Educational Workshop EW18: Human Parasites of the Gut: Epidemiology and Diagnostic Approaches in the Molecular Era

Educational Workshop EW18: Human parasites of the gut: epidemiology and diagnostic approaches in the molecular era

Arranged with the ESCMID Study Group for Clinical Parasitology (ESGCP) and the ESCMID Study Group for Molecular Diagnostics (ESGMD)

Convenors: Titia M. Kortbeek (Bilthoven, NL) Paul Savelkoul (Maastricht, NL)

Faculty: Marjan van Esbroeck (Antwerp, Belgium) Theo Mank (Haarlem, The Netherlands) Spencer Polley (London, United Kingdom) - no handout available Simone Caccio (Rome, Italy)

2 Van Esbroeck - General introduction and epidemiology of common parasites of the human gut

Human parasites of the gut: epidemiology and diagnostic approaches in the molecular era General introduction and epidemiology of common parasites of the human gut

Marjan van Esbroeck Institute of Tropical Medicine Antwerp, Belgium

Influence of study population

Protozoa • Giardia lamblia •E. histolytica • Cryptosporidium

Helminths • warm t, humidity, poor sanitation, dirty  nematodes water, substandard and crowded housing  soil‐transmitted helminths •Sub‐Saharan Africa > Asia > Latin America • Ancylostoma duodenale •Behavior: more active in the environment, rarely employ good sanitation, crowd • Necator americanus together (schools, orphanages, slums) • Ascaris lumbricoides • helminths = immunomodulators • Trichuris trichiura

Influence of study population

Distribution of intestinal parasites in internationally adopted children in ITM. Number of children: 120 ‐ Period: 1/1/2008‐30/6/2009

3 Van Esbroeck - General introduction and epidemiology of common parasites of the human gut Influence of study population

• HIV/AIDS • solid/bone marrow transplantation • malnutrition

• Cryptosporidium > Cyclospora > Cystoisospora • microsporidia •(Blastocystis)

Influence of study population

Study of 2591 travelers ITM

4/2005 – 5/2006

Giardia lamblia 4,7% 6,0% Cryptosporidium 0,5% 1,3% E. histolytica 0,3% 0,5% Strongyloides 0,1% 0,8%

Influence of study population

• Prolonged diarrhoea (> 14 days) •1‐3% of travellers • Protozoal parasites >> helminth parasites

• Giardia lamblia • Cryptosporidium •E. histolytica

4 Van Esbroeck - General introduction and epidemiology of common parasites of the human gut Influence of study population

Protozoa •E. histolytica Helminths • Giardia lamblia • Strongyloides •Cyclospora • Ascaris lumbricoides • Cryptosporidium • hookworms •Cystoisospora • Trichuris trichiura • microsporidia •tapeworms

Greater burden of intestinal protozoan infections globally

Worldwide increase in • immunocompromised individuals •travel • medical tourism • immigration

Pathogenic Non‐pathogenic • Entamoeba histolytica • Entamoeba dispar, E. moshkovskii, • Giardia E. coli, E. polecki, E. hartmanni • Cyclospora cayetanensis • Iodamoeba butschlii • Endolimax nana • Cryptosporidium parvum • Chilomastix mesnili • Cystoisospora (Isospora) belli • Sarcocystis sp. • microsporidium

• Dientamoeba fragilis • Blastocystis hominis

5 Van Esbroeck - General introduction and epidemiology of common parasites of the human gut Giardia lamblia (G. intestinalis, G. duodenalis)

Developed regions of the world  transmission via contaminated water Ubiquitous distribution  person‐to‐person transmission in settings of poor fecal‐oral hygiene Food transmission is increasingly recognized Denmark, Norway, Finland, Sweden ‐ asymptomatic: 3% (2,6‐3,3%) ‐ symptomatic: 6% (5,3‐6,3%) 6% to 8% of children 15‐30% of stools in endemic areas Developing world: 200 M symptomatic infections 500.000 new cases each year ITM Antwerp: 4,6 (6%)

Giardia lamblia (G. intestinalis, G. duodenalis)

Waterborne tranmission

common agent identified in water‐borne outbreaks of diarrhea ‐ widely distributed in humans and other mammalian species  contamination of surface water ‐ cysts can survive for weeks in cold, fresh water ‐ cysts are relatively resistant to chlorination ‐ few parasites are necessary to establish infection

Giardia lamblia (G. intestinalis, G. duodenalis)

Person‐to‐person transmission Situations of poor fecal‐oral hygiene ‐ developing regions  15‐30% in children < 10 y ‐ day‐care centers  20‐50% ‐ oral‐anal sexual contact

Transmission by food infected food handler > enviromental contamination of food

Travel to areas of poor fecal‐oral hygiene

6 Van Esbroeck - General introduction and epidemiology of common parasites of the human gut Cryptosporidium

Worldwide distribution ‐ developed countries  sporadic outbreaks ‐ developing countries  endemic infections  southeast Asia (India) Species Major host Minor host C. parvum Cattle, sheep, goats, humans Deer, mice, pigs water and foodborne C. hominis Humans, monkeys Dugongs, sheep pathogen C. muris Rodents, bactrian camels Humans, rock hyrax, moutain goats C. andersoni Cattle, bactrian camels Sheep selflimiting  HIV/AIDS C. felis Cats Humans, cattle C. canis Dogs Humans C. meleagridis Turkeys Parrots, humans C. suis Domestic pigs Humans C. baileyi Chicken, turkeys Cockatiels, quails, ostriches, ducks C. galli Finches, chicken, capercalles C. serpentis Snakes, lizards C. wrairi Guinea pigs C. molnari Fish C. bovis Domestic cattle C. scophthalmi Fish C. varanii Lizards

Cryptosporidium

Most common method of transmission: drinking and recreational water  oral ingestion of oocysts

‐ the inoculum required to establish infection is very low (one ocoyst) ‐ excretion of large numbers of organisms into the environment  105‐7 oocysts per gram feces ‐ smal size  escape common water filters ‐ outer shell: cysts can survive for months in the water soil cysts are relatively resistant to chlorine ‐ the oocyst is immediately infectious on ingestion

Cryptosporidium

7 Van Esbroeck - General introduction and epidemiology of common parasites of the human gut E. histolytica

• Globally distributed •Most morbidity/mortality in developing world –low hygienic standards, high population density – Indian subcontinent, Central/South America, Africa •50 M invasive amebiasis • 40.000‐100.000 deaths annually

• Outbreaks –Mexico, Vietnam, Egypt –Chicago 1933, sewage and tap water mixed  800 cases • Amebic liver abscess <1% (potentially fatal illness)

E. histolytica

E. histolytica/dispar, ITM, Belgium, 2005‐2013

2005 2006 2007 2008 2009 2010 2011 2012 2013

N % N %N N % % N %N% N%N %N %

E. histolytica/dispar 152 189 194 214 214 212 248 248 266

E. histolytica 10 6,6 13 6,9 9 4,6 4 1,9 4 1,9 7 3,3 15 6 13 5,2 7 2,6

Strongyloides stercoralis

•nematode • (sub)tropical areas (+ moderate climate) •global prevalence unknown • estimation 3 – 100 M infected persons – travel to endemic areas – consuming contaminated water –contact with infected soil (barefoot travel) •no intermediate host  auto‐infection • cell mediated immunity defect  hyperinfection (↑ Mt) –corticoƟ – organ transplantation –HTLV‐1

8 Van Esbroeck - General introduction and epidemiology of common parasites of the human gut Dientamoeba fragilis

cosmopolitan distribution Prevalence 0,3%‐ 52% no consensus on ‐ age distribution ‐ gender distribution ITM: 2,8%

5 µm tot 15 µm

Dientamoeba fragilis

•Cyststage? Bimodal age distribution in patients positive for D. fragilis •E. vermicularis as vector for transmission? – epidemiological link – higher incidence of coinfection than expected –DNA of Dientamoeba found in Enterobius

Proportion of patients positive for E. vermicularis

Blastocystis species (B. hominis)

• cosmopolitan distribution •> 1.000.000.000 colonized • genetic diversity  Blastocystis sp. • unclear virulence factors, pathogenicity, risk factors • Significant difference in distribution of the ST – across host species – across geographical regions •virulence~ subtypes (~ species) (ST3) intrasubtype variation •virulence~ morpholigical forms ~ amoeboid forms

9 Van Esbroeck - General introduction and epidemiology of common parasites of the human gut Cystoisospora belli – Cyclospora cayetanensis

•global distribution • predominance in (sub‐)tropical areas •ingestion of contaminated food or water • endemic areas: children • developed countries: outbreaks

• travellers to endemic areas • immunocompromised individuals –more severe disease –disease may spread beyond the intestines

Cystoisospora Cyclospora 22

Schistosoma

fresh water contact poor sanitation school‐age children

S.mansoni S. haematobium

•Southern and sub‐Saharan Africa •Southern and sub‐Saharan Africa the great lakes the great lakes • Nile River valley in Sudan and Egypt • Nile River valley in Egypt, Magreb •South America: Brazil, Suriname, region of North Africa Venezuela • Also found in areas of the Middle • Caribbean (low risk) East

10 Van Esbroeck - General introduction and epidemiology of common parasites of the human gut S. japonicum S. intercalatum

Indonesia and parts of China Parts of Central and West Africa and Southeast Asia

Schistosoma mekongi

Cambodia, Laos

Soil transmitted helminth infections

11 Van Esbroeck - General introduction and epidemiology of common parasites of the human gut

Ascaris lumbricoides

• cosmopolitan distribution • (sub)tropical areas ‐ poor sanitation and personal hygiene • 807‐1221 M people infected

Fertilized eggs Unfertilized eggs

Trichuris trichiura

•whipworm • cosmopolitan distribution •more frequently in areas with tropical weather and poor sanitation • 604‐795 M people infected

Ancylostomidae

• Hook worm • 576‐740 M people infected • cosmopolitan distribution, worldwide in areas with warm, moist climates • difficult differentiation  no detailed numbers on the distribution ‐ Necator americanus: America, tropical Africa, Australia ‐ Ancylostoma duodenale: Mediterranean region, Far East

12 Van Esbroeck - General introduction and epidemiology of common parasites of the human gut Enterobius vermicularis

• Cosmopolitan distribution, temperate climate •no intermediate host  auto‐infestation • approximately 200 million people infected •most common helminth infection in the USA and Western Europe • children  adults (households where infected children transmit the infection to the rest of the family) •prevalence in children in certain communities up to – 61% in India – 50% in England – 39% in Thailand – 37% in Sweden – 29% in Denmark

Taenia

Taenia saginata Taenia solium • beef tapeworm •pork tapeworm • consumption of raw beef • under‐developed communities • cosmopolitan distribution with poor sanitation •Eastern Europe, Russia, eastern • consumption of raw or Africa and Latin America undercooked pork •Latin America, Eastern Europe, sub‐Saharan Africa, India, and Asia

Taenia solium

human as intermediate host  (neuro)cysticercosis

developing countries pork as main food

Latin America Asia Sub Saharan Africa parts of Oceania

virtually disappeared from Western Europe

13 Van Esbroeck - General introduction and epidemiology of common parasites of the human gut Hymenolepis nana Hymenolepis diminuta

•dwarf tapeworm •worldwide distribution • wide distribution, warm areas (rodents) • children in countries with poor •rare in humans sanitation and hygiene • intermediate host: several • institutional settings arthropods •no intermediate host  autoinfection

Fasciolidae

Fasciola hepatica •worldwide distribution (not in Antarctica) in > 50 countries ‐ sheep and cattle • geographic distribution is very patchy • consumption of raw watercress or other water plants •> 2M people infected • hyperendemic in the Andean highlands of Bolivia and Peru

Fasciolidae

Opisthorchis spp. Clonorchis spp. •raw or undercooked fish • Chinese or oriental liver fluke from endemic countries •Asia: Korea, China, Taiwan, • Thailand, Laos, Cambodia, Vietnam, Japan, and Asian Viet Nam, and other areas of Russia  Asian immigrants Asia, Eastern Europe, and •raw or undercooked fish, the former Soviet Union crab, crayfish

outbreaks in Italië O. Felineus carpaccio van Tinca Tinca

14 Van Esbroeck - General introduction and epidemiology of common parasites of the human gut Diphyllobothrium spp.

• cosmopolitan distribution •raw or undercooked fish • Northern Hemisphere: Europe, independent states of the Former Soviet Union, North America, Asia) • Uganda and Chile

15 Mank - Classical diagnostic methods for detection of parasites

Human parasites of the gut: epidemiology and diagnostic approaches in the molecular era

Classical diagnostic methods for detection of parasites

Theo Mank

“classical”

• relating to the first developed form or system of a science

• relating to, or being music in established European styles and forms (as the symphony and opera)

• having recognized and of permanent value

“classical”

• relating to the first developed form or system of a science

• relating to, or being music in established European styles and forms (as the symphony and opera

• having recognized and of permanent value

16 Mank - Classical diagnostic methods for detection of parasites

What are we looking for?

• adult worms / proglottides •larvae • helminth eggs •cysts • trofozoites / vegetative stages • oöcysts • spores

• Adult worms and eggs are to be found in the definitive host only!

• Protozoans • Helminths

– Cryptosporidium sp – Ascaris lumbricoides – Cyclospora cayetanensis – Diphyllobotrium sp – Cystoisospora belli – Enterobius vermicularis – Dientamoeba fragilis – Fasciola hepatica – Entamoeba histolytica – Hymenolepis nana – Giardia lamblia – Hymenolepis diminuta – Microsporidium sp – Hookworm – Schistosoma sp – Blastocystis hominis – Strongyloides stercoralis – Trichuris trichiura – Chilomastix mesnili – Taenia sp – Endolimax nana – Clonorchis sinensis – Entamoeba coli – Opisthorchis sp – Entamoeba dispar – Cappilaria phillipinensis – Entamoeba hartmanni – Iodamoeba bűtschlii

Classical diagnostic methods for detection of intestinal parasites

• macroscopic examination

• microscopic techniques

• non microscopic (non morphological) techniques • ELISA / copro-immunochemistry / Ag testing

• serology

17 Mank - Classical diagnostic methods for detection of parasites

Ascaris lumbricoides

male and female

Ascaris lumbricoides

male and female

18 Mank - Classical diagnostic methods for detection of parasites

11 Eg,Em & Tsol

19 Mank - Classical diagnostic methods for detection of parasites

Taenia solium cysticercose

– CT scan brain laesions < 2cm – X –ray muscles (eg upperleg)

Serodiagnosis 15 Molecular techniques

20 Mank - Classical diagnostic methods for detection of parasites

Enterobius vermicularis

Cellulose tape (Scotch tape) • Size 10 mm

microscopic diagnosis of intestinal parasites

S. stercoralis

Ascaris C. cayetanensis G. lamblia

T. trichiura E. histolytica/ D. fragilis hookworm dispar

21 Mank - Classical diagnostic methods for detection of parasites

Microscopic techniques

• fresh / preserved stool sample • multiple sampling • concentration techniques / Ridley and Hawgood • wet smear / Iodine stained smears / eosin • permanent staining • eg chlorazol black / IHK / Giemsa / Trichrome • Acid fast staining (ZN ) • Autofluorescence • Optical Bright staining / mod Trichrome

22 Mank - Classical diagnostic methods for detection of parasites

cave

• well trained and experienced technicians are essential for reliable microscopy (sensitivity and specificity)

• anamnestic data are mandatory for a parasitological workup eg travelhistory; complaints, immuunstatus, eosinophilia, elevated IgE

• epidemiological knowledge / priori chance

Microscopy

Improved diagnosis of intestinal parasites

T.F.T. = Triple - Faeces – Test

T van Gool, R Weijts, E Lommerse T Mank Triple Faeces Test: an effective tool for detection of intestinal parasites in routine clinical practice. Eur J Clin Microbiol & inf diseases 2003:22(5);284-90

Characteristics of the TFT

• Use of fixative: – SAF

• Use of multiple sampling: – 3 consecutive days

• Use of permanent stain: – Chlorazol black or Iron Haematoxylin Kinyoun

23 Mank - Classical diagnostic methods for detection of parasites

Evaluation of the TFT in routine clinical practice

462 patients AMC, Amsterdam

24 Mank - Classical diagnostic methods for detection of parasites

Increased recovery of intestinal protozoa in TFT (462 patients)

Organism One stool TFT Increase with sample(NF) TFT pathogen G. lamblia 18 24 6 E. histolytica 14 18 4 D. fragilis 0 45 45

apathogen E. coli 52 65 13 E. hartmanni 10 23 13 E. nana 42 47 5 C. mesnilii 7 10 3 I. bütschlii 6 12 6 B. hominis 0 124 124

Results with one, non fixed, sample compared to TFT (no. patients = 462)

One, non fixed, sample TFT

Conclusions TFT in routine clincial practice:

• High sensitivity and specificity

• Multiple sampling with high compliance

• Low cost sampling and laboratory material

• Additional labour-time acceptable

T van Gool, R Weijts, E Lommerse TMank Triple Faeces Test: an effective tool for detection of intestinal parasites in routine

clinical practice. Eur J Clin Microbiol & inf diseases 2003:22(5);284-90

25 Mank - Classical diagnostic methods for detection of parasites

ELISA (copro-immunochemistry)

• 65kD Giardia Specific Antigen (GSA) monoclonal • Single sample (fresh or SAF preserved) • Less intermittent shedding • “excreted” during encystation

• 96 wells format • Immunocards (ICT test) – often combined with Cryptosporidium sp & E hist/dispar • Dipsticks

26 Mank - Classical diagnostic methods for detection of parasites

Name Company Remarks

Triage parasite Biosite Giardia lamblia Cryptosporidium spp E. histo/dispar

X/pect Remel Giardia solo Giardia / Crypto combi

ImmunoCardSTAT Meridian Giardia lamblia Cryptosporidium spp

Rida Quick r-biopharm Giardia solo Giardia / Crypto combi

Litt + comparative studies with TFT

Naam Species

Triage parasite Giardia lamblia sens: >93% Cryptosporidium sp spec: >98%

X/pect Giardia lamblia sens: >95% Cryptosporidium sp spec: >98%

ImmunoCardSTAT Giardia lamblia sens: >95% Cryptosporidium sp spec: >98%

Rida Quick Giardia lamblia sens: >95% Cryptosporidium sp spec: >98%

cave

• well trained and experienced technicians are essential for reliable microscopy (sensitivity and specificity)

• anamnestic data are mandatory for a parasitological workup eg travelhistory; complaints, immuunstatus, eosinophilia, elevated IgE

• epidemiological knowledge / priori chance

27 Mank - Classical diagnostic methods for detection of parasites

Diarrhea I presume

Diarrheal complaints

• Protozoans

• In case of immuundeficiency – Cryptosporidium! – Microsporidium sp

• Protozoans • Helminths

28 Mank - Classical diagnostic methods for detection of parasites

E. histolytica and E. dispar

Entamoeba histolytica trophozoite

fagositized Red blood cells

nucleus

29 Mank - Classical diagnostic methods for detection of parasites

E. histolytica and E. dispar

• E. histolytica/dispar

• High priori chance of E. histolytica – Travelling endemic country – Blood in feces

– Specific E. histolytica serology – copro-ELISA no discrimination / low sensitivity –PCR

Cryptosporidium sp

• Oocysts are small 3-5 μm • Are easily missed in standard O&P • Specific methods are necessary

• Many Medical doctors are unfamiliar with the parasite

Cryptosporidium sp

• Acid fast staining – Mod ZN, Kinyoun, IHK – Ridley sediment

• Auramin staining

•IFA

•ELISA

30 Mank - Classical diagnostic methods for detection of parasites

Travelhistory (sub) tropics

• Helminth infections

• Schistosoma sp – prepatency! it takes several weeks after possible exposition / infection before eggs can be found – serology!

• Strongyloides stercoralis - Fresh stool sample (<12 hrs) is mandatory - serology!

31 Mank - Classical diagnostic methods for detection of parasites

Nematodes

• Strongyloides stercoralis – Fresh stool sample – Baermann – Culture (filariform larvae)

–PCR

Differentiation Strongyloides stercoralis / hookworm

Filariform larvae (after culture)

short mouth long mouth S. stercoralis hookworm opening opening forked tail tapered tail large genital small/no genital unsheathed sheated primordium primordium

New developments

• Introduction of rt-multiplex PCR as a screening tool for parasitological stoolexaminations

32 Mank - Classical diagnostic methods for detection of parasites

PCR

• rt-multiplex PCR – Giardia lamblia Short Subunit ribosomal RNA gen (62 bp) – C parvum/hominis DNAJ like protein gen (138 bp) – E. histolytica SSU rRNA gen (172 bp) • Verweij et al 2004

• rt-multiplex PCR – Dientamoeba fragilis 5.8S rRNA (98 bp) –PhHV • Verweij et al 2007

rt-PCR Giardia: Verweij et al 2004 microscopy: TFT

rt-PCR Giardia: Verweij et al 2004 Microscopy: TFT Giardia:ELISA: Ridaquick Giardia Crypto Entamoeba combi R-biopharm

33 Mank - Classical diagnostic methods for detection of parasites

Molecular techniques

• PCR – multiplex rt PCR – Giardia lamblia, Cryptosporidium, D fragilis, E histolytica

- microscopic confirmation of positive PCR result is mandatory since the clinical relevance of unconfirmed positive PCR results is (still) unclear

• Protozoans • Helminths

34 Mank - Classical diagnostic methods for detection of parasites

Parasitic species detected in 2000 TFT sets by rt-PCR and microscopy Species rt-PCR TFT Giardia/ Crypto/ E. histo/ D.fragilis microscopy Cryptosporidium sp 85 62 D. fragilis 245 186

E. histolytica 11 (E his/dis) Giardia lamblia 202 184

Blastocystis hominis 483 Non pathogenic protozoal 63 species like E.coli, E. nana

Ascaris lumbricoides 1 Diphyllobotrium latum 1 Hymenolepis nana 2

Taenia saginata 3 (2x proglottides)

“the best of both worlds”

- Molecular screening rt-PCR e.g.Giardia lamblia, Cryptosporidium parvum/hominis, E. histolytica en D. fragilis TFT-2

- Confirmation of positive PCR results

- Additional test based on request of physician

- Additional test based anamnestic data (lab)

Laboratory form

Parasitology Questionnaire parasites

-Intestinal parasites -foreign travel / tropics? -Schistosoma spp -specific skin disorders? -Strongyloides -eosinofilia? -Amebic abces -immunocompromised / deficient? -Leishmania spp -helminths seen? -Pneumocystis (PCP) -therapy? -Malaria

-other: -other:

35 Mank - Classical diagnostic methods for detection of parasites

“the best of both worlds”

DFT plus Duo Feces Test 1 day stool sample 1 part in tube 1 (SAF) 1 part in tube 2 (fresh)

Conclusions

• It is of major importance to be familiar with the possibilities / impossibilities of the different diagnostic methods used in parasitological stoolexaminations

• anamnestic data are mandatory for a parasitological workup (eg travelhistory; complaints, immuunstatus, eosinophilia, elevated IgE)

• well trained and experienced technicians are essential for reliable microscopy (sensitivity and specificity)

• epidemiological knowledge on parasitic infections is mandatory

36 Mank - Classical diagnostic methods for detection of parasites

Conclusions

• parasitological stoolexaminations based on molecular techniques only will result in loss of microscopy skills of well trained and experienced technicians and would be to the detriment of parasitology services worldwide

37 Caccio - Molecular typing of parasites

Molecular typing of gut parasites

Simone M. Cacciò

European Union Reference Laboratory for Parasites, Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità

24rd ECCMID, Barcelona 10-13 May 2014

A broad topic...

 The human gut is the ecological niche of many parasites

Protozoa  Helminthes  Blastocystis  Ancylostoma  Dientamoeba  Enterobius  Giardia  Ascaris  Cryptosporidium  Trichuris  Entamoeba  Cyclospora

What to do for this short talk?

 Instead of reviewing the various methods and their current applications, I have selected a few examples, starting with some biological questions and then show how molecular typing can provide answers and help formulate new hypotheses.

 Let’s move from simple questions and approaches to more complex questions and thus more complex approaches

38 Caccio - Molecular typing of parasites

Example 1: Dientamoeba

Anaerobic flagellate that lacks flagella

Life cycle not established

Transmission routes still unclear (cyst? Eggs of helminth?)

Highly prevalent in humans

Host range still unclear

Question 1: non-human hosts?

 Microscopic diagnosis in stools is not easy, due to the fragile nature of the trophozoites and the fact that morphological feateures overlap with those from other organisms like Endolimax and Entamoeba.  Surveys of stools from mammals and birds have identified only gorillas and pigs as possible hosts, but many other gastrointestinal parasites were also observed in the stools. ?

Question 1: molecular approach

 We investigated the presence of D. fragilis in stools of pigs from farms in Central Italy and included samples from farm workers. We used qPCR as a diagnostic tool and sequencing of 18S rDNA PCR products for genotype identification

39 Caccio - Molecular typing of parasites

Question 1: answer

 We demonstrated that pigs are infected with D. fragilis and that the same genotype (genotype 1) is present in pigs and farm workers. Cacciò et al., Emerging Infectious Diseases , 18, 5, 2012

 While these results did not prove zoonotic transmission of Dientamoeba, we now have a suitable animal model to study various aspects of the biology of this poorly known parasite

Question: emergence of new zoonotic agents Can phylogeny help?

 We are staying for this example within the Parabasalia, that comprises also Dientamoeba  In this large group of organisms, the traditional view supported a strict host specificity of at least some species (e.g., Trichomonas vaginalis or T. tenax), while others have a wider host range (e.g., Dientamoeba and Pentatrichomonas), compatible with zoonotic potential.  The phylum includes important animal pathogens, like Tritrichomonas foetus, the causative agent of the venereal cattle disease,trichomonosis.

What’s new?

1) Demonstration of parasites in atypical locations  T. tenax ,T. vaginalis, P. hominis in respiratory tracts (not in immunosuppressed individuals)

2)Demonstration of unexpected species in humans  Tetratrichomonas gallinarum or T. gallinarum-like organisms in human lungs or oral cavity (they are usually found of the digestive tract of domestic and wild birds)  Trichomonas foetus or T. foetus-like organisms again in the lungs of human patients

40 Caccio - Molecular typing of parasites

What about phylogeny?

Tree based on Rbp, largest subunit of RNA polymerase II

Human parasites

Avian parasites

Human parasites Avian parasite common ancestor

Note that some avian parasites, like those from doves, are closer to human parasites than to other avian parasites

Hypothesis: new zonotic agents?

 Molecular phylogenies reveal close relationships between human and avian trichomonads and suggest the zoonotic transfer of parasites from humans to birds and/or vice versa.  Future studies, based on wider sampling and more informative molecular typing (such as through comparative genomic investigations), will facilitate identifying the closest relatives of human trichomonad pathogens, providing a better understanding of how these diseases have emerged

Example 2: Cryptosporidium

Small, environmentally resistant stage (oocyst)

Large animal and human reservoirs

Ubiquitous

Able to multiply to very large numbers in a single host

Low infective dose

Oocysts immediately infectious upon excretion

41 Caccio - Molecular typing of parasites

Cryptosporidium: so many species

That show very little morphological variation

Question: can we learned about epidemiology from population genetics studies ?

 The analysis of highly polymorphic markers (mini- and micro-satellites) from parasites collected from different hosts and geographical origin has been used in a number of studies.  PCR and fragment typing (determination of the size of the amplicon) of markers allow assignment of alleles and the combination of alleles at a number of loci (7) defines Multi Locus Genoypes (MLGs).  MLG data are then analyzed using tools for genetic analysis (LD, PCoA, Clustering, eBURST, Structure ect)

What have we learned? Many things…  First, that there is extensive genetic variability in the population (> C. parvum, < C. hominis)

 Second, that both mutation and recombination are important for parasite differentiation

 Third, that population structure can be clonal, panmictic or epidemic, and that this is related to ecological factors that vary between countries

 Fourth, that high resolution genotyping allows to distinguish recently introduced strains

42 Caccio - Molecular typing of parasites

eBURST network

C. hominis isolates from UK C. hominis isolates from Uganda

MLGs organized in a The MLG network is star-like cluster with straggly and shows the most abundant in long chaining of the centre. single locus variants

Notice the presence of singletons not connected to the main cluster. These correspond to infections acquired abroad

Evidence of C. parvum strains that are found in humans but not in animals Are there non-zoonotic strains within a species considered as zoonotic?

Mallon et al., Infection, Genetics and Evolution 3 (2003) 207–218

 The concept of human-adapted C. parvum strains has been further substantiated by sequence analysis of the highly polymorphic GP60 gene, which identifies many “allelic families”, including family IIc that appears to be transmitted anthroponotically.

 Therefore, in this example, genetic typing has been instrumental not only --for understanding the complexity of population structures, but also

--for providing a rationale to distinguish zoonotic from non-zoonotic parasite strains

43 Caccio - Molecular typing of parasites

Question: host specificity One step further: genome-wide analysis

 We have seen that some C. parvum strains apparently circulate only in humans.  How can we identify potential determinants of this host specificity?

 One approach is to compare informative parasite isolates at the level of the whole genome, with the objective of identifying the genetic variations that may be responsible for observed phenotypic differences

This type of study is now possible thanks to so-called Next Generation Sequencing (NGS) approaches, which allow to generate hundred millions of sequence (reads) from a given DNA (or RNA) sample.

In the case of Cryptosporidium, the genome sequences of a zoonotic C.parvum strain and of a C. hominis strain were determined before the introduction of NGS.

Thus, NGS sequencing (Illumina) of the genome of a non-zoonotic C. parvum strain (IIc family) was performed and the results compared to the two reference genomes

What to look for?

The hypothesis is that some genetic signatures involved in host adaptation may be identified by genome comparison.

One can look at many things in the genome, e.g.: General organization (synteny) Presence/absence of specific genes Distribution of SNPs (in genes, in intergenic regions) Distribution of synonymous versus non-synonymous substitutions (genes under selection) Regulatory elements Repetitive sequences

44 Caccio - Molecular typing of parasites

Where are the differences?

 Among the 22 most divergent genes identified through genome comparison, a statistically higher frequency of ATP binding cassette (ABC) transporters and of genes encoding proteins with a signal peptide was noticed.

 This suggests that, in a parasite which is highly dependent on host cell metabolites like Cryptosporidium, transporters play an important role in mediating the import of metabolites that the parasite is unable to synthesize.

Other important features?

A comparison of homologous genes in the 3 genomes indicate that there are regions where the non-zoonotic C. parvum is more similar to C. hominis than to the zoonotic C. parvum.

 This was futher corroborated by PCR and sequencing these regions in other non-zoonotic C. parvum strains and by performing a phylogenetic analysis:

Non-zoonotic C. parvum and C. hominis

Zoonotic C. parvum

45 Caccio - Molecular typing of parasites

Hypothesis for future studies

 Sequencing of species with distinct and with similar host range can be useful to test the hypothesis that sympatric speciation is driven by the divergent evolution of a small number of loci  Together with genes involved in host cell invasion and immune evasion, we should consider genes encoding for transporters that control the exchange of metabolites between the host cell and intracellular developmental stages of the parasite, as important factor influencing the ability to establish an infection in a particular host species.

These were not just arbitrary examples

 I may have chosen Giardia or Blastocystis to show how other biological questions (e.g., correlation between clinical symptoms and parasite genetics) have beed studied using similar molecular approaches.

Conclusions

 While biological meaningful questions should continue to drive research, the amazing advances in technology (the “omics” revolution) has changed radically the way such questions can be addressed  On top of the applications I just showed you, we can now study gut eukaryotic organisms in the context of the microbiota, and try to understand their role in healthy individuals and in patients suffering from gastrointestinal diseases and syndromes

46 Caccio - Molecular typing of parasites

Thank you for your attention!

Questions?