Table 2.

Specimen types and applications of Cryptosporidium diagnostic tests

Specimen type	Relevant patient or animal group	Sample preparation and standard test options	Appropriate number of specimens	Test availability; key findings
Direct detection of parasite
Faeces (the most commonly examined specimen).	Patients/animals with diarrhoea. Local policy may not include examination specifically for Cryptosporidium in all samples, even if a request for ‘ova, cysts, and parasites’ is made. Common selection criteria for examination include: • Young age (for farmed animals, usually <1 month of age); • Recent travel to an endemic country; • Other risk factor; for example farm visits, local outbreak; immunocompromise. Clinicians should familiarise themselves with local laboratory practice, and specify Cryptosporidium on the request form to ensure that appropriate testing is carried out.	Fresh, frozen or preserved faeces [e.g., in 10% formalin, merthiolate–iodine–formaldehyde; sodium acetate–acetic acid–formalin; other preservatives: e.g., polyvinyl alcohol (PVA) may optimise detection of other parasites, especially for the morphological examination of trophozoites, and preservation in different vials may be required]. Preservation pros: • Preserves other parasite morphology or stages. • Allows transport at high ambient temperature. • Allows prolonged storage. Preservation cons: • May impede PCR analysis, Strongyloides larvae concentration, and examination for trophozoites of other protozoans of interest. • Many immunoassays cannot be performed on PVA preserved samples. • Preserved faeces require concentration by a recognised method before microscopy. Some faeces may need additional processing, for example very liquid stools may be concentrated, high-fat faeces may require de-fatting, mucoid samples may need treatment with KOH or dithiothreitol, high-fibre faeces may need sieving to remove fibres. Test faeces or concentrate as shown in Figure 2.	For farm animals, ideally, collect faecal samples from a representative number of affected animals in the herd/flock. Repeat sampling may be required if samples are negative and Cryptosporidium is still suspected because shedding can be intermittent: • Ideally, for humans, three stool specimens collected over no more than a 10 day period, usually collected every other day, before being deemed negative. • For animals, 2–3 consecutive negative faeces before confirmed to have cleared the infection.	Available locally. Prevalence among patients with gastrointestinal symptoms is 1–4% in Europe and North America and 3–20% in Africa, Asia, Australia, South and Central America [82]. Incidence rates may be underestimated because not all infections are symptomatic and persons with symptoms do not always seek medical attention, laboratory diagnosis may not be sought, laboratories may not include Cryptosporidium testing, and case reports or notifications may not be complete. In the UK, where most laboratories test all diarrhoeic stools for Cryptosporidium, the ratio of disease rates in the community and presenting to general practice relative to the rate of reported diagnoses to national surveillance) has been estimated to be 8.2 (95% CI 2.1–31.7) [42]. One of the most frequent causes of calf diarrhoea [25].
Bowel content collected postmortem.	Animals that died with signs of diarrhoea.	As above.	As discussed with clinicians. Sampling guided by gross pathology.	Available locally.
Small bowel scrapings or aspirates.	Severely immunocompromised patients with persistent idiopathic gastrointestinal symptoms. Animals that died with clinical signs consistent with cryptosporidiosis.	Prepare a smear on a well slide and stain with immunofluorescent stains for oocysts and/or extract DNA and perform PCR.	As discussed with clinicians.	May be available locally; more likely refer for specialist testing.
Small bowel biopsy or tissue sections collected postmortem.	As above.	Examine haematoxylin and eosin (H&E) histology sections for endogenous stages and pathology consistent with cryptosporidiosis and/or extract DNA and perform PCR. DNA quality from fixed tissue sections is reduced but can be used for PCR.	As discussed with clinicians. Sampling guided by gross pathology.	As above.
Tracheal scrape collected postmortem.	Birds that died with clinical signs consistent with respiratory cryptosporidiosis.	As above.	As above.	As above.
Bile from endoscopic retrograde cholangio-pancreatography.	Severely immunocompromised patients with symptoms of cholangitis whose stool test is negative for Cryptosporidium.	Prepare a smear on a well slide and stain with immunofluorescent stains for oocysts and/or extract DNA and perform PCR.	As discussed with clinicians.	As above.
Liver biopsy, preferably in saline.	Severely immunocompromised patients with symptoms of liver disease.	Examine H&E stained sections for endogenous stages and pathology consistent with infection and/or extract DNA and perform PCR. DNA quality from fixed tissue sections is reduced but can be used for PCR.	As discussed with clinicians. Sampling specific lesions can be ultrasound or computerised tomography scan guided.	As above.
Sputum or bronchoalveolar lavage.	Severely immunocompromised patients with unexplained respiratory symptoms.	May require pretreatment with dithiothreitol. Prepare a smear on a well slide and stain with immunofluorescent stains for oocysts and/or extract DNA and perform PCR.	As discussed with clinicians.	As above.
Antral washout.	Severely immunocompromised patients with unexplained sinusitis.	Prepare a smear on a well slide and stain with immunofluorescent stains for oocysts and/or extract DNA and perform PCR.	As above.	As above.
Indirect detection of infection
Blood serum.	Human populations for seroconversion, prevalence or sero-epidemiological analysis.	Large and mini-format Western blot, enzyme immunoassay (EIA), or microsphere assay incorporating recombinant proteins (e.g., 15/17 kD and 27 kD) for detection of Cryptosporidium-specific IgG and IgM and sometimes IgA.	Depends on study design.	Specialist test for research purposes. Strong IgG responses elicited to the 15/17 kD antigen occur within 6 months of infection; response to 27 kD antigen is usually much weaker but is detectable for longer [83].
Oral fluid.	Populations for seroconversion, prevalence, or sero-epidemiological analysis.	Dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) or microsphere assays incorporating recombinant proteins for detection of Cryptosporidium-specific IgG and IgA.	Depends on study design.	A specialist test is under development for population monitoring [50]. Anti-Cryptosporidium IgG seroconversion reported in infected children [84]; detection of anti-Cryptosporidium IgG in oral fluid has been associated with recent diarrhoea [85]
Faeces/stool.	Indication of active infection.	Copro-antibody EIA for detection of Cryptosporidium-specific IgA.	Depends on study design.	Used for research purposes.