COCCICI

Coccidia

The intestinal coccidia that are pathogenic to man belong to the genera Cryptosporidium, Cyclospora, Isospora and Sarcocystis within the phylum Apicomplexa. Because their classification and identification are still in progress, their taxonomic designations may change in the future.

Coccidia affecting man

Cryptosporidium sp.

This protozoan parasite can infect many vertebrates: mammals (including man), birds, reptiles and fish. The name Cryptosporidium refers to the absence of sporocysts within the oocyst. Recent studies indicated that this genus is more phylogenetically related to gregarines than to coccidia. If this is confirmed, Cryptosporidium will no longer be considered an intestinal coccidian, while Isospora, Cyclospora and Sarcocystis will still be.

Cryptosporidium is an obligate intracellular monoxenous parasite that carries out its entire biological cycle in a single host. The infectious stage is represented by the oocysts, each containing four sporozoites that are accidentally transmitted to man in various ways: man-to-man, animal-to-man, or from contaminated water, food or air. Oocysts are partially digested in the stomach and the free sporozoites cross the microvillous brush border and enter epithelial cells of the jejunum and duodenum, where they complete their biological cycle with both asexual and sexual phases.

Asexual reproduction is repeated several times, enabling multiplication of the parasite. Sexual reproduction, which ends with the formation of oocysts, always occurs at the luminal apex of the enterocytes, where the parasites collect in an extracytoplasmatic parasitophorous vacuole whose membrane is gradually formed from the enteric epithelium. About 20% of these oocysts have a thin wall and the other 80% have a thicker, strong wall. The oocysts with a thicker wall are excreted in the feces and are immediately infective. The oocysts with a thin wall rupture in the intestine, releasing their four sporozoites: this gives rise to a new cycle of infection (endogenous autoinfection). This latter process explains the severe chronic forms of cryptosporidiosis in immunocompromised persons, even in the absence of exogenous re-infection. In respiratory tract infections, reported in immune-deficient patients, the infective oocysts spread into the environment via nasal secretions.

Cryptosporidium hominis and Cryptosporidium parvum are the species most responsible for human infection, but other species can also infect man, including Cryptosporidium meleagridis, Cryptosporidium muris, Cryptosporidium felis, and Cryptosporidium canis. In fact, there is no host specificity for the species that live in animals.

Cryptosporidiosis is a frequent cause of severe diarrhea in humans and represents an important public health problem worldwide, in both for immunocompromised and immunocompetent individuals. The disease is probably of great impact on children of developing countries due to the malnutrition and stunted growth caused by diarrhea. In developed countries, epidemics of cryptosporidiosis can result from contaminated food or drinking water (due to lack of treatment, filtering or chlorination) or from contaminated recreational water (as in swimming pools).

In HIV/AIDS patients, Cryptosporidium causes profuse and prolonged diarrhea, resistant to chemotherapy. In the last 10 years, the intensive use of anti-retroviral therapy, which supports immune function, has reduced the disease burden. In immunocompetent persons with cryptosporidiosis, diarrhea is less severe and usually self-limiting.

Cryptosporidium hominis, Cryptosporidium parvum

These two species are morphologically indistinguishable by light microscope examination.

Oocysts

Size: 4-6 μm.

Morphology: round, oval.

The detection of oocysts in wet mount preparation is often difficult due to their small size, their frequent presence in low numbers, and their resemblance to yeast or fungal spores.

The formalin-ether or formalin-ethyl acetate sedimentation concentration procedures require a 10-min centrifugation at 500 x g. Sheather’s flotation-concentration method (using a sucrose gradient) gives excellent results.

Oocysts examined by phase-contrast microscopy are highly refracting and contain prominent dark granules, unlike yeast which are not refractile and contain no granules. Moreover, Cryptosporidium oocysts do not stain with Lugol's iodine solution, while yeast do. Oocysts are not stained with merbromine or nigrosin but they are highly refractile and clearly visible against the stained background.

Specimen positivity must always be confirmed either with a modified Ziehl-Neelsen (hot) or Kinyoun (cold) staining to demonstrate the acid resistance of the oocysts. Oocysts fluoresce yellow-orange when stained with auramine-rhodamine; autofluorescence in UV light is negative.

Microscopic investigation for oocysts in feces can be substituted by many modern methods: direct immunofluorescence assays (DFA), which employs monoclonal antibodies specific for an oocyst surface antigen; immunoenzymatic tests (e.g. ELISA, EIA), to detect specific antigens; PCR followed by DNA sequencing to identify exact species; restriction fragment length polymorphism (RFLP) analysis. However, genotyping with these particular methods remains an activity restricted to specialized laboratories.

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