Microscopic Identification of Cyclosporacayetanensis and other coccidia

Henry Bishop

December 11 2019

Center for Global Health Division of Parasitic Diseases and Malaria Specimen collection and processing for detecting coccidian parasites • Fresh, unpreserved, C & S: fo r m a l- ethyl acetate concentration, wet mount*, Acid -fast or Safranin stain, DFA (Cryptosporidiumspp.), UV fluorescence (Cyclospora,Cystoisospora )

• 10% percent formalin : formal ethyl acetate concentration and subsequent wet mount*, Acid-fast or Safranin stain

• Ecofix: formal ethyl acetate concentration and subsequent wet mount*, Acid- fast or Safranin stain

• Total-fix : centrifugation only (10 min x 500g), wet mount*, UV fluorescence, Acid -fast or Safranin stain.

* (wet mount examination for Cryptosporidiamspp is not recommended.) Microscopy

Light microscope : • Bright Field • DIC • Phase Contrast

Fluorescent microscope : • DFA (Cryptosporidiumspp specific kits only – FITC filter) • UV fluorescence Cyclospora( , Cystoisospora, Sarcocystis– DAPI filter) Cryptosporidiumspp .

Tridimensional model of an oocyst ofCryptosporidium sp. containing 4 sporozoites Cryptosporidiosis • Caused byCryptosporidium sp p. (Primarily C. hominisand C.parvum ; other rare infections with animal-associated species are known). • Diarrheal illness that can range from self-limiting to life-threatening depending on host immunity • Incubation period of 2—10 days (7 days average) • Duration of illness: 1—2 weeks (shedding persists 1-4 weeks) • Oocysts are shed on average 9—12 days post infection • Autoinfective cycle • Numerous outbreaks in daycare centers, swimming pools, water parks, drinking water, and ap p le cider • Outbreaks in several US states in community swimming pools and splash/spray pools • One of the leading causes of waterborne disease in humans in the US Cryptosporidiosis: Clinical Presentation

• In immunocompetent humans: • Profuse watery diarrhea, anorexia, cramping, abdominal pain, nausea, vomiting, mild fever, fatigue or asymptomatic individuals

• In immunocompromised humans: • As above, often more severe and protracted, risk of extraintestinal infection

• Extraintestinal infection • Gall bladder, hepatobiliary ducts, pancreas, respiratory tract

Cryptosporidiumspp.

• Two main species in the human host:C. parvum and C. hominis • Species cannot be differentiated morphologically. • Small oocysts (4 – 6 micrometers, transmissible stage) penetrate treatment processes • Environmentally-hardy oocyst resists disinfection (chemical vs. p hysical) • Ubiquitous in environment • Anthroponotic and zoonotic strains/species with many isolates not infective for humans (but present in water sources) • Small infectious dose • No effective chemotherapeutic agents (nitazoxanide may be used in children to reduce length of illness) Diagnostic Considerations

• Ova and Parasite Examination (O&P): – Wet concentrated material (detection of oocysts difficult unless heavy infection) – Centrifugation: 10 minutes at 500 xg. • Stains: – Modified Kinyoun’sAcid-Fast (hot or cold) – Hot Safranin – Fluorescence auramine( O, auramine /rhodinine) • Microscopy: – Bright Field – DIC – Phase Contrast – Merifluor DFA Cryptosporidiumspp. Acid-Fast Stain Cryptosporidiumand Cyclospora Acid-Fast stain

Cyclospora

Cryptosporidium CryptosporidiumOocysts Hot Safranin Cryptosporidiumvs. Yeast in Acid-Fast Cryptosporidiosis: Key Points

• Caused primarily byCryptosporidium hominisand C.parvum , t h e latter a ruminant/livestock parasite zoonotic in the human host. • Diagnosis is made by the finding of oocysts in stool specimens. • Not readily detected by routine O&P exams. Specialty stains (Kinyoun’s acid-fast, safranin) or sp ecific kits (DFA, ELISA) p referred methods of diagnosis. • Oocysts 4—6 μm in feces. Oocysts usually sporulated, containing four naked sporozoites. • Oocysts do not autofluoresce like other coccidians. Cystoisospora(=Isospora) belli Cystoisosporabelli

• Formerly calledIsospora belli • Essentially the only Cystoisosporaspecies that infects humans, apart from extremely rare cases of Cystoisosporanatalensis • Found in the human intestinal tract • Self limiting enteritis in immunocompetent individuals • In patients with AIDS may produce a chronic diarrhea associated with considerab le weight loss

Cystoisosporabelli

• Oocysts measure 20—33 µm long by 10—19 µm wide.

• Ovoid, taper at the ends, and have a smooth, double layered hyaline wall; Unsporulated when passed in the feces.

• Diagnosed by wet mount, acid- fast stain, andautofluorescence .

• Repeated stool examinations and concentration procedures are recommended (oocysts may be passed in small amounts and intermittently,) Cystoisosporabelli

Acid-Fast Safranin Cystoisosporabelli autoflourescence Cystoisosporiasis: Key Points

• Caused byCystoisospora (=Isospora) belli. • Largest human coccidian. • Diagnosis is made by finding oocysts in stool. • Not readily detected by routine O&P exams. • Preferred diagnostic methods include Kinyoun’s modified acid fast, safranin, and UV microscopy. • Oocysts 20—33 µm long by 10—19 µm wide and unsporulated (or in a very early stage of sp orulation) in fresh stool. Cyclospora cayetanensis Cyclospora cayetanensis • C. cayetanensisonly species currently known from human host. • Other species are associated with simian infections, morphologically identical to C. cayetanensis.

• Outbreaks occurring from food products such as raspberries, basil, and lettuce in the US and Canada.

• Usually seasonal with warmer months predominating. 2018 Cyclosporiasis Outbreak

Multistate Outbreak of Cyclosp oriasis Linked to Fresh Exp ress Salad Mix Sold at McDonald’s Restaurants — United States, 2018

Multistate Outbreak of Cyclosp oriasis Linked to Del Monte Fresh Produce Vegetab le Trays Kwik Trip /Kwik Star— United States, 2018

Highlights • Multistate outbreak of Cyclosporacayetanensisinfections. • Several laboratory-confirmed cases of Cyclosporainfections in people from 15 states and New York City • Linked to the consumption of a variety of salads/vegetables from McDonald ’s or Kwik Trip /Kwik Star. • Several people were hospitalized. No deaths were reported. • McDonald’s voluntarily stopped selling salads at over 3,000 locations in 14 states. Cyclosporiasis

• Symptoms • Watery diarrhea, nausea, anorexia, abdominal cramping, prolonged fatigue, weight loss, and flu-like illness. • In immunocompromised patients diarrhea may last for months in some patients. • Microscopic diagnosis • Stool examination of multiple samples collected over several days • The sediment can be examined microscopically with different techniques: • Wet mounts (by conventional light microscopy/UV fluorescence microscopy • Stained smears (using modified acid-fast stain or a modified safranin stain) • Molecular diagnosis • Using conventional and real-time PCR protocols. • CDC utilizes a real-time PCR that targets a region of the 18SrRNA gene using a species-specific TaqManprobe. Cyclosporiasis

• Ova and Parasite Examination (O&P): – Wet concentrated material (detection of oocysts difficult unless heavy infection) – Centrifugation: 10 minutes at 500 x g (Total-fix without formalin and ethyl acetate) • Stains: – Modified Kinyoun’sAcid-Fast (hot or cold) – Hot Safranin • Microscopy: – Bright Field – DIC – Phase Contrast – UV fluorescence Cyclospora cayetanensis

• Oocyst size: —8 10 µm • Thick oocyst wall and many refractile globules • Excreted as noninfectious, in an unsporulated state, takes ~7—10 days to become infective outside of host, though occasionally sporulated oocysts may be found • Depending on method, staining may be highly variable • Does not multiply/develop outside of host but probably very hardy and chlorine resistant.

C. cayetanensisModified Kinyoun’sAcid-Fast C. cayetanensisModified Kinyoun’sAcid-Fast C. cayetanensisModified Kinyoun’s Acid-Fast C. cayetanensisHot Safranin C. cayetanensisHot Safranin C. cayetanensisHot Safranin C.cayetanensis Total-fix trichrome stain C.cayetanensis Total-fix AF stain C.cayetanensis Ecofixtrichrome stain C.cayetanensis EcofixAF stain Epifluorescence forCyclospora What’s needed?

• A microscope with fluorescence capabilities • A DAPI UV b lue excitation filter set (wavelength 330—365 nm ) Total-fix wet prep Artifacts: Spores (?) Acid-Fa st Cyclosporiasis: Key Points

• Caused byCyclospora cayetanensis. • Diagnosis made by the finding of oocysts in stool. • Not readily detected by routine O&P examinations • Preferred diagnostic methods include Kinyoun’s modified acid-fast stain, safranin stain, and UV microscopy. • Oocysts 8—10 μm in diameter and unsporulated in fresh stool. • Usually contain globular masses and not mature sporozoites. Human Coccidiosis Acid Fast vs. Hot Safranin Stain

Acid Fast

Hot Safranin

Unresolved issues ofCyclospora cayetanensis

• The number of species infecting humans • Median infective dose • Comp lete intracellular life cycle and sexual rep roduction • Involvement of animal reservoir hosts • Maximum rate of oocyst sporulation • Factors that affect sporulation rate • Factors influencing seasonality of outbreaks • Suscep tib ility to chlorine • The CDC needs your assistance to solve some of the issues! • If any lab would like to assist the CDC withour Cyclosporaresearch effort, they are encouraged to contact [email protected] about specimen submission. • We accept samples in most preservatives except formalin (Total- fix, Eco fix, P VA ) as well as unpreserved. If unpreserved, we aske that the specimen be placed in an 2.5% aqueous solution of potassium dichromate (1:1 with ¼ airspace) • 500uL is the minimum sample volume required for molecular analysis. However, larger specimen volumes are welcomed (and encouraged!) • Ep i-related inquires – [email protected] • Diagnostic lab oratory inquiries – [email protected] • Research laboratory inquires – [email protected] DPDx: Telediagnosis

Center for Global Health Division of Parasitic Diseases and Malaria What is Telediagnosis?

Using a camera, microscope, and computer with Internet access, laboratorians can transmit images of suspected parasites to CDC via- mail…e DPDx Team members reviewing telediagnosis images sent by PHL

Email message with digital images attached

Email message with CDC’s diagnosis based on analysis of images sent Blaine Mathison at the Arizona State Health Laboratory at one telediagnosis station capturing/transmitting digital images …and receive diagnostic assistance in minutes to hours. Cost and Time Effectiveness of Telediagnosis Traditional route for specimen submission Microscopic exam (PHL) Approximately 1 hour $10.00 labor* 1 specimen costs at least $95.00 to send and evaluate. Avg time of 48 hours Package and ship specimen Diagnostic assistance 48-72 hours Approximately 1 hour $70.00 for shipping and packaging** $15.00 labor†

Using DPDx for specimen identification 1 “specimen” (images of Microscopic exam (PHL) the specimen + patient Approximately 1 hour information) costs close to $10.00 labor* $20.00 to send and evaluate. Avg time of minutes Capture image and DPDx assistance send email Approximately 30 minutes Approximately 15 minutes $7.50 labor † $2.50 labor*

* Estimating labor as 10.00/hour † Estimating labor as 15.00/hour ** Including labor Equipment

• Dedicated microscope with camera computer with internet access • Hand-held digital camera with applicable adaptors • Smartphone with applicable adaptors • PhotoShop(optional)

• IT support recommended to address internet security issues Camera-Olympus DP74 Hand-held digital camera Smartphone adaptor alternative to dedicated digital camera Telediagnosis: What to Submit?

• Request a ShareFile link (HIPA compliant) • Images • CDC 50-34 form for formal reporting (required) • Stain used and magnification (preferred) • Measurements when relevant • Travel History (preferred) • Other pertinent medical information (blood transfusions, asplenicpatient, history of tick bites,etc ). From: John Doe Sent: Tuesday, December 10, 2019 2:39 AM To: CGH DPDM DPDx Web Inquiries (CDC) Subject: Parasite diagnostic assistance I need diagnostic assistance regarding Plasmodium speciation. Thank you very much. John Doe, MD MS Pathologist 123-456-7890 (office) | 123-456-7899 (cell) Please use the following link to download and complete the current version of the submission form (CDC form 50.34) https://www.cdc.gov/laboratory/specimen- submission/pdf/form-50-34.pdf. Aft e r then please use this secure Sharefile link https://centersfordiseasecontrol.sharefile.com/rr16cf129da584698a to submit your CDC 50.34 Form and any static images you have of your suspect organism. The link is to protect patient information. To avoid unnecessary delay in the processing and review of your case please make sure that the following sections are fully comp leted: 1. To begin, select “Human” as the specimen origin in the upper left-most box of the form 2. Laboratory Examination Requested - Use the “Parasite – Morphologic Identification ” option for “Test Order Name” 3 . Patient Information 4 . Specimen Information 5 . Original Submitter and if ap p licab le Intermediate Sub mitter 6. Travel History if known – this is very imp ortant 7. All relevant information on Patient History, Ep idemiological Data, etc. 8 . STATE PHL/New York…Section – use the dropdown arrow in the “Institution Name” box to choose your State’s PHL. Foreign submitters may leave this section b lank. *Preferred image file format – JPEG 50.34 Specimen Submission Form https://www.cdc.gov/laboratory/specimen-submission/form.html Telediagnosis: Summary

Advantages Challenges • Rapid identification or • Submission of images preliminary screening (usually biased by expertise and within one day, sometimes less ‘pre-diagnosis’ of submitter. than 15 minutes!). • Does not allow to see • Inexpensive. multiple focal planes. • Reported under guidelines of • Subject to ‘user error’: poor CLIA (U.S.A.). images (focus), wrong • Allows for information sharing magnification, poorly- (DPDx Image Library, Monthly cropped images, Case Studies). overexposed. • Some organisms inherently difficult (amastigotes, coccidians, microsporidia).