ESCMID Online Lecture Library © by Author

ESGCP, Amsterdam, June, 2012

Cryptosporidium, Isospora and Cyclospora

Francis DEROUIN

Laboratoire de Parasitologie-Mycologie Faculté Paris Diderot/Hôpital© by author Saint-Louis, Paris, France

[email protected] Online Lecture Library Phylum Apicomplexa Large group of protists, characterized by the presence of a unique set of organelles called an apical complex (rhoptries, micronemes). Most members have a complex life-cycle, involving both asexual and sexual reproduction. Apicomplexan cells contain a single plastid, called the apicoplast.

Haemosporida Plasmodium Babesia © by author Coccidia Toxoplasma ESCMIDCryptosporidium Online Lecture Library Isospora Cyclospora Emeria Several common features between Cryptosporidium, Cyclospora and Isospora

• Protozoa, Apicomplexa, Coccidia • Sexual and asexual replication in the intestinal mucosa • Shedding of oocysts in stools • Resistance of oocysts in the environment

In humans • Oral contamination © by author • Responsible for outbreaks (Cryptosporidium, Cyclospora) • Disease: diarrhea, opportunistic infections (Cryptosporidium IsosporaESCMID) Online Lecture Library • Diagnosis in fecal samples Cryptosporidium spp and cryptosporidiosis

• Discovered by Ernest Edward Tyzzer in 1907 • Was thought to be only a veterinary problem • First found in humans in 1976 • The number of human cases rose along with the AIDS epidemic • First spread through a water system in Carollton, GA (USA) in 1987 (13,000 ©cases) by author • In 1993 was the world’s largest municipal water supply outbreak in Milwaukee, WI (USA): 400,000 cases • In 2001ESCMID EPA water Onlinerequirements Lecture were increased Library to control Cryptosporidium contamination of water resources Specificities of intestinal life cycles

Cryptosporidium Shedding of sporulated (infective) Intracellular but extracytoplasmic replication oocysts

Source: Chen et al. NEJM. 2002 Source: ANOFEL CD Rom 3 Cryptosporidium species infecting humans

Species Major hosts Minor hosts

C. hominis Humans Dugong, sheep

C. parvum Cattle, livestock, Deer, mice, pigs humans C. muris Rodents Humans, rock hyrax, mountain goat C. suis Pigs Humans

C. felis Cats Humans, cattle

C. canis Dogs Humans

Cryptosporidium Deer, cattle© by authorHumans cervine genotype Cryptosporidium Rabbit Humans cuniculusESCMID Online Lecture Library C. meleagridis Turkeys Humans, parrot

Source: Xiao L et al. Clin Microbiol Rev, 2004; Caccio & Pozzio, Exp. Rev. Anti Infect. Ther, 2006 = zoonosis © by author

SimilarityESCMID in morphometric Online measurements Lecture of oocysts of LibraryC. parvum and C. parvum -related Cryptosporidium spp. Size: 4.5/5.5 µµµm

Source: Xiao L et al. Clin Microbiol Rev, 2002 Distribution of Cryptosporidium spp. in immunocompetent individuals (%)

England -Wales 2000-2003 (8000 isolates) Peru (533 children) Chalmers et al. 2009 Cama et al. 2008

0,5 3,5 9,6 2,1 4,3 2,1 49,2 9,6

45,9 64,9

C. hominis C. parvum Other species C. hominis C. parvum C. meleagridis Dual infections Not typable © by authorC. canis C. felis Dual infections In Europe: almost equivalent distribution of C. parvum and C. hominis. Less than 5% of other species In some other countries (e.g. Peru) : high prevalence of C. hominis and other species.ESCMID India: 90% of C. hominis Online Lecture Library In immunocompromised patients. Apparent overrepresentation of species other than C. hominis and C. parvum. Not confirmed in large genotypic studies in England and Wales. Source: Cama et al EID 2008, Chalmers et al. Eurosurveillance 2009, Ajjampur JCM 2010 Clinical symptoms 1. Immunocompetent individuals

Incubation time: 7 +/- 2 days

Signs: Non-bloody watery diarrhea, abdominal pain, asthenia, nausea, vomiting. Moderate fever in 40-60% of cases Weight loss (mean 4.5 kg), 50-75% of cases

Non gastrointestinal symptoms (joint pain, eye pain, recurrent headaches, dizzy spells, fatigue)

Duration of symptoms: 12 +/- 6 days (up to 40 days) Transient relapses (2 days) may© occur by in author up to 30% of cases. Sequelae: intestinal, non gastrointestinal, impact on nutritional status and growth.ESCMID Online Lecture Library Oocyst shedding : 3-8 days (up to 90 days)

Sources: Chen et al. NEJM 2002, Chalmers ett la. 2010, Hunter et al. JID 2004, Caccio et al. 2009 Genotype and illness Duration of symptoms

Hunter at al, EID 2004 427 patients, 191 isolates were genotyped; 115 C.hominis, and 76 C. parvum.

Duration of illness: All patients : 12.7 days and median of 11 days. C. hominis, 13.5 days median 12.5 © by authorC. parvum , 11.3 days median 10.5) Variance of duration for C. parvum was significantly lower than for C. hominis (F = 8.312, p = 0.005). However, the difference in ESCMID Online Lecturemedian Libraryduration was not significant.

Source: Hunter et al. EID 2004. Genotype and illness Sequelae Presence of symptoms following acute infection with Cryptosporidium hominis or Cryptosporidium parvum*

Intestinal symptoms Non gastrointestinal symptoms •weight loss, •joint pain, •decrease in appetite, •eye pain, •recurrent vomiting, •recurrent headaches, •abdominal pain, •dizzy spells, •diarrhea •fatigue

*The questionnaire included information on the presence, location, and severity of various symptoms experienced since onset of the diarrheal illness or in the previous 2 months).

Source: Hunter et al. JID 2004. Clinical symptoms 2. HIV infected patients

Intestinal symptoms Severe and persistent diarrhea leading to chronic malabsorption Abdominal pain, vomiting Weight loss +++ Severity and duration of symptoms: increase with CD4 counts decrease.

Biliary involvement Gallbladder cryptosporidiosis In up to 30% of cryptosporidiosis cases Source: Brown Medical School Papillary stenosis, sclerosing cholangitis, acalculous cholecystitis.

Gastric involvement may present as nausea and vomiting, with or without diarrhea. © by author Cryptosporidial pneumonitis OccasionallyESCMID reported : probable Online aspiration ofLecture organisms from Library the GI tract. Outcome: In patients who fail to respond to therapy, death usually occurs in 3-6 months.

Source: Pozio, E., et al JID, 1997 Cama JID 2007 Geographical distribution of cryptosporidiosis

Worldwide distribution Prevalence/incidence : poorly documented. Up to 10% in developing countries with poor sanitary conditions •0.5 to 2% in developed countries

HIV infected patients •Before HAART:HAART 3 to 25% according countries •50-90% reduction of incidence after HAART

In Europe: Collected and recorded by health agencies in several European countries Confirmed cases© fromby 16 author countries are reported to the European Basic SurveillanceESCMID Network (BSN) Onlineand ECDC Lecture Library No “official” report from many European countries Information lacking for HIV-infected patients

Source: Semenza JC, Nichols G, Eurosurveillance, 2007, Dillingham Microbes Infect, 2002, ECDC 2011 Estimated prevalence in europe (2008): 2.74 per 100 000 population The disease is likely to be underdiagnosed and underreported in several EU countries. Reports were provided by 21 out of 31 EU and EEA/EFTA countries. Most case reported: © by author UK, Germany, Ireland, Belgium Zero cases were ESCMID Online Lecture Libraryreported by eight countries. Age distribution (Europe)

Source: ECDC Surveillance report 2011 Age distribution (France)

Cas de cryptosporidiose en France (2006-2009). 70 Répartiton en fonction de l'âge et du statut immunitaire Immunocompetents 60 VIH 50 Greffés/Transplantés

40 autres immunodépressions 30 Nombre de cas de Nombre 20 © by author 10

ESCMID 4 9 4 9 4 Online9 4 9 4 Lecture9 4 9 4 9 Library4 9 4 9 - - 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 0 5 ------0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 Age

Source: Crypto-ANOFEL Network Seasonal variations (Europe)

Source: ECDC Surveillance Report 211 Risk factors Strongly associated with illness (general population)*: •Travel outside the United Kingdom (C. hominis), •Contact with another person with diarrhea, •Helping a child <5 years of age to use the toilet (C. hominis) •Number of glasses of tap water drunk at home each day •Contact with cattle (C. parvum).

Associated with illness in HIV infected patients : Contact with children <5 years of age (C. hominis) Increased susceptibility according to CD4 counts: x 2 for CD4 counts of 500-1000/mm3 x 3.6 for CD4 of 100-200/mm3 , x 6 for CD4 counts <100/mm3 © by author Other susceptible immunocompromised patients Transplant patients: case reports in renal, liver, intestinal and SCT recipients but no prospectiveESCMID study on prevalence Online Lecture Library Primary immunodeficiencies: X-linked Hyper IgM syndrome with CD40 ligand deficiency: higher susceptibility to Cryptosporidium infection

Source: *Hunter et al. EID 2004, Caccio et al. 2009, Cama JID 2007 Outbreaks of cryptosporidiosis 1983-2001: 165 waterborne outbreaks of cryptosporidiosis Number of cases: 5- 407 000 >90% in Europe and USA >70% of European outbreaks occurred in UK

Large variation according to the nature of the surveillance systems and to the quality of the public water supply. Probable underestimation in many countries

Major outbreaks: contaminated drinking water Others causes: fecal contamination of swimming pools or recreational areas, foods, person to person transmission© by (child author care facilities ), educational farm visit. England and Wales : 149 cryptosporidiosis outbreaks between 1983 and 2005* 55 linked to municipal drinking water supply 6 to ESCMIDprivate water supplies, Online Lecture Library 43 to swimming pools 16 to contact with animals

Source: Semenza Eurosurveillance, 2007; *Nichols G, et al. Drinking Water Directorate 2006, Karanis et al. J. Water and Health 2007. Outbreaks of cryptosporidiosis associated with water. USA 1984-2007 (n=147)

Predominance of outbreaks associated with recreational water

Source: Yolder Exp. Parasitol. 2010 Impact of waterborne outbreaks of cryptosporidiosis in HIV-infected patients

Milwaukee outbreak (1993) 400,000 cases in the general population, •100 deaths in patients with AIDS •73% mortality at one year in patients with AIDS when baseline CD4 count was less than 50 cells/mm3 •36% mortality when baseline count was between 50 and 200 cells/mm3

Las Vegas outbreak (1994) 78 cases of cryptosporidiosis in AIDS patients, 41 deaths by 1 year after the presumed date of contamination. © by author

Very limited data from Europe: ItalyESCMID: Higher attack rate inOnline HIV-infected patientsLecture (30.7%) comparedLibrary to HIV-negative patients (13.6%) (Pozio et al JID, 1997) Isospora belli and Cyclospora cayetanensis

Isospora, Cyclospora (e.g) Shedding of unsporulated oocysts Intracytoplasmic replication

Source: Ortega & Sanchez CML, 2010 Source: ANOFEL 4 Clinical symptoms

•Incubation time: estimated at 1 week for Isospora

•Immunocompetent Acute, watery diarrhea, abdominal pain and cramps. Low grade fever. More severe in children and in elderly patients. Duration of symptoms: Usually self limited within 2-3 weeks; oocyst shedding may persist for 2 to 3 weeks after the patient recovers.

•Immunocompromised Isospora: Profuse diarrhea. Long persistence of symptoms; several months or more. Malabsorption, weight loss, dishydratation© by author (possible life-threatening). Alithiasic cholangitis and cholecystitis, reactive arthritis, and rare disseminated infection. High rate of recurrences (50%). CyclosporaESCMID Online Lecture Library More severe symptoms, higher weight loss and longer duration than in immunocompetent patients. Cases report of biliary involvement (acalculous cholecystitis). Epidemiology

Geographical distribution Worldwide, but largely predominant in tropical and subtropical countries. High prevalence in Haiti, Guatemala, Peru, Nepal, Southeast Asia. Identified risk factors in endemic countries: consumption of untreated water, lack of adequate sanitation. In Europe: mainly observed in travelers returning from endemic countries and AIDS patients. Isosporiasis more frequent than cyclosporiasis. Rare QuickTime™décompresseursont requis pour et TIFF unvisionner (LZW) cette image. Cyclospora outbreaks.

Outbreaks Isospora: 3 limited waterborne ©outbreaks by author Cyclospora: 32 reported outbreaks between 1990 and 2009 (12 to 1475 patients) Most areESCMID classified as « foodborne Online » outbreaks Lecture (raspberries, Library berries, salad, fruits, fresh basil). Probably related to contamination of field watering systems and parasite retention on the fruit/leaf

Source: Karanis J. Water Health 2007, Ortega & Sanchez 2010 Laboratory diagnosis and treatment

Microscopy 4.5-5.5 µm., spherical 23-36x12-17 µm, ovoid 8-10 µm, spherical

Modified Ziehl-Neelsen Unstained, light Autofluorescence acid fast, safranin, microscopy. Alternative :Phase contrast auramine, Alternative: MZN microscopy, MZN, safranin immunofluorescence

Antigen Immunochromatography. 70-90% sensitivity for© C. by authorNo No parvum, C. hominis infections. 20-30% for other species* PCR ESCMIDYes. Developed Online but not Yes (specializedLecture LibraryYes (specialized commercialized. laboratories) laboratories) RT-PCR, genotyping

* Agnamey et al. J Clin Microbio, 2011 Staining methods for Cryptosporidium

Method Fixation Concentration Oocyst Background Comment appearance Bright field stain Modified Kynouin None Formalin/ethyl Red Green Good acid fast acetate differentiation Modified Ziehl- None Formalin/ethyl Red Green Good Neelsen acid fast acetate differentiation Safranin None None Orange-pink Blue Fast and rapid Negative stain Carbol fuschin None None Unstained Red Fast and simple Nigrosin Formalin Formalin/ethyl Unstained unstained Fast and acetate simple Auramine-rhodamine None or None or Orange Non fluorescent Sensitive. Risk formalin formalin/ethyl (fluorescent) of false pos. acetate© by author Fluorescent antibody tests DAF tests using None or None or Bright green Dark Sensitive monoclonal ESCMIDformalin Onlineformalin/ethyl Lecture LibraryCommercial antibodies acetate tests

Source: Caccio & Pozzio, Exp. Rev. Anti Infect. Ther, 2006, Sensitivity of antigen detection according to infecting species

100

% positive/test 60 •RIDA Quick: 55% % •X-Pect: 62% •Immunocard: 64% 40 20 © by author

0 ESCMID OnlineRIDA quick Lecture X-Pect Library Immunocard C. parvum C. hominis Autres espèces

Agnamey et al. J. Clin Microbiol, 2011 (source: ANOFEL Cryptosporidium Network) Comparative sensitivity and specificity of 7 Cryptosporidium assays (UK)

Crypto-Cel IFM 97,4 Crypto/giardia CHEK EIA 93,4 IVD Crypto/Giardi Combo EIA 92,8 Auramine Phenol microscopy 92,1

Remel ProSpecT EIA 91,4 ICL Ridaquick © by author 84,9 ZN microscopy 75,7 ESCMID 50Online 55 60 Lecture 65 70 75 80Library 85 90 95 100 Gold standard: real-time PCR + oocyst detection after IMS

Chalmers RM J Medical Microbiol, 2011 Some examples Cryptosporidium in stools

Staining •oocysts are rounded and measure 4.5 to 5.5 µm in diameter.

•Sporozoites are sometimes visible inside the oocysts, indicating that sporulation has occurred. © by author •Staining of Cryptosporidium spp. oocysts can be variable (non-acid-fastESCMID oocysts “ghosts.”) Online Lecture Library

Source: ANOFEL 4 Some examples Cryptosporidium in biopsies

Histologic features associated with Cryptosporidium infection

•neutrophilic infiltrate in the stomach,

•villus blunting in the duodenum,

•reactive epithelial changes in the stomach andESCMID duodenum. Online Lecture Library Source: ANOFEL 4

Source: Lamadue et al. AIDS 1998 Some examples Isospora in stools Wet mounts •Large immature oocysts (25 to 30 µm)

•Typical ellipsoidal shape.

•Containing one sporoblast (sometimes 2 after 1 or 2 days)

Staining •Red stain ESCMIDwith safranin or Ziehl Online Nielsen Lecture Library

•Autofluorescence at UV microscopy.

Source: ANOFEL 4 Some examples Isospora in biopsies

Histologic features associated with Isospora infection

•Villous shortening,

•Crypt hyperplasia,

•IntraepithelialESCMID lymphocytosis Online Lecture Library

Source: ANOFEL 4 Some examples Cyclospora in stools

Wet mounts •Phase contrast microscopy: spherical, refractile (8-10 µm)

•Containing one sporoblast

•Sporulation if the sample is stored at 23 to 30°C for 7 to 15 days, © by author Staining •Autofluorescence at UV microscopy +++ ESCMID Online Lecture Library

•Variable red/pink stain with Ziehl Nielsen Source: ANOFEL 4 Some examples Cyclospora biopsies

Histologic features associated with Isospora infection

•Villous shortening,

Source: ANOFEL 4 Treatment Cryptosporidiosis Isosporiasis, cyclosporiasis Few drugs have some activity and Cotrimoxazole: none is curative. Trimethoprim-sulfamethoxazole (TMP-SMX): Nitazoxanide: Reduction of symptoms and first line treatment :960mg twice daily for 7 parasitic load in immunocompetent individuals days

No evidence for a significant reduction in the Ciprofloxacin duration or frequency of diarrhea in patients Ciprofloxacin is an acceptable alternative if with AIDS (only used as compassionate TMP-SMX is contra-indicated :500mg twice treatment) daily for 7 days Paromomycin: Anticryptosporidial© activity by in authorHigh rate of relapses in HIV-infected patients: vitro and in animal models need for secondary prophylaxis as CD count Reduce clinical symptoms without eradicating remain low the parasites in immunocompetent patients. In AIDS patients,ESCMID not more effective Online than LectureOther alternative Library placebo Pyrimethamine (50mg-75mg per day)

Source: Derouin & Lagrange,-Xelot, Exp. Rev. Anti Infect Ther, 2008 Prevention and control

Preventing contamination : • Preventing contamination of water resources and foods • Individual recommendations (especially for immunocomprised patients): – Food and hand hygiene +++ –Avoid contact with human feces (diaper changing), infected animal , farm animals (for Cryptosporidium) – Avoid drinking untreated raw water – Be aware that bathing in lakes, rivers, outdoor recreational areas, (swimming pool) might be contaminated – Precautions with drinking tap water, ice, fruits, fresh fruit juice. Preferential consumption of bottle water and pasteurized juices – Reinforced recommendations when traveling (especially in countries with low sanitary conditions) © by author Chimioprophylaxis: Not recommended In immunocompromisedESCMID patient:Onlineimmune Lecture reconstitution Library has resulted in a marked decrease of prevalence in HIV- infected patients = probable best preventive measure

Source: adapted from IDSA, 2002 Impact of Water Regulation (UK)

Drinking Water Inspectorate: Section 70 of the 1991 Act makes it a criminal offence for a water company to supply water that is unfit for human consumption. It is a regulatory duty on water companies to notify DWI of any event which has the potential to give rise to a significant risk to © by author public health Total number of case patients associated with waterborne outbreaks of infectious intestinal disease. Public water supplies. ESCMIDPrivate water supplies Online Lecture Library Other water supplies Swimming pools

Source: Smith et al. EID 2006 Conclusions

Probable underreporting of cryptosporidiosis, isosporiasis, cyclosporiasis and consequences of infection. Improve diagnostic practice in laboratories Develop educational information for the population and for practitioners (misknowledge diseases) Investigate long term consequences of infection : sequeleas, nutritional status, link with inflammatory diseases (cancer ?)

Risk assessment of environmental and food related risks Need for cost-effective methods© by to authoridentify oocysts in water and in foods Better case reporting at a national /European level PreventionESCMID and treatment Online Lecture Library Education of risk factors and hygiene Promote research on anti-Cryptosporidium drugs and disinfectants Cephalic Carnage Cryptosporidium lyrics

Septicemia, Yersinia Pestis Load a bowl... fire it up Trying to control nausea Delusional Hyponatremia No more bloody stool, I hit the bong H2O poisoning without knowing Internal anal hemorrhaging, bleeding sphincter Eating to constipate Tap water infects my throat Hoping I won't defecate Nausea, retching, and constantly wiping, Lying in pain slowly dying Water epidemic deploys town pallor bowel movements A defective filter is what was found Epigastric discomfort Wandering in disbelief Yearning at the terror inside me At the water treatment plant, Consume water from the sink Intake/Outtake valves Trying to rehydrate myself Cramping in unbearable I want to cry Somehow this has to end Were somehow crossed, Virulent liquid maggots produce sending untreated sewage Rapid progressive condition Same bacteria eats my friends Drink Pepto-Bismal to remiss Into the home, community infected... Instant depletion of body fluids Cryptosporidium infecting my body, Electrolyte balance precarious Smoke a joint to progress Pale and pink... acid vomit sprays Living to tell the this purulent story Secretion of intestinal polypeptide Smoking some kind and feeling alive Dilation of blood vessels Dizziness erupts... I slip away Asiatic cholera secreting fluid and salt Immune system starts to fade In the intestinal tract, viremia, vipoma, A fight with time Water born epidemic Antibiotics will save my life Confusion in thought Cryptosporidium© by killing insideauthor Intestinal rot, Bacterial Fate Burning and churning, intestines writhe Trying to rehydrate I am still breathing, but barely alive Leading to hypovolemia... Parasites dying, I come back to life chronic renal dysfunction Cheating death, smoke another bowl Massive loss of potassium,ESCMID RegainingOnline hope, bless Lecture my soul Library ion (K) from diarrhea and vomiting, The body is gaining strength Reaching for the stash tray Load a bowl... fire it up