Intestinal cryptosporidiosis is considered a major public health issue. The parasite responsible for this condition is transmitted through food or water contaminated by the sporulated oocysts of parasites from the genus Cryptosporidium. This enteric apicomplexan protozoan organism infects intestinal epithelial cells and causes transient diarrhea in healthy individuals. However, it can be responsible for chronic and serious diarrhea in patients with acquired immunodeficiency disorder (AIDS) or other immunodeficiencies.
A myriad of techniques have been employed to detect infection with Cryptosporidium parasites in humans and animals. These include:
- examination of feces for the presence of oocysts
- histology and detailed ultrastructural examination of biopsy material for the identification of life-cycle stages
- the detection of Cryptosporidium antigens
- the detection of its genetic material by molecular techniques
Classically, the laboratory diagnosis of intestinal cryptosporidiosis is achieved by one or more of the following techniques:
- the demonstration of Cryptosporidium oocysts in stool specimens
- the demonstration of Cryptosporidium parasites in biopsy specimens of a small bowel or in the intestinal fluid
- by finding Cryptosporidium antigen in stool specimens
The mature oocysts of Cryptosporidium are round, 4-6 µm in size, and easily identifiable by modified Kinyoun’s acid-fast staining method; nevertheless, some Cryptosporidium oocysts cannot be stained appropriately by using this method, and consequently present as “ghost-like bodies” that can be evaluated only by experienced laboratory personnel.
In order to maximize the recovery of oocysts, it is recommended that stool samples be concentrated prior to microscopic evaluation. At least three stool specimens should be interrogated before a negative result is reported, even though studies have shown that the first sample is enough for accurate diagnosis in most cases.
Small bowel biopsy and intestinal fluid are seldom used for the diagnosis of intestinal cryptosporidiosis, but enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay are routinely employed. The latter two techniques are highly sensitive and specific methods, and are therefore convenient for screening large number of material samples within a short period of time.
Methods of diagnosis of parasitic infestation that are based on staining and microscopic examination are being widely replaced with molecular techniques that are specific for a pathogen of interest. One of these is the polymerase chain reaction (PCR) that has the benefit of enhanced sensitivity and specificity in the diagnosis of intestinal cryptosporidiosis. This has restricted applicability at present in low-resource or point-of-care settings due to its cost, the infrastructure requirements, as well as the need for high technical expertise.
When PCR was employed to detect Cryptosporidium species in clinical samples, the prevalence in the stool samples of South African children with diarrhea was found to be 20%. The organism was found in three-fourths of AIDS patients with diarrhea from Uganda. In general, the diagnosis of Cryptosporidium by using PCR has a sensitivity which is between 97% and 100% and a specificity of 100%.
Combination of techniques as an optimal approach
ELISA and immunofluorescence assays are highly sensitive and specific, and PCR is even better in these respects. However, the staining method has the advantage of detecting active infection, unlike these methods which may not discriminate between active and non-active infections. In addition, the staining methods are less complicated and less costly, although their use means that some cases of intestinal cryptosporidiosis will go undiagnosed due to their inherently lower sensitivity.
Therefore a combination of staining methods with either ELISA or PCR techniques would be the “gold standard” and would ensure that infections with Cryptosporidium parasites do not go undiagnosed. Finally, as the incidence of intestinal cryptosporidiosis is evidently higher in HIV-positive patients, monitoring Cryptosporidium oocysts in those individuals helps in providing more effective treatment.