Waterborne protozoan zoonoses: Giardia

Transcription

1 Waterborne protozoan zoonoses: Giardia Simone M Cacciò European Union Reference Laboratory for Parasites, Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità 7th Workshop of National Reference Laboratories for Parasites, Rome May, 22 And in the summer, when I feel disposed to look at all manner of little animals, I just take the water that has been standing a few days in the leaden gutter up on my roof, or the water out of stagnant shallow ditches: and in this I discover marvellous creatures from a letter of Antoni van Leeuwenhoek to Constatijn Huygens, Dec. 26, 678 Structure of this talk Introduction to the organism Giardia: one of these creatures Giardia is a flagellated, unicellular eukaryote, that has a simple life cycle which comprises two stages: the trophozoite and the cyst Taxonomy Diagnostics Descriptive epidemiology Environmental contamination Outbreak investigations Conclusions Trophozoite Cyst

2 Life cycle of Giardia Taxonomy of Giardia More than 5 species of Giardia have been described on the basis of morphology. At that time, the assumption was: one host, one parasite In the 5s, Filice proposed a taxonomical scheme with only 3 valid species, on the basis of the morphology of the trophozoites. G. agilis G. muris G. duodenalis Source: CDC Species Taxonomy of Giardia Host G. agilis Amphibians G. ardeae Birds G. psittaci Birds G. muris Rodents G. microti Rodents G. duodenalis Mammals (including man) However, G. duodenalis is not a single species. If we look at the morphology of cysts from different hosts, there is no variability, but when cysts are compared at the level of protein (isoenzymes) or DNA polymophism, a large amount of genetic variation is observed Genetic relationships based on isoenzyme data Genetic groups or assemblages Ass. C and D in dogs Ass. G in rats Ass. B in various mammals Ass. E in livestock Ass. F in cats Ass. A in various mammals 2

3 Giardia duodenalis Genetic relationships based on DNA (gdh) Genetic groups Assemblage A Assemblage B Assemblages C, D Assemblage E Assemblage F Assemblage G Assemblage H Host Man, primates, livestock, pets, wildlife Man, primates, livestock, dog, beaver, horse, rat, muskrat dog, canids livestock cat rat, hamster marine mammals Biological features of Giardia which influence the epidemiology Small, environmentally resistant cyst stage Large animal and human reservoirs Ubiquitous Low infective dose (- organisms) Cysts immediately infectious upon excretion Ability to multiply to large numbers in a single host General considerations on the epidemiology of human giardiasis Most cases are waterborne Person-to-person transmission Zoonotic transmission (?) Foodborne transmission 3

4 Prevalence / rate of infection Europe: 59.6 per,, comparable to Campylobacteriosis (44. per,) and Salmonellosis (29.75 per,). The highest rates of giardiasis were reported by Romania (69 per, population, responsible for 89% of all the reported cases) Bulgaria (28 per,) Estonia (2 per,) Sweden (7 per,). Prevalence / rate of infection USA: 2.2 to 33.8 cases per, New Zealand: 49.4 per, Other regions of the world: only prevalence data are available (Thailand 37%, Nepal 73%, Malaysia 25%) Data from the ECDC, Annual epidemiological report on communicable diseases in Europe, 2 Giardiasis is a pediatric infection Seasonal trends in giardiasis Europe Age-specific notification rates of giardiasis cases in EU and EEA/EFTA countries in 28 (n = 6,734). Europe Data from the ECDC, 2 Annual Report (data for 28) USA Age-specific notification rates of giardiasis cases in USA in 23, 24 and 25. Numbers in. USA Data from the CDC, (Yoder and Beach, 2) AUTUMN PEAK? 4

5 Clinical manifestations of giardiasis Very variable. Three situations can be distinguished: Asymptomatic giardiasis: this represents a considerable percentage of infections. The role of carriers in the epidemiology of giardiasis is not well understood Symptomatic giardiasis: the most common symptom is diarrhea, followed by nausea and abdominal pain. In severe cases, a malabsorption syndrome can occur Chronic giardiasis: in a small number of cases, the infection tends to be chronic, and resistance to treatment has been observed Risk factors: sporadic giardiasis UK: study of 232 cases and 574 controls Swallowing water while swimming Recreational fresh water contact Drinking treated tap water Eating lettuce USA: study of 7 cases and 68 controls Use of shallow water sources Travelling abroad Daycare center exposure Germany: study of 2 cases and 24 controls Gender (males more likely to be infected than women) Immunocompromised more likely to be infected Daily consumers of green salad Italy: study of 52 cases and 72 controls Exposure to surface water Travelling abroad High educational level New Zealand: study of 83 cases and 386 controls Changing nappies is a risk factor (4-fold increase) Summary Very common infection with a global distribution Children represent the main risk category Multiple transmission routes Infection usually self-limited Clinical symptoms highly variable DIRECT and INDIRECT Risk factors identified 5

6 Diagnosis: staining techniques Staining techniques have a relatively good sensitivity and specificity for the detection of Giardia cysts (and trophozoites) However, these techniques cannot be used to identify species (or assemblages) of the parasite, because of the lack of morphologic variation between cysts Giardia cysts Giardia trophozoite Detection of cysts by immunofluorescence This method offers higher sensitivity and specificity compared to staining techniques. However, autofluorescent algae and other debris can cause cysts to be obscured when the sample is fixed to a slide, making confirmation by phase contrast or differential interference contrast (DIC), difficult. The antibodies do not differentiate between cyst of Giardia belonging to different species Detection using molecular techniques How to characterise Giardia Cysts DNA/RNA isolation Amplification by PCR Post-PCR assays RFLP Sequencing Real-time PCR 8S rdna (genus-specific) ITS region and 5.8S rdna Beta-giardin Triose phosphate isomerase Glutamate dehydrogenase Actin Tubulin Histone Ferredoxin Platforms: PCR-RFLP Sequencing Real-time Microarray 6

7 % % AI Calf, sheep,cat, deer, human (4) Multilocus data for assemblage B 8 isolates Multilocus data for assemblage A isolates Human, wildlife, monkeys Human, cat () AII AIII Varioys species of deer, wild boar, cat Phylogenetic relationships of assemblage A isolates based on 3 concatenated genes. The 3 main groups are strongly supported (boostrap values >98) Zoonotic transmission is not supported by these data No evident clusters in terms of hosts or sample origin are observed for assemblage B Sprong, Cacciò, van der Giessen (PLOS Neglected Tropical Diseases 29) We are in the genomic era The genomes of representative of Giardia duodenalis assemblage A (WB), ass. B (GS) and ass. E (P5) have been sequenced. All the information is available in the EUPATHDB (Eukaryotic Pathogens Database Resource), thus it is possible to identify novel genetic markers with desider characteristics Technical challenges Large number of different matrices to analyse Necessity to detect very low number of cysts Necessity to distinguish between viable (infectious) and non-viable organisms Necessity to distinguish between human and nonhuman pathogens 7

8 Molecular tools: to do what? To establish the identity of the parasites in humans and animals (zoonosis?) To identify infection or contamination sources To correlate parasite s genetic diversity and the clinical manifestation in the host To detect Giardia cysts pathogenic to humans in water or food samples Is giardiasis a zoonosis? Questions of interest Is there a link between the genetic diversity of the parasite and the clinical manifestation of giardiasis? Is there sex in Giardia? What controls drug resistance in Giardia? What controls host specificity? Is giardiasis a zoonosis? Giardia in domestic animals Assemblage E is prevalent Assemblage A mainly AI Assemblage B almost absent Assemblage F is prevalent Assemblage C rare Assemblage A (mainly AI) Assemblage B rare Assemblage C / D prevalent Assemblage B rare Assemblage A more common Mixed infections common Assemblage-specific strains are more common in their respective hosts than zoonotic strains 8

9 Giardia in domestic animals Wild carnivores Giardia in wildlife Hoofed animals Dog/Cat cycle Frequency of transmssion UNKNOWN Human cycle Direct or waterborne Direct or waterborne Frequency of transmssion UNKNOWN Livestock cycle Well designed studies are needed to understand the role of domestic animals in the epidemiology of human infection. Few studies in endemic regions (India, Thailand) have provided some indications for a zoonotic role of dogs. Coyotes, wolves, foxes, ferrets Assemblages C / D commonly found. Assemblage A (mainly AI) also present. Rodents Beavers, muskrats, voles, wild mice Giardia microti is commonly found, beavers and muskrats also infected with assemblage B Fallow deer, roe deer, reindeer, moose wild boars, water buffaloes Assemblage A, zoonotic subtype A I present but subtype AIII is more common and very distinct from those found in humans. Non-human primates Non-human primates are infected with assemblages A and B, but most isolates are from captive animals Giardia in wildlife The data indicate a minor role of wild animals in the transmission of Giardia to humans. Environmental transmission Frequency of transmssion LOW Human cycle Direct or waterborne Giardia Standardised methods available USEPA method 623 The Water Supply (Water Quality) Regulations 2 (SI 2/384 ) Wildlife cycle(s) 9

10 Overview of the method Filtration Immunomagnetic separation Microscopy Giardia and water Surveys performed in many countries from all over the world. Cysts detected in wastewaterwater, surface water, ground water, springs and drinking water samples. In short, cysts are ubiquitous in the aquatic environment Tongariro National park, New Zealand SEWAGE RAW WATER SURFACE DRINKING Yes, Giardia is just everywhere! Number of positive samples and number of cysts decrease

11 Wastewater in Europe Country Cyst/liter % positive method France 3 2, Light microscopy France.23-25, IF France 25, IF France IF+RT-PCR Italy 2-42, IF+PCR Norway 3,6 93 IF Spain 67 IF Spain 2 4,4 98 IF Scotland IF Scotland -3,6 7 9 IF How to reduce this load? Wastewater treatment plants are designed to produce effluents that carry low health risk levels for the exposed population Wastewater treatment consists of a set of subsequent stages which usually involve a combination of physical (sedimentation, retention and filtration, inactivation by solar radiation and UV) biological (activated sludge and organic matter assimilation and removal) chemical (coagulation and inactivation by oxidation) stages Almost all sample are positive, and very high number of cysts Removal efficiency of WWTP Country Treatment Reduction % Cyst per liter France Stabilization pond France Stabilization pond Spain Stabilization pond 85-5 Germany Constructed wetland 99.7 Not reported Ireland Constructed wetland Scotland Activated sludge Scotland Biological filtration Italy Ultrafiltration Italy Active oxidation Data from Nasser et al. Journal of Applied Microbiology, 22 Surface water in Europe Country Sampling Cyst/l % positive method Finland 39 sites, 39 samples Not reported 4 IMS+IF+PCR France Paris, 5 sites, 57 samples IMS+IF Netherlands Spain Amsterdam, canals Recreational water Rivers, 56 samples Reservoirs, 36 samples Switzerland Recreational water, 2 sites, 4 samples Portugal Hungary Italy Surface water, 3 samples Groundwater, 39 samples Rivers, 5 samples Reservoirs, samples Rivers, 39 samples Watershed, 48 samples IMS+IF IMS+IF IMS+IF Not reported Not reported IMS+IF+PCR IMS+IF+PCR IMS+IF Positivity still high, but number of cysts much lower

12 Genetic identity of Giardia cysts in water Surprisingly, only 65 sequences have been deposited in GenBank (compared to >4 from feces) Country Type of water No of samples % positive Range cysts/liter Assemblage Hungary Surface A and B Portugal Surface A Spain Surface A and E Norway Sewage A and B France Wastewater A and B France Wasterwater slaughterhouse Italy Wastewater A and B A E Waterborne outbreaks A total of 6 outbreaks were associated with contaminated drinking water systems, the majority have been reported in the USA (77%) and Canada (3%). The main cause was deficiencies in water treatment process (insufficient barrier and inadequate or poorly operated treatment and disinfection systems) Another 6 outbreaks were associated with contaminated recreational water. Giardia and Cryptosporidium are responsible for almost all waterborne outbreaks of gastroenteritis caused by protozoa Investigation of outbreaks Despite the large number of waterborne outbreaks, only a few have been investigated to include genetic characterization of cysts from suspected sources and confirmed cases What have we learned from those few studies? Beaver fever? Ann Intern Med. 98 Feb;92(2 Pt ):65-7. Municipal waterborne giardiasis: an epidemilogic investigation. Beavers implicated as a possible reservoir. The beaver has gained attention as a potential source of Giardia contamination of lakes, reservoirs and streams, but human fecal wastes are probably as important Small aquatic or semi-aquatic wild mammals, such as beavers, muskrats, and small rodents harbor water-born cysts of Giardia and serve as important reservoir hosts 2

13 Outbreak investigation 994 Outbreak in the community of Temagami (Canada) 3% affected by illness High concentration of Giardia cysts in treated water sampes A BWA was issued 2 2 human samples, sample of filtered water and beaver sample from the outbreak analysed by PCR (SSU-rRNA) Only assemblage A found in 6 human samples, all other samples negative Lesson? In the developed world, waterborne transmission is usually the result of contamination with Giardia of human origin or a process failure by water utilities, industry or in swimming pools. Such contamination may impact negatively on ecosystem health leading to infections in aquatic wildlife which may then establish reservoirs of human infection. The role of the beaver as a spill back reservoir of Giardia in North America is the best known example This is the only outbreak caused by assemblage A No involvement of beavers? (when positive, beavers harbor assemblage B) The outbreak in Bergen, Norway Giardiasis is mainly considered as an imported disease in Norway, with >9% of cases linked to travel or detected at immigrant screenings. A survey of surface water showed that Giardia is present in 8.5 % of sources sampled, albeit in small numbers (8% of the samples had only cyst/ l, and the highest concentration observed was 3 cysts/ l) Due to the geography and low population density of Norway, drinking water frequently has minimal treatment This was the largest waterborne outbreak of giardiasis, with almost 3 laboratory confirmed cases, and 25 persons receiving medical treatment. The municipality of Bergen is supplied with drinking water from six different waterworks, all using surface water sources Case distribution indicated that water contamination of water supply A was the likely source of infection Map of giardiasis cases in Bergen municipality 3

14 The age distribution of cases can be explained by the larger consumption of water (>5 glasses per day) of young women, and the low attack rate among children and elderly suggests that the contamination was small and that large amounts were needed for infection. Genotyping of Giardia from human cases Genotyping of cysts from 2 randomly selected patient samples taken over an 8 day period during the peak of the outbreak revealed that they were from all Assemblage B, irrespective of the gene used for analysis. Interestingly, extensive sequence variability was found at the b-giardin and gdh genes among different isolates. Further genotyping of 63 isolates from patients diagnosed in the 2 months subsequent to the outbreak peak showed that particular sequences within Assemblage B gradually predominated over time These sequences had not been the most frequently identified amongst 2 isolates from the outbreak peak. Sources? Sewage leakage or discharge from a septic tank was first considered as a possible source of contamination Samples were taken from the tank (5 liters) and from the soil (2 samples) from the vicinity of the septic tank The sample from the tank was positive, as were 4 of th 2 soil samples, but in most cases the cysts were breoken or distorted and DAPI negative Sources? Despite the fact that epidemiologic data supported water supply A as the source of contaminated water causing the outbreak, water from that source was not investigated to detect and characterize Giardia cysts PCR and sequencing of the b-giardin and gdh genes showed that the cysts from the septic tank were Assemblage A 4

15 Lesson? Unexpected complexity of genotyping results illustrates the difficulties in tracing the source(s) of infection Unexpected long-term health sequelae of giardiasis: a prospective cohort study demonstrated that 32% (4/24) of patients with persistent symptoms had chronic Giardia infection, with mean disease duration of 7 months Conclusions Giardiasis is a very common infection worldwide Human infection is caused by assemblages A and B Transmission through contaminated water is a key element in the epidemiology of infection Water contamination is widespread We lack information on the circulation of cysts of assemblages A and B in the water, on their relative ability to persist in the environment and associated risk Many waterborne outbreaks have occurred but few of them have been investigated Thank you for your attention! Questions? 5