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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 28-29 May, 2012

� And in the summer, when I feeldisposed to look at all manner oflittle animals, I just take the water that has been standing a few daysin the leaden gutter up on my roof, or the water out of stagnant shallowditches: and in this I discovermarvellous creatures

from a letter of Antoni van Leeuwenhoek to ConstatijnHuygens, Dec. 26, 1678

Structure of this talk

� Introduction to the organism

�Taxonomy

�Diagnostics

�Descriptive epidemiology

�Environmental contamination

�Outbreak investigations

�Conclusions

Giardia: one of these creatures

Trophozoite Cyst

Giardia is a flagellated, unicellular eukaryote, that has a simple life cyclewhich comprises two stages: the trophozoite and the cyst

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Source: CDC

Life cycle of Giardia Taxonomy of Giardia�More than 50 species of Giardia have been described on the

basis of morphology. At that time, the assumption was: one host, one parasite

� In the ’50s, Filice proposed a taxonomical scheme with only 3valid species, on the basis of the morphology of thetrophozoites.

�G. agilis�G. muris�G. duodenalis

Taxonomy of GiardiaSpecies 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 variationis observed

Genetic relationships based on isoenzyme data

Ass. C and D in dogs

Ass. G in rats

Ass. B in various mammals

Ass. E in livestock

Ass. A in various mammals

Ass. F in cats

Genetic groups or assemblages

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Genetic relationshipsbased on DNA (gdh)

Giardia duodenalis

• Genetic groups Host

• Assemblage A Man, primates, livestock, pets, wildlife

• Assemblage B Man, primates, livestock, dog,beaver, horse, rat, muskrat

• Assemblages C, D dog, canids

• Assemblage E livestock

• Assemblage F cat

• Assemblage G rat, hamster

• Assemblage H marine mammals

Biological features of Giardia which influence the epidemiology

�Small, environmentally resistant cyst stage

� Large animal and human reservoirs

�Ubiquitous

� Low infective dose (1-10 organisms)

�Cysts immediately infectious upon excretion

�Ability to multiply to large numbers in a single host

General considerations on the epidemiology ofhuman giardiasis

�Most cases are waterborne�Person-to-person transmission�Zoonotic transmission (?) �Foodborne transmission

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Prevalence / rate of infection�Europe: 59.6 per 100,000, comparable to

Campylobacteriosis (44.1 per 100,000) and Salmonellosis (29.75 per 100,000).

�The highest rates of giardiasis were reported by�Romania (691 per 100,000 population, responsible for

89% of all the reported cases)�Bulgaria (28 per 100,000)�Estonia (20 per 100,000) �Sweden (17 per 100,000).

� Data from the ECDC, Annual epidemiological report on communicable diseases in Europe, 2010

Prevalence / rate of infection

�USA: 2.2 to 33.8 cases per 100,000

�New Zealand: 49.4 per 100,000

�Other regions of the world: only prevalence data are available (Thailand 37%, Nepal 73%, Malaysia 25%)

Giardiasis is a pediatric infection

Age-specific notification rates ofgiardiasis cases in EU and EEA/EFTAcountries in 2008 (n = 161,734).

Age-specific notification rates of giardiasiscases in USA in 2003, 2004 and 2005.Numbers in 1000.

Europe

USA

Seasonal trends in giardiasis

Data from the ECDC, 2010 Annual Report (data for 2008)

Data from the CDC, 2003-2005 (Yoder and Beach, 2010)

Europe

USA

AUTUMN PEAK?

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Clinical manifestations of giardiasisVery variable. Three situations can be distinguished:

Asymptomatic giardiasis: this represents a considerablepercentage of infections. The role of carriers in theepidemiology of giardiasis is not well understood

Symptomatic giardiasis: the most common symptom isdiarrhea, followed by nausea and abdominal pain. In severecases, a malabsorption syndrome can occur

Chronic giardiasis: in a small number of cases, the infectiontends to be chronic, and resistance to treatment has beenobserved

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 171 cases and 681 controls• Use of shallow water sources• Travelling abroad• Daycare center exposure

Germany: study of 120 cases and 240 controls• Gender (males more likely to be infected than women)• Immunocompromised more likely to be infected• Daily consumers of green saladItaly: study of 52 cases and 72 controls• Exposure to surface water• Travelling abroad• High educational levelNew Zealand: study of 183 cases and 386 controls• Changing nappies is a risk factor (4-fold increase)

DIRECT and INDIRECT

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

�Risk factors identified

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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 ofmorphologic variation between cysts

Giardia cysts Giardia trophozoite

�This method offers higher sensitivity and specificitycompared 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 differentialinterference contrast (DIC), difficult.

�The antibodies do not differentiate between cyst of Giardiabelonging to different species

Detection of cysts by immunofluorescence

Detection using molecular techniques

Amplification by PCR

Post-PCR assays

DNA/RNA isolationCysts

RFLP Sequencing Real-time PCR

How to characterise Giardia

Platforms: �PCR-RFLP�Sequencing �Real-time�Microarray

� 18S rDNA (genus-specific)� ITS region and 5.8S rDNA

� Beta-giardin� Triose phosphate isomerase� Glutamate dehydrogenase

� Actin� Tubulin� Histone

� Ferredoxin

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Varioys species of deer, wild boar, cat

Human, cat (1)

Calf, sheep,cat, deer, human (4)AI

AII

AIII

1%

111 isolates

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

Multilocus data for assemblage AHuman, wildlife, monkeys

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37

56

64

6

1

11

0

0

98

35

7

93

2

1

0

24

0

0

0

12

23

0

1

6

4

0

0

43

1

0

21

2

0

20

3

0

0

0

18

2

0

11

0

0

17

0

0

0

13

3

9

2

0

0

0

0

0

0

100

0

33

21

18

99

99

26

1

1

0

45

39

1

0

20

15

10

63

68

2

0

51

41

13

41

24

5

25

14

57

57

57

51

29

29

23

17

17

14

7

19

1

5

1

0

18

2

0

28

14

0

12

0

0

0

0

1%

118 isolatesMultilocus data for assemblage B

No evident clusters in terms of hosts or sample origin are observed for assemblage B

Sprong, Cacciò, van der Giessen (PLOS Neglected Tropical Diseases 2009)

We are in the genomic era

� The genomes of representative of Giardia duodenalisassemblage A (WB), ass. B (GS) and ass. E (P15) have been sequenced.

� All the information is available in the EUPATHDB(Eukaryotic Pathogens Database Resource), thus it ispossible to identify novel genetic markers with desidercharacteristics

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 non-human pathogens

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Molecular tools: to do what?

� To establish the identity of the parasites in humansand 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

Questions of interest

� Is giardiasis a zoonosis?

� 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 rareAssemblage A more commonMixed infections common

Assemblage-specific strains are more common in their respective hosts than zoonotic strains

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Livestock cycle

Dog/Cat cycle

Human cycle

Direct orwaterborne

Direct orwaterborne

Frequency oftransmssionUNKNOWN

Giardia in domestic animals

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.

Frequency oftransmssionUNKNOWN

Giardia in wildlifeWild carnivores Hoofed animals

Coyotes, wolves, foxes, ferretsFallow deer, roe deer, reindeer, moose

wild boars, water buffaloes

Rodents

Beavers, muskrats, voles, wild mice

Assemblage A, zoonotic subtype A I presentbut subtype AIII is more common and verydistinct from those found in humans.

Assemblages C / D commonly found. Assemblage A (mainly AI) also present.

Giardia microti is commonly found, beavers and muskrats also infected withassemblage B

Non-human primates

Non-human primates are infected withassemblages 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.

Wildlife cycle(s)

Direct orwaterborne

Frequency of transmssionLOW

Human cycle

Environmental transmission

Giardia Standardised methods availableUSEPA method 1623

The Water Supply (Water Quality) Regulations 2000 (SI 2000/3184 )

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Overview of the method

Filtration Immunomagnetic separation Microscopy

Giardia and water

�Surveys performed in many countries from all overthe world.

�Cysts detected in wastewaterwater, surface water, ground water, springs and drinking water samples.

�In short, cysts are ubiquitous in the aquaticenvironment

Tongariro National park, New Zealand

Yes, Giardia is just everywhere!

Number of positive samples and number of cysts decrease

SEWAGE RAW WATER SURFACE DRINKING

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Country Cyst/liter % positive methodFrance 1300–12,000 100 Light microscopy

France 0.23-25,000 100 IF

France 1000–25,000 100 IF

France 330-4300 100 IF+RT-PCR

Italy 2100-42,000 100 IF+PCR

Norway 100–3,600 93 IF

Spain 67 100 IF

Spain 2–14,400 98 IF

Scotland 5-940 96 IF

Scotland 10-3,600 70 – 91 IF

Wastewater in Europe

Almost all sample are positive, and very high number of cysts

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

Removal efficiency of WWTP

Country Treatment Reduction%

Cyst per liter

France Stabilization pond 99.7-100 0-30

France Stabilization pond 99.1 0-16

Spain Stabilization pond 85 0-5

Germany Constructed wetland 99.7 Not reported

Ireland Constructed wetland 95.4 11-111

Scotland Activated sludge 75.3 345

Scotland Biological filtration 91.6 40

Italy Ultrafiltration 100 0

Italy Active oxidation 94.5 200-300

Data from Nasser et al. Journal of Applied Microbiology, 2012

Surface water in EuropeCountry Sampling Cyst/l % positive methodFinland 39 sites, 139 samples Not reported 14 IMS+IF+PCR

France Paris, 5 sites, 57 samples 0-16 66 IMS+IF

Netherlands Amsterdam, canalsRecreational water

0-160-1

9737

IMS+IF

Spain Rivers, 56 samplesReservoirs, 36 samples

0-2500-13

9255

IMS+IF

Switzerland Recreational water, 2 sites, 40 samples

0-22 97 IMS+IF

Portugal Surface water, 30 samplesGroundwater, 39 samples

Not reportedNot reported

5759

IMS+IF+PCR

Hungary Rivers, 5 samplesReservoirs, 11 samples

0-30-9

10045

IMS+IF+PCR

Italy Rivers, 39 samplesWatershed, 48 samples

1-4650-0.1

1000-50

IMS+IF

Positivity still high, but number of cysts much lower

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Genetic identity of Giardia cysts in water� Surprisingly, only 65 sequences have been deposited in

GenBank (compared to >4000 from feces)

Country Type of water No ofsamples

% positive

Rangecysts/liter

Assemblage

Hungary Surface 16 62.5 0.1-17.4 A and B

Portugal Surface 69 58 - A

Spain Surface 116 67.2 2-722 A and E

Norway Sewage 72 53 100-51333 A and B

France Wastewater 24 100 530-11000 A and B

France Wasterwaterslaughterhouse

12 58.592

1400-4100-

A E

Italy Wastewater 16 100 2100-42000 A and B

�A total of 106 outbreaks were associated with contaminated drinking water systems, the majority have been reported in the USA (77%) and Canada (13%).�The main cause was deficiencies in water treatment process (insufficient barrier and inadequate or poorly operated treatment and disinfection systems)

�Another 16 outbreaks were associated with contaminated recreational water.

�Giardia and Cryptosporidium are responsible for almost all waterborne outbreaks of gastroenteritis caused by protozoa

Waterborne outbreaks

Investigation of outbreaks

� Despite the large number of waterborne outbreaks, only a few have been investigated to include genetic characterization of cysts from suspectedsources and confirmed cases

� What have we learned from those few studies?“”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”

Ann Intern Med. 1980 Feb;92(2 Pt 1):165-70.Municipal waterborne giardiasis: an epidemilogic investigation.

Beavers implicated as a possible reservoir.

Beaver fever?

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1994Outbreak in the community of Temagami (Canada)30% affected by illnessHigh concentration of Giardia cysts in treated water sampesA BWA was issued

200012 human samples, 1 sample of filtered water and 1 beaversample from the outbreak analysed by PCR (SSU-rRNA)Only assemblage A found in 6 human samples, all other samples negative

This is the only outbreak caused by assemblage ANo involvement of beavers?

(when positive, beavers harbor assemblage B)

Outbreak investigation 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

The outbreak in Bergen, Norway

� Giardiasis is mainly considered as an imported disease in Norway, with >90% of cases linked to travel or detected at immigrant screenings.

� A survey of surface water showed that Giardia is present in 18.5 % of sources sampled, albeit in small numbers (80% of the samples had only 1 cyst/10 l, and the highest concentration observed was 3 cysts/10 l)

� Due to the geography and low population density of Norway, drinking water frequently has minimal treatment

� This was the largest waterborneoutbreak of giardiasis, with almost 1300 laboratory confirmed cases, and 2500 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

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� The age distribution of cases can be explained bythe larger consumption of water (>5 glasses per day) of young women, and the low attack rate amongchildren and elderly suggests that the contaminationwas small and that large amounts were needed forinfection.

Genotyping of Giardia from human cases

�Genotyping of cysts from 21 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 12 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 21 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 (12 samples) from the vicinity of the septic tankThe sample from the tank was positive, as were 4 of th 12 soil samples, but in most cases the cystswere breoken or distorted and DAPI negative

PCR and sequencing of the b-giardin and gdh genes showedthat the cysts from the septic tank were Assemblage A

Sources?

� Despite the fact that epidemiologic data supportedwater supply A as the source of contaminated water causing the outbreak, water from that source wasnot investigated to detect and characterize Giardiacysts

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Lesson?

� Unexpected complexity of genotyping results illustratesthe difficulties in tracing the source(s) of infection

� Unexpected long-term health sequelae of giardiasis: a prospective cohort study demonstrated that 32% (40/124) 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 ofthem have been investigated

Thank you for your attention!

Questions?