Educational Workshop

Transcription

1 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) 1

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3 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 nematodes soil transmitted helminths Ancylostoma duodenale Necator americanus Ascaris lumbricoides Trichuris trichiura warm t, humidity, poor sanitation, dirty water, substandard and crowded housing Sub Saharan Africa > Asia > Latin America Behavior: more active in the environment, rarely employ good sanitation, crowd together (schools, orphanages, slums) helminths = immunomodulators 2 Influence of study population Distribution of intestinal parasites in internationally adopted children in ITM. Number of children: 120 Period: 1/1/ /6/

4 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) 4 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% 5 Influence of study population Prolonged diarrhoea (> 14 days) 1 3% of travellers Protozoal parasites >> helminth parasites Giardia lamblia Cryptosporidium E. histolytica 6 4

5 Van Esbroeck - General introduction and epidemiology of common parasites of the human gut Influence of study population Protozoa E. histolytica Giardia lamblia Cyclospora Cryptosporidium Cystoisospora microsporidia Helminths Strongyloides Ascaris lumbricoides hookworms Trichuris trichiura tapeworms 7 Greater burden of intestinal protozoan infections globally Worldwide increase in immunocompromised individuals travel medical tourism immigration 8 Pathogenic Entamoeba histolytica Giardia Cyclospora cayetanensis Cryptosporidium parvum Cystoisospora (Isospora) belli microsporidium Non pathogenic Entamoeba dispar, E. moshkovskii, E. coli, E. polecki, E. hartmanni Iodamoeba butschlii Endolimax nana Chilomastix mesnili Sarcocystis sp. Dientamoeba fragilis Blastocystis hominis 9 5

6 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 new cases each year ITM Antwerp: 4,6 (6%) 10 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 11 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 12 6

7 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) water and foodborne pathogen selflimiting HIV/AIDS Species Major host Minor host C. parvum Cattle, sheep, goats, humans Deer, mice, pigs C. hominis Humans, monkeys Dugongs, sheep C. muris Rodents, bactrian camels Humans, rock hyrax, moutain goats C. andersoni Cattle, bactrian camels Sheep 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 13 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 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 14 Cryptosporidium 15 7

8 17 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 deaths annually Outbreaks Mexico, Vietnam, Egypt Chicago 1933, sewage and tap water mixed 800 cases Amebic liver abscess <1% (potentially fatal illness) 16 E. histolytica E. histolytica/dispar, ITM, Belgium, N % N % N N % % N % N % N % N % N % E. histolytica/dispar E. histolytica 10 6,6 13 6,9 9 4,6 4 1,9 4 1,9 7 3, ,2 7 2,6 Strongyloides stercoralis nematode (sub)tropical areas (+ moderate climate) global prevalence unknown estimation 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

9 5 µm tot 15 µm 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% 19 Dientamoeba fragilis Cyststage? E. vermicularis as vector for transmission? epidemiological link higher incidence of coinfection than expected DNA of Dientamoeba found in Enterobius Bimodal age distribution in patients positive for D. fragilis Proportion of patients positive for E. vermicularis 20 Blastocystis species (B. hominis) cosmopolitan distribution > 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 21 9

10 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 23 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 24 10

11 Van Esbroeck - General introduction and epidemiology of common parasites of the human gut S. japonicum S. intercalatum Indonesia and parts of China and Southeast Asia Parts of Central and West Africa 25 Schistosoma mekongi Cambodia, Laos 26 Soil transmitted helminth infections 27 11

12 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 M people infected Fertilized eggs Unfertilized eggs 28 Trichuris trichiura whipworm cosmopolitan distribution more frequently in areas with tropical weather and poor sanitation M people infected 29 Ancylostomidae Hook worm 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 30 12

13 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 31 Taenia Taenia saginata beef tapeworm consumption of raw beef cosmopolitan distribution Eastern Europe, Russia, eastern Africa and Latin America Taenia solium pork tapeworm under developed communities with poor sanitation consumption of raw or undercooked pork Latin America, Eastern Europe, sub Saharan Africa, India, and Asia 32 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 33 13

14 Van Esbroeck - General introduction and epidemiology of common parasites of the human gut Hymenolepis nana Hymenolepis diminuta dwarf tapeworm wide distribution, warm areas children in countries with poor sanitation and hygiene institutional settings no intermediate host autoinfection worldwide distribution (rodents) rare in humans intermediate host: several arthropods 34 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 35 Fasciolidae Opisthorchis spp. raw or undercooked fish from endemic countries Thailand, Laos, Cambodia, Viet Nam, and other areas of Asia, Eastern Europe, and the former Soviet Union Clonorchis spp. Chinese or oriental liver fluke Asia: Korea, China, Taiwan, Vietnam, Japan, and Asian Russia Asian immigrants raw or undercooked fish, crab, crayfish outbreaks in Italië O. Felineus carpaccio van Tinca Tinca 36 14

15 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 37 15

16 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

17 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 Cryptosporidium sp Cyclospora cayetanensis Cystoisospora belli Dientamoeba fragilis Entamoeba histolytica Giardia lamblia Microsporidium sp Blastocystis hominis Chilomastix mesnili Endolimax nana Entamoeba coli Entamoeba dispar Entamoeba hartmanni Iodamoeba bűtschlii Helminths Ascaris lumbricoides Diphyllobotrium sp Enterobius vermicularis Fasciola hepatica Hymenolepis nana Hymenolepis diminuta Hookworm Schistosoma sp Strongyloides stercoralis Trichuris trichiura Taenia sp Clonorchis sinensis Opisthorchis sp Cappilaria phillipinensis Classical diagnostic methods for detection of intestinal parasites macroscopic examination microscopic techniques non microscopic (non morphological) techniques ELISA / copro-immunochemistry / Ag testing serology 17

18 Mank - Classical diagnostic methods for detection of parasites Ascaris lumbricoides male and female Ascaris lumbricoides male and female 18

19 Mank - Classical diagnostic methods for detection of parasites 11 Eg,Em & Tsol 19

20 Mank - Classical diagnostic methods for detection of parasites Taenia solium cysticercose CT scan brain laesions < 2cm X ray muscles (eg upperleg) 15 Serodiagnosis Molecular techniques 20

21 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 D. fragilis T. trichiura hookworm E. histolytica/ dispar 21

22 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

23 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); 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

24 Mank - Classical diagnostic methods for detection of parasites Evaluation of the TFT in routine clinical practice 462 patients AMC, Amsterdam 24

25 Mank - Classical diagnostic methods for detection of parasites Organism Increased recovery of intestinal protozoa in TFT (462 patients) One stool sample(nf) TFT Increase with TFT pathogen G. lamblia E. histolytica D. fragilis apathogen E. coli E. hartmanni E. nana C. mesnilii I. bütschlii B. hominis 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);

26 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

27 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

28 Mank - Classical diagnostic methods for detection of parasites Diarrhea I presume Diarrheal complaints Protozoans In case of immuundeficiency Cryptosporidium! Microsporidium sp Protozoans Cryptosporidium sp Cyclospora cayetanensis Cystoisospora belli Dientamoeba fragilis Entamoeba histolytica Giardia lamblia Microsporidium sp Blastocystis hominis Chilomastix mesnili Endolimax nana Entamoeba coli Entamoeba dispar Entamoeba hartmanni Iodamoeba bűtschlii Helminths Ascaris lumbricoides Diphyllobotrium sp Enterobius vermicularis Fasciola hepatica Hymenolepis nana Hymenolepis diminuta Hookworm Schistosoma sp Strongyloides stercoralis Trichuris trichiura Taenia sp Clonorchis sinensis Opisthorchis sp Cappilaria phillipinensis 28

29 Mank - Classical diagnostic methods for detection of parasites E. histolytica and E. dispar Entamoeba histolytica trophozoite Entamoeba histolytica trophozoite fagositized Red blood cells nucleus 29

30 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

31 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

32 Mank - Classical diagnostic methods for detection of parasites Strongyloides stercoralis Fresh stool sample Baermann Culture (filariform larvae) Nematodes PCR Differentiation Strongyloides stercoralis / hookworm Filariform larvae (after culture) short mouth opening large genital primordium long mouth opening small/no genital primordium S. stercoralis hookworm forked tail tapered tail unsheathed sheated New developments Introduction of rt-multiplex PCR as a screening tool for parasitological stoolexaminations 32

33 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

34 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 Cryptosporidium sp Cyclospora cayetanensis Cystoisospora belli Dientamoeba fragilis Entamoeba histolytica Giardia lamblia Microsporidium sp Blastocystis hominis Chilomastix mesnili Endolimax nana Entamoeba coli Entamoeba dispar Entamoeba hartmanni Iodamoeba bűtschlii Helminths Ascaris lumbricoides Diphyllobotrium sp Enterobius vermicularis Fasciola hepatica Hymenolepis nana Hymenolepis diminuta Hookworm Schistosoma sp Strongyloides stercoralis Trichuris trichiura Taenia sp Clonorchis sinensis Opisthorchis sp Cappilaria phillipinensis Protozoans Cryptosporidium sp Cyclospora cayetanensis Cystoisospora belli Dientamoeba fragilis Entamoeba histolytica Giardia lamblia Microsporidium sp Blastocystis hominis Chilomastix mesnili Endolimax nana Entamoeba coli Entamoeba dispar Entamoeba hartmanni Iodamoeba bűtschlii Helminths Ascaris lumbricoides Diphyllobotrium sp Enterobius vermicularis Fasciola hepatica Hymenolepis nana Hymenolepis diminuta Hookworm Schistosoma sp Strongyloides stercoralis Trichuris trichiura Taenia sp Clonorchis sinensis Opisthorchis sp Cappilaria phillipinensis 34

35 Mank - Classical diagnostic methods for detection of parasites Parasitic species detected in 2000 TFT sets by rt-pcr and microscopy Species rt-pcr Giardia/ Crypto/ E. histo/ D.fragilis TFT microscopy Cryptosporidium sp D. fragilis E. histolytica 1 1 (E his/dis) Giardia lamblia 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 -Schistosoma spp -Strongyloides -Amebic abces -Leishmania spp -Pneumocystis (PCP) -Malaria -other: -foreign travel / tropics? -specific skin disorders? -eosinofilia? -immunocompromised / deficient? -helminths seen? -therapy? -other: 35

36 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

37 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

38 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 24 rd ECCMID, Barcelona May 2014 A broad topic... The human gut is the ecological niche of many parasites Protozoa Blastocystis Dientamoeba Giardia Cryptosporidium Entamoeba Cyclospora Helminthes Ancylostoma Enterobius Ascaris Trichuris 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

39 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

40 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

41 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

42 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

43 Caccio - Molecular typing of parasites eburst network C. hominis isolates from UK MLGs organized in a star-like cluster with the most abundant in the centre. C. hominis isolates from Uganda The MLG network is straggly and shows long chaining of 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) 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

44 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

45 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

46 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

47 Caccio - Molecular typing of parasites Thank you for your attention! Questions? 47