Comments Hand washing Prior to eating and food preparation; after touching children in diapers; after touching clothing, bedding, toilets, bed pans from anyone with diarrhea; after gardening; after touching pets or other animals; after touching or coming in contact with anything contaminated with human or animal stool (includes dirt); after removing gloves to perform any of the above activities. Practicing safe sex Always wash hands after touching partner’s anal or rectal area. Avoiding touching farm animals If HIV infected, avoid cleaning litter boxes or cages or stool disposal (very important if animals are younger than 6 months); have puppies or kittens (if younger than 6 months) tested for presence of Cryptosporidium before bringing them home; have any pet tested that has diarrhea. Swimming Avoid swallowing water when in lakes, rivers, pools, or hot tubs; organisms are not killed by routine chlorination; avoid swimming in polluted ocean water, since oocysts can survive for several days in salt water

79. Preventive measure Comments Washing and cooking food Thoroughly wash all fruits and vegetables if eating uncooked; use safe water for washing food; peel fruit; avoid unpasteurized milk or dairy products. Cooking kills Cryptosporidium, so cooked or packaged food is safe unless handled by someone infected with the organism. Drinking water Do not drink directly from lakes, rivers, streams, or springs; may want to avoid tap water (including refrigerator ice-maker ice and chilled water); boil water (rolling boil for 1 min is sufficient to kill organisms); filter with appropriate filters; a home distiller can be used. Store filtered water as for boiled water. Bottled water Bottled water labels reading “well water,” “artesian well water,” “spring water,” or “mineral water” do not guarantee that the water does not contain Cryptosporidium. However, water that comes from protected well or spring water sources is less likely to be contaminated than bottled water from rivers and lakes. Drinks Canned or bottled soda, seltzer, fruit drinks, and steaming hot tea and coffee are safe; fountain drinks, fruit drinks mixed with tap water from concentrate, and iced tea or coffee may not be safe.

80. SUMMARY IN IMAGE GALARY

81. Be Safe- Be Free From Cryptosporidiosis