12/06/2012
Background
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
Taxonomy
The first description of the parasite was made by
Jepps and Dobell in 1918; they initially considered it
as a non-pathogenic commensal
The name Dientamoeba fragilis refers to the fact
that it is an enteric ameba with the curious
characteristic of being binucleate and that it tends
to degenerate rapidly after excretion in stool
Phylogenetic position of Dientamoeba fragilis
It has taken a long time to prove that Dientamoeba
fragilis is a flagellate that has lost its flagella and not
an ameba
The parasite is classified within the Parabasalia,
and its closest relatives are the Tritrichomonadea
(e.g., species of Monocercomonas and
Tritrichomonas)
18 S rRNA based phylogeny of Parabasalia (Malik et al. PLoS ONE 2011 6: e20774)
1
12/06/2012
They are nice!
What are Parabasalia?
A monophyletic but complex assemblage of flagellated
protists with a characteristic parabasal apparatus (Golgi
complex associated with striated fibers) and anaerobic
energy-generating organelles called hydrogenosomes.
Most parabasalids inhabit the digestive tract of animal
hosts as commensals, parasites, or symbionts.
Several are of considerable medical and veterinary
importance, like Trichomonas vaginalis and
Tritrichomonas foetus
Structure of Dientamoeba
Fine structure of the trophozoite
Digestive vacuole
Trophozoites showing phagocytosis and
engulfing rice starch (Rs) or bacteria (b)
Engulfed bacterium
Engulfed rice starch
Trophozoites dividing by binary fission.
Only mononucleated cells divide.
Hydrogenosome
Pseudopodia
Trophozoites appear round after 48-72 h,
when nutrients became less abundant
Smooth and ruffled populations of
trophozoites observed.
Banik et al, IJP 2012, 42:139–153
Dientamoeba shows some of the typical structures seen in other parabalids
Banik et al, IJP 2012, 42:139–153
2
12/06/2012
A rather mysterious organism
Life cycle / transmission routes
Life cycle: unknown
Transmission mode: unknown
Natural host range: poorly invesigated
Diagnosis: difficult
Pathogenicity: controversial
Animal model: unavailable
Axenic culture: unavailable
Epidemiology
Worldwide distribution of the infection.
High prevalence in developing and industrialised countries
INDIRECT TRANSMISSION
The role of Enterobius as a
mechanical vector has been proposed
but it is no longer considered as a
valid hypothesis
DIRECT TRANSMISSION
The high frequency of co-infection with
other enteric protozoa transmitted through
the fecal-oral route suggests that direct
transmission could occur.
Host range
Non-human primates (Stark et al., 2008)
Between 6 and 30% of people suspected of suffering from
intestinal parasitosis will harbor Dientamoeba.
Pigs (Crotti et al., 2007)
The limited host range detected suggests human infection
may not involve transmission from other animal species.
From Barrat et al., Gut Microbes 2011, 2:3-12
3
12/06/2012
Diagnostic methods
Microscopy
Pleiomorphic trophozoite, 5 to 15 µm in diameter,
one to four nuclei, with fragmented chromatin
and pale grey-blue finely vacuolated cytoplasm.
Diagnostic methods
Molecular methods
In the last few years, several methods based on PCR
have been developed.
PCR-RFLP PCR and sequencing Real-time PCR
The infection is difficult to diagnose, because of
the “fragile” nature and intermittent shedding of
the trophozoites
Both for diagnostics and for
identification of genotypes
Mostly for diagnostics
The most commonly employed fixative is SAF (sodium acetate-acetic acid-formalin),
whereas staining with iron-haematoxylin or Giemsa is routinely used
Two genotypes can be distinguished. Genotype 1 is by far the
most commonly found in humans
Pathogenicity
Controversial
A rather mysterious organism
Life cycle: unknown
We lack an animal model to understand this aspect
Transmission mode: unknown
Natural host range: unknown
Two lines of evidence support the concept that D.
fragilis is a true pathgen:
Pathogenicity: controversial
Most patients have diarrhea and abdominal pain
Treatment of patients resolves clinical symptoms
Animal model: unavailable
Axenic culture: unavailable
4
12/06/2012
Results of microscopy
Our study
152 fecal samples from pigs raised in 9 indoor farms of
central Italy, 7 in the Umbria region and 2 in the Marche
region.
Samples from piglets (n = 74, on 8 farms), fattening pigs
(n = 14, on 2 farms) and sows (n = 64, on 7 farms).
Samples from 21 pig farmers were collected from 5 of
the 9 farms,
Farm
Herd type
Piglet
positive /
tested
10 / 10
Fattening pig
positive /
tested
-/-
Sow
positive /
tested
1 / 10
Human
positive / tested
1
2
Weaner
production
Farrow-to-finish
9 / 10
-/-
3 / 10
0/4
3
4
Farrow-to-finish
10 / 10
7 / 10
0 / 10
2/8
Farrow-to-finish
1 / 10
-/-
0 / 10
-/-
5
Farrow-to-finish
4 / 10
-/-
0 / 10
0/2
6
Farrow-to-finish
4 / 10
-/-
1 / 10
-/-
7
Fattening
-/-
-/-
3/4
-/-
8
Fattening
10 / 10
-/-
-/-
2/3
9
Farrow-to-finish
Total
-/-
4/4
4/4
-/-
0/4
52 / 74
11 / 14
8 / 64
4 / 21
Blastocystis spp. (42%), Endolimax nana (32%) and
Iodoamoeba buetschli (25%) were also identified in pigs
Molecular typing
Diagnostic real-time PCR
Only ribosomal sequences are known
Of the 17 human fecal samples, 13 were positive, with cycle
threshold (Ct) values ranging from 29 to 40.
Diagnostic real-time PCR
18 S rRNA
ITS 1
5.8 S
ITS 2
All 24 microscopically positive pig samples were amplified,
28 S
with Ct values ranging from 30 to 34, whereas the 14
microscopically negative pig samples were all negative by
qPCR.
Conventional PCR
Single and nested PCR for the amplification of various fragments of:
18S rRNA
ITS 1 + 5.8 S
ITS 2 + 5.8 S
followed by sequencing of PCR products
5
12/06/2012
Results at the 5.8S rRNA gene
The expected size (98 bp) was confirmed by capillary gel
electrophoresis
Sequence analysis revealed genotype 1 in both pigs (n=11)
and humans (n=4) isolates
Results at the 18S rRNA gene
Fixed differences between genotypes 1 and 2 were
confirmed by sequencing 18S rRNA from 4 human isolates
Sequence analysis of nested PCR (366 bp) revealed
genotype 1 in all samples, and a very limited amount of
polymorphism among isolates from both hosts
In 3 pig samples microscopically negative for Dientamoeba,
nested PCR and sequencing identified another flagellate,
possibly Trichomitus rotunda. Therefore, this PCR assay
needs to be optimized in terms of specificity
Sequence comparison by
BLAST identified genotype 1
in all samples
Results at the ITS locus
Windsor et al (2006) showed extensive variation between
copies of the Internal Transcribed Sequence (ITS) within
the same strain. Complex and aberrant chromatograms are
observed after direct sequencing of PCR products.
Results at the ITS 1 locus
To circumvent this problem, it has been proposed to
visualise only the Cytosine residues of the ITS1 sequence,
and to compare the so-called C-profiles from different
strains
Overlapping sequences
Variable number of T residues due to the presence of several alleles
6
12/06/2012
Results at the ITS1 locus
However, we have been unable to generate reliably
specific amplification products of the ITS 1 locus
from pig samples, due to interference of DNA from
other flagellates present in the feces
Nevertheless, the two ITS1 sequences from pigs that
could be analyzed were identical to ITS1 sequences
obtained from human cases in the United Kingdom
and in the Netherlands.
Unfortunately, a direct comparison of parasites from
pigs and humans from a single Italian farm was not
possible.
Results at the ITS2 locus
Results at the ITS2 locus
On the contrary, specific amplification of the ITS2 locus was
obtained from all human and pig samples tested (n=26). Like
for ITS1, the ITS2 sequences from PCR products cannot be
compared directly.
We generated “G profiles” in the same way as the “C
profiles”, and compared profiles of D. fragilis from humans
and pigs
P44
c
P56
c
P26
c
Conclusions
In this analysis, most isolates appear to have unique G
profiles.
We have shown that pigs are a natural host of
Dientamoeba fragilis
However, comparative genotyping using ITS1 and ITS2
sequences does not appear, in our opinion, to be a
robust method. C or G profiles, indeed, are nut fully
reproducible.
We confirmed that piglets are more susceptible to
infection
Moreover, it is unclear on what basis two isolates can be
considered “genetically identical”. There are both
qualitatitive (number of alleles) and quantitative
(frequency of alleles) aspects
Molecular analyses of 5.8S and 18S rRNA
sequences demonstrated that pigs harbor
genotype 1, as humans
Comparative analyses of ITS1 and ITS2 sequences
are difficult
7
12/06/2012
Perspectives
THANK YOU FOR YOUR ATTENTION!
The possibility of zoonotic transmission raised by
our results deserves future investigations
There is a need to develop molecular markers
suitable for comparative studies
QUESTIONS?
The availability of an animal model will be very
useful to study various aspects of the biology of
this elusive organism
7th Workshop of National Reference Laboratories
for Parasites, Rome 28-29 May, 2012
8