Lecture on parasitological risk in drinking water for the faculty of Veterinary Medicine, University of Helsinki; Course in Environmental Health

1. WATERBORN
E PARASITES
Crytosporidium and Giardia

2. CRYPTOSPORIDIUM Biology and epidemiology

3. TAXONOMY
Apicomplexan parasite – it was classified as Coccidia but new
evidences put this group of organisms as Gregarine
Class – Gregarinomorphea; Subclass – Cryptogregaria; Genus –
Cryptosporidium
30 valid Species – C. parvum; C. hominis  responsible of the
majority of human infections (in human also C. meleagridis, C. felis,
C. canis)
In addition there are several genotypes (some of them detected in
humans)
C. parvum is zoonotic  wide range of animal hosts
Differs from other apicomplexas
 lost apicoplast and mitochondrion

4. LIFE CYCLE
Primary site of infection with C.
hominis and C. parvum is the small
intestine
Endogenous phase begins after the
oocyst is ingested by a suitable host
 excystation
Motile sporozoites approach a host-
cell apical end and actively invade
Parasitophorous vacuole are
intracellular but extracytoplasmic
Asexual and sexual multiplication
Oocysts sporulate in situ  contains
4 sporozoites
 Thin walled  autoinfection
 Thick walled  excretion
Excystation
Invasion
Internalization
Asexual Multiplication (Merogon
Sexual Multiplication
Sexual MultiplicationSporogony
C. parvum has two meront types
Fayer 2008

5. LIFE CYCLE – IMPLICATION IN
WATERBORNE TRANSMISSION
Cryptosporidium has been shown to have the capacity to multiply
epicellularly and extracellularly
Cryptosporidium has been shown to survive, multiply and develop in
biofilms  free-living potential
Ability to complete its life cycle in the absence of host cells 
epicellular and extracellular multiplication and development and they
may both occur simultaneously in the host for mass production of
new oocysts
Extracellular gamont-like stages (as observed in other Gregarine)

6. EXTRACELLULAR
STAGES
Extracellular stages (intestine
lumen  in water after
contamination)
 Oocysts
 Gamont-like (detected in faecal
samples in large numbers  not
abnormal development due to the
lack of host-cells but probably
normal part of the life cycle)
 Gametocyst (multi-nucleated
mass)-like (after the fusion of
gamont-like)
Current IFAT methods cannot
identify all stages = problem
for water industries (Hijjawi et al., 2002; 200

7. CLINICAL FEATURES IN HUMAN AND
PATHOGENESIS
Warren & Guerrant 2008

8. CLINICAL FEATURES IN HUMAN AND
PATHOGENESIS
Significant diarrhea (≥ 1 L/day)
with minimal histopathology in
the gut
Pathogenesis unclear – in part
explained by malabsorption
secondary to blunted or inflamed
villi coupled with fluid secretion
from the crypts
In contrast to C. parvum, C.
hominis elicited a serum IgG
response in most infected
persons
Oocyst shedding ranged from 1
Warren & Guerrant 2008

9. DETECTION AND IDENTIFICATION
Sporocyst absent; a single spore with four
sporozoites
Microscopic examination of faecal samples for
detecting oocysts is difficult and operator-
dependent
 Staining techniques have been developed  suffer sensitivity,
sensibility and reproducibility
 Species identification is not possible  oocysts of many
species lack unique features and are indistinguishable from
one another
 Immunofluorescent antiobody testing (IFAT)  standard
method for water
Molecular approach offers greater sensitivity and
specificity
fluorescent stain
Ziehl-Neelson modified
acid-fast
Images from cdc

10. Confirmed case
 Clinical:
 Diarrhoea + Abdominal
pain
 Laboratory:
 Demonstration of
Cryptosporidium oocysts in
stool
 Demonstration of
Cryptosporidium in
intestinal fluid or small-
bowel biopsy specimens
 Detection of
Cryptosporidium nucleic
acid in stool
 Detection of
Cryptosporidium antigen in
stool

11. EPIDEMIOLOGY
More than one genotype/species is responsible for human disease
and individuals can be co-infected
Distribution of species differs among countries (e.g. C. meleagridis
more common in Peru)
Host range for species and genotypes varies
 C. parvum = wide range of animal hosts; C. hominis is not limited to human but
also detected in cattle
 Some subtypes of C. parvum may be uncommon in agricultural animals but can
occur in humans
The change in distribution of cases over time differs according to
seasons
The global burden of disease associated with water, sanitation, and
hygiene is estimated to be around 4% of all deaths and 5.7% of the
disease burden as measured by disability-adjusted life years (DALYs)

12. TRANSMISSION
Direct transmission via faecal/oral route by the oocyst stage
Person-to-person  common within family homes, day-care centers,
hospitals, and in urban environments (high density)
Transmission between immunocompromised individuals is higher than
between immunocompetent individuals
Required dose to cause infection in 50% of subjects (=ID50)
 C. homins 10 – 83 (but even 1 could be sufficient); C. parvum 132
Water is an essential vehicle of transmission:
 60% of waterborne outbreaks worldwide (2004-2010)
 Surface water most likely contaminated than ground water
Viability of Cryptosporidium parvum oocysts  is infectivity in an animal
model

13. TRANSMISSION PATHWAYS

14. ENVIRONMENTAL FACTORS
AFFECTING CRYPTOSPORIDIUM
SURVIVAL
In the absence of freezing conditions, colder water temperatures tend
to promote the survival
Oocyst stability under freezing conditions dependent upon the
surrounding matrix (cryoprotection)
Physical shear forces affect oocyst viability: effects of sand and gravel
particles or fast-flowing waters (sands filtering)
Microbial predation

15. RISK FACTORS
Drinking water outbreaks  breakdowns in water treatment and/or its
operation
↑ Cryptosporidiosis have been associated with heavy rainfall and flooding;
dry period that ends with heavy rain
Outbreaks of cryptosporidiosis have been associated with swimming pools
due to sewage contamination:
 broken drain
 fecal accidents
 defective filtration
 backwashing during pool use rather than at the end of a day
Better regulation and drinking water treatment improvements can
substantially reduce disease attributed to drinking water, but disease
associated with swimming may be more difficult to tackle

16. GIARDIA Biology and epidemiology

17. TAXONOMY Microaerophilic flagellates  two nuclei, each with
four associated symmetrical flagella
Lack both mitochondria and a Golgi apparatus
The two nuclei are inherited independently during
mitosis
In the cyst, karyogamy facilitate homologous
recombination
Sex is possible but still understudied
Giardia duodenalis, G. intestinalis & G. lamblia are
synonymous for the same organisms responsible for
human giardiasis
 G. duodenalis & G. intestinalis are used in referring to Giardia
species infecting mammals
 In the medical field G. lamblia is still used to refer to Giardia
species infecting humans
Kingdom Protista
Subkingdom Protozoa
Phylum Sarcomastigophora
Subphylum Mastigophora
Class Zoomastigophora
Order Diplomonadida
Family Hexamitidae
Genus Giardia

18. LIFE CYCLE
In the small intestine, excystation
releases trophozoites (each cyst
produces two trophozoites) (3)
Trophozoites multiply by
longitudinal binary fission,
remaining in the lumen of the
proximal small bowel where they
can be free or attached to the
mucosa by a ventral sucking disk
(4)
Encystation occurs as the
parasites transit toward the colon
(5)
Ankarklev et al. 2010 Nature Review Microbiology

19. CLINICAL MANIFESTATION
Majority of individuals are
asymptomatic
Within the diarrhea spectrum:
 Persistent diarrhea with or without
malabsorption and enteropathy
(bottom right)
 Protection against acute diarrhea with
normal histopathology (bottom left)
The clinical status is related to:
 Differences in virulence between
Giardia strains
 Host genetics and immunity
 Host nutritional intake
Chronic sequelae have been
reported
Bartelt & Sartor 2015 F1000Prime Reports 2015,

20. SYMPTOMS OF INFECTION AND
TREATMENT
Onset typically begins after 6-15 days from ingestion
Clinical symptoms:
 Steatorrhea & watery diarrhea
 Nausea & epigastric pain
 Weight loss for malabsorption of vitamins A & B12, xylose, iron and zinc
 Lactase deficiency in 20-40% of symptomatic cases
Usually treated with metronidazole or other nitroimidazoles
 treatment failure and drug resistance have been reported
http://healthfixit.com/steatorrhea/

21. PATHOGENESIS
Giardiasis is multifactorial disease:
complex interplay between the host
and parasite
The parasite is non-invasive and
replicates attached to the intestinal
epithelium
Disease mechanism:
 Apoptosis of enterocytes
 Loss of epithelial-barrier function
 Hypersecretion of Cl-
 Malabsorption of glucose, water & Na+
 Diffuse microvillus shortening
 Immune reaction
 Interference with bile salt metabolism Bartelt & Sartor 2015 F1000Prime Reports 2015,

22. EPIDEMIOLOGY
G. duodenalis has a global distribution; most common cause of
protozoan diarrhea worldwide
Majority of cases are unreported and the true burden is unknown
In Nordic countries: 254 to 867 unreported cases for each reported
one
 Estimated incidence 4 670/100 000 !!!!!!
Infection rates generally low in developing countries and in
asymptomatic
Infection rate in Nordic countries: 2.9% (asymptomactic) & 5.8%
(symptomatic) [similar trend reported in The Netherland]

23. Confirmed case
 Clinical:
 Diarrhoea; Abdominal pain;
Bloating; malabsorption
(e.g. steatorrhoea, weight
loss)
 Laboratory:
 Demonstration of Giardia
lamblia cysts or
trophozoites in stool,
duodenal fluid or small-
bowel biopsy
 Demonstration of Giardia
lamblia antigen in faces

24. EPIDEMIOLOGY
Cysts are immediately infectious when excreted in feces (allowing
person-to-person transmission)
Twofold increase in the transmission of giardiasis occurred during the
summer (increased outdoor activities)
Human giardiasis – two broad settings:
 outbreaks
 endemic transmissions (sporadic infections)
Outbreaks are most frequently waterborne: drinking or recreational
water contamination (+ also demonstrated person-to-person
transmission during outbreaks)
Waterborne giardiasis outbreaks occur even when drinking water has
been treated by chlorination or in the absence of faecal indicators

25. Study Cases/cont
rol
Risks
Retrospective case-control study
in rural New England
171/684 1) Household use of shallow water sources
2) Foreign travel
3) Daycare center exposure
4) Household case contact
Matched case-control study in
the United Kingdom
232/574 1) Swallowing water while swimming
2) Recreational fresh water contact
3) Drinking treated tap water
4) Eating lettuce
Case-control study in Germany 120/240 1) Sex (male)
2) Immune status (immunocompromised)
3) Daily consumers of green salad
(!!contact with animals & exposure to surface water
were not associated!!)
Case-control study in Italy
(Catholic Jubilee)
52/72
all
residents of
Rome
1) Traveling abroad
2) Exposure to surface water
3) High educational level
Auckland, New Zealand, explored
the risk of nappy changing
183/336 Nappy changing = fourfold increased risk; infection
associated strongly with childcare center attendance
RISK FACTORSCacciò & Sprong, 2011

26. GIARDIASIS IN FINLAND: TRAVEL-
RELATED AND NON TRAVEL-
RELATED CASES
15-month period, from 1 June 2005 to 31 August 2006 based on
Giardia notifications in the FIDR
One third of Finnish citizens with Giardia infection had not travelled
abroad
The travel-related and non travel-related cases did not differ in terms
of symptoms, severity or duration of the symptoms or medication
Among non travel-related cases included in the study the major risk
factors for infection included drinking water from nature, drinking
well-water and swimming in a lake
Rimhanen-Finne R, Kuusi M. Giardiasis in Finland: A
Comparison Between Travel-Related and Non Travel-
Related Cases. EpiNorth 2010; 11

27. G. DUODENALIS GENOTYPES IN
HUMANS
G. duodenalis is a multispecies complex and major genetic groups
are now known as assemblages  A & B are infecting humans
Other assemblages are sometime detected in humans but their
identification is sometime unreliable
Genotypes may correspond to distinct species
Recently
detected
in Human
in Brasil
[Fantinatti,
JID 2016]
(Ryan & Cacciò 2013)

28. G. DUODENALIS GENOTYPES IN
HUMANS
(In general) assemblage B has a higher prevalence than assemblage A
worldwide
 58% vs 37%
However, there are geographical variations in the distribution of
assemblages  reasons unclear
Mix infection A+B are more common in developing countries
Assemblages A & B are potentially zoonotic and genetic differences
may provide relevant information for source tracking and outbreak
investigation
Assemblage-specific reservoirs and transmission routes may exist 
the knowledge of which is still rather limited

29. G. DUODENALIS ASSEMBLAGE
SPECIFIC RISK FACTORS
Study Assemblage A Assemblage B
Malaysia, 2011
[Anuar 2014 BMC Infect Dis]
Keeping pets in the household Presence of children in the
household and other family
members with Giardia infection
England, 2012
[Minetti 2015 J Clin Microbiol]
Dog ownership Contact with young children
and diarrhoeic people
The Netherland, 2010 -
2013 [Pijnacker 2016 Eur J
Clin Microbiol Infect Dis]
Contamination of indoor
sandpits by cats in Day Care
Centers (DCC)
Associated with factors related
to hygiene practices within the
DCC
Zoonotic transmission Anthroponotic transmission

30. ZOONOTIC ORIGIN OF
ASSEMBLAGES A
Cacciò & Sprong, 2011

31. FACTOR ASSOCIATED TO
WATERBORNE TRANSMISSION OF
GIARDIA
1. Environmental contamination (ex from sewage treatment plants):
 Large numbers of cysts are excreted into the environment + prolonged excretion period of several
days or even weeks
 2 × 106 cysts/gram of human faeces
2. Infectious dose is relatively low
 ~10–100 cysts
3. Robustness of the cyst transmission stage (related to the filamentous cyst
wall)
 Cysts are remarkably stable and can survive for weeks to months (in absence of freeze-thaw cycles;
if they are not desiccated)
 In low temperatures cysts may remain viable or infective for at least one month
 Resistant to environmental pressures and to many of the disinfectant regimes commonly used in the
water industry
4. Zoonotic nature of this parasite
 Low host specificity of those genotypes that are infectious to humans
 Onward contamination by transport hosts (Aquatic birds; Filter-feeding molluscs)

32. ENVIRONMENTAL TRANSMISSION
OF GIARDIAS
Robertson & Lim 2011
Similar to
Cryptosporidium
Assemblage B (?)
Assemblage A (?)

33. OCCURRENCE OF GIARDIA IN
DRINKING WATER
Giardia cysts occur in potable waters  concentrations of cysts are
very low <1 cysts/L
In surface water: data from North America, South America, Europe
and Asia
 Above 30% (few reaching almost 100% of sampled water
 Concentrations  <0.01 to <100 cysts/L (higher in developing countries – Asia)
The presence of cysts in groundwater is alarming as it is considered
to be at a relatively low risk of being contaminated
 Bulgaria: 18 well-water 22% positive (0.016 to 127.5 cysts/L)  run-off of faecal
contaminated surface water
 Finland: 4/20 well-water positive  wastewater treatment activities in the
neighborhood

34. WATER MANAGEMENT Detection of parasites and
water treatment

35. DETECTION IN WATER SAMPLES
Cryptosporidium and Giardia are reference pathogen for the assessment
of drinking water safety
1) Concentration
 10-1000 L of water (filter membrane ≤ 2 µm)
2) Isolation of oocyst or cyst by immunomagnetic separation (IMS)
3) Detection and enumeration by fluorescence microscopy (IFAT)
 based on cell wall antigens of Cryptosporidium oocyst or Giardia cysts
Three standard methods
 ISO 15553:2006(E) – Water quality: Isolation and identification of Cryptosporidium
oocysts and Giardia cysts from water
 US EPA 1623.1 – Cryptosporidium and Giardia in water filtration/IMS/FA
 UK Env. Agency: Drinking Water (2010); Part 14 – Methods for the isolation, identification
and enumeration of Cryptosporidium oocysts and Giardia cysts
Very expensive ≈ 500€ sample
EnvirocheckTM
IDEXX Filta-Max®

36. TRETMENT OF WATER
Cryptosporidium oocysts and Giardia cysts are
particularly resistant to removal and
inactivation by conventional water treatment
(Crypto > Giardia)
 coagulation, sedimentation, filtration and chlorine
disinfection
(oo)cysts are very resistant to environmental
pressures and to many of the disinfectant
regimes commonly used in the water industry
The multiple-barrier approach is generally
accepted as the guiding principle for
providing safe drinking water
Low (oo)cysts concentrations are problematic
The process may prove inadequate in the face
of poor source water quality or deficient

37. FILTRATION
Cryptosporidium oocysts are much smaller than Giardia cysts, which
could affect their ability to be filtered
 Giardia cysts were more readily removed than Cryptosporidium oocysts during
filtration
Conventional Filtration:
 Coagulation, flocculation, sedimentation, and filtration (3 log or 99.9% removal of
Cryptosporidium)
Largely influenced by the effectiveness of coagulation pretreatment
Physical removal is ultimately achieved by properly functioning
conventional filters
 a bed of sand (sand filtration)
 a layer of diatomaceous earth (diatomaceous earth filtration)
 a combination of coarse anthracite coal overlying finer sand (dual- and tri-media
filtration)

38. CHEMICAL DISINFECTION
(oo)cysts are much more resistant than bacteria and viruses to
chlorine based disinfectants
 Concentrations well above those generally employed in routine water treatment
practices
 (Giardia) Resistant to chlorination at low temperatures and high pH
Chloramines as an alternative to chlorine  reduced excystation of
Giardia cysts at lower concentration and relative shorter contact
Chlorine dioxide:
 limited ability to inactivate Giardia cysts
 the efficacy of chlorine dioxide for the disinfection of Cryptosporidium is strongly
dependent on temperature.

39. OZONE TREATMENT
Exposure of cysts to ozone causes extensive protein degradation and
profound structural modifications to the (oo)cyst wall
Efficacy apparently reduces as (oo)cyst concentration decreases
Environmental factor most affecting the efficacy of a given
concentration × time (CT) is temperature
 reaction rates—and so inactivation kinetics—decreasing steadily with temperature
The requirement for higher activation energy for protozoa compared
with bacteria  result in the formation of disinfection by-products
 bromate is considered to be of most concern due to its potentially carcinogenic
effects
 Treatment options include addition of ammonia, pH reduction, scavenging of
hydroxyl radicals and scavenging or reduction of HOBr (hypobromous acid)

40. UV TREATMENT
(oo)cysts are susceptible to UV
disinfection at doses that are
applicable to water
improved protozoa reduction
while minimizing the disinfection
by-products resulting from the
use of chlorine and ozone
Cryptosporidium oocysts do not
have the capacity for repair
following UV irradiation
 DNA repair after UV radiation was
observed in Giardia

41. PHOTOCATALYTIC DISINFECTION
Environmentally friendly
technology for water disinfection
Low concentrations can be
efficiently disinfected in
continuous flow by using a
commercial fibrous ceramic TiO2
photocatalyst
Efficiency was enhanced by
addition of a small concentration
of chlorine
Lee & Park 2013
Navalon et al., 2009