Many laboratories do not routinely test for Cryptosporidium, and in many instances, the tests used to look for this organism are insensitive.  Studies in the United States have documented cryptosporidiosis in about 4% of stools sent for parasitologic examination, while overall, about 13% of stool studies submitted for parasitologic studies in developing countries reveal Cryptosporidium oocysts. However, these numbers likely underrepresent the true number of infections, owing to the poor sensitivity of commonly used techniques.
Cryptosporidium can be difficult to diagnose and usually is missed unless specific tests are performed. Most often, stool specimens are examined microscopically using different techniques (eg, acid-fast staining, direct fluorescent antibody [DFA], enzyme immunoassays, or immunochromatographic tests for detection of Cryptosporidium species’ antigens). (See the image below.) 
Urea, electrolyte, and liver function tests
Urea and electrolyte tests are used to assess electrolyte replacement requirements and the presence of prerenal uremia.
Elevated alkaline phosphatase and glutamyl transpeptidase without hyperbilirubinemia are typical signs of biliary infection.
Imaging studies are not indicated as a first-line diagnostic approach in cryptosporidiosis. Abdominal radiography and computed tomography (CT) scanning are nonspecific but may reveal distended loops of bowel, air-fluid levels, and disrupted bowel motility.
When indicated, as guided by symptoms, ultrasonography or CT scanning may reveal an enlarged gallbladder with a thickened wall, dilated or irregular intrahepatic and extrahepatic biliary ducts, and a normal or stenotic distal common bile duct. Cholangiography may reveal beading of the common bile duct or papillary stenosis.
In cases of respiratory involvement, chest radiography is unremarkable, with modest infiltrates or increased bronchial markings.
Unconcentrated, fresh specimens can be examined by wet mount preparations. Concentration by the formalin ethyl acetate method is preferable. Optimal centrifugation time and speed, 10 minutes at 500 X, are critical for concentrating Cryptosporidium oocysts.
Commercial fecal concentration tubes are available that decrease processing time and supplies needed for concentrating specimens (eg, Fecal Parasite Concentrator, Evergreen Scientific). Polyvinyl alcohol (PVA)-preserved specimens are not acceptable for modified acid-fast staining or antigen-detection assays for detection of Cryptosporidium.
Types of tests
Modified acid-fast staining procedure is useful for the identification of oocysts of the coccidian species, including those of Cryptosporidium (which may be difficult to detect with routine stains, such as trichrome). Cryptosporidium species stain a pinkish-red color. The background should stain uniformly green. Unlike the modified Ziehl-Neelsen acid-fast (MZN-AF) stain, this stain does not require the heating of reagents for staining. (See the images below.)
Chalmers et al demonstrated that enzyme-linked immunosorbent assays and immunofluorescent tests have sensitivities above 90% and were significantly higher than that of modified Ziehl-Neelsen stains (75%).  Molecular tests such as PCR are even more sensitive and are increasingly being used for diagnosis. Immunochromatographic tests are less sensitive, especially for zoonotic species.
Concentrated sediment of fresh (within 30 min after passage of stools) or formalin-preserved stool may be used. Other types of clinical specimens, such as duodenal fluid, bile, and pulmonary samples (induced sputum, bronchial wash, biopsies) may also be stained.
The formalin ethyl acetate method is used to concentrate stool before staining with a modified acid-fast stain, because routine laboratory examination of stool for ova and parasites does not detect Cryptosporidium. [1, 24] This technique stains oocysts pink or red, whereas fecal debris or yeast assumes the color of blue or green counterstain. Oocysts are small (4-6 μm in diameter) and can be missed without a very careful examination of the slide.
Because shedding may be intermittent, examine at least 3 stool specimens collected on separate days before considering the test results negative. Fecal leukocytes are not found in stool specimens, because invasion does not occur below the epithelial layer of the mucosa. Other testing strategies include the following:
GI biopsy specimens can be used instead of stool specimens; a high concentration of oocysts is seen in the jejunum
Electron microscopy of stool or biopsy specimens can also be performed for direct visualization of oocysts
For research purposes and for species identification, PCR assays are used
Serologic detection of specific anti- Cryptosporidium antibodies is primarily used as a research or epidemiologic tool
Evaluation of Immune Function
Lymphocyte subset analysis
CD4+ lymphocyte counts predict the duration of disease in patients infected with HIV. When the counts are greater than 150 cells/μL, the diarrhea is likely to resolve spontaneously. With lower counts, however, the diarrhea may be chronic. Counts are typically less than 50 cells/μL in patients with either biliary involvement or choleralike syndromes.
Prolonged diarrhea caused by cryptosporidiosis may warrant HIV testing.
Children with chronic diarrhea from cryptosporidiosis should be screened for primary immunodeficiencies associated with depressed cellular immune function. The most commonly identified immunodeficiency is hyper-IgM syndrome, which can be identified by antibody screening. T-cell deficiencies can be identified by examining lymphocyte numbers and subsets. 
Abdominal Ultrasonography and ERCP
Dilated or irregular intrahepatic and extrahepatic bile ducts, along with a thickened gallbladder, as detected with abdominal ultrasonography, indicate biliary involvement.
Endoscopic retrograde cholangiopancreatography (ERCP) is often needed to diagnose sclerosing cholangitis or papillary stenosis.
ERCP identification of Cryptosporidium oocysts in bile or intracellular forms on biopsy confirms the diagnosis of biliary cryptosporidiosis. Papillary stenosis may be present and responds symptomatically to endoscopic sphincterotomy, often with stent placement.
Biopsy and Lavage
GI or liver biopsy
GI or liver biopsy may be indicated in cases of diagnostic uncertainty. Different parts of the intestinal tract may be affected. Liver biopsy findings may reveal the organism attached to bile duct epithelial cells. Concurrent infection with cytomegalovirus (CMV), Enterobacter cloacae, and microsporidia is common.
Bronchoalveolar lavage and lung biopsy
In patients with related symptoms, bronchoscopy may reveal the parasite in lavage fluid, in brushing specimens, and in biopsy specimens, attached to the surface of bronchial mucosal cells, or in macrophages. In most instances, another pulmonary pathogen, such as CMV or Pneumocystis (carinii) jiroveci, is concurrently detected; however, in a series of 4 patients infected with HIV, Cryptosporidium was the only pathogen identified in the respiratory tract. Clear association with intestinal cryptosporidiosis or diarrhea has not been shown in these cases.
Histologic examination of the small intestine is not required to confirm the diagnosis of cryptosporidiosis, although the small intestine does show the parasite projecting from the brush border of the mucosal surface. Parasites may also be identified in bile or biliary tract biopsies.
Villous atrophy with blunting, epithelial flattening, and an increase in lamina propria lymphocytes are seen in patients with persistent cryptosporidiosis. In patients with heavier infection, crypt hyperplasia and marked infiltration with lymphocytes, plasma cells, and neutrophils are also noted.
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