Phylogenetic Analysis and Pathogenic Potential of Blastocystis sp ...

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Phylogenetic Analysis and Pathogenic Potential of Blastocystis sp ...

Tamalee Roberts 1,2 , J. HARKNESS 1 , J. ELLIS 2 , D. STARK 1

1 SydPath, St. Vincents Hospital, Sydney, NSW, Australia

2 University of Technology, Sydney, NSW, Australia


Blastocystis is an enteric parasite that has been

isolated from humans and animals

Originally described as a yeast by Brumpt in

1912

1967 Zeirdt concluded it was not a yeast but actually

a protozoa due to morphological and physiological

aspects

Recently placed within the Stramenopiles group


Blastocystis has a world-wide distribution

Higher rates of infection found in developing

countries

Transmission-

Faecal-oral

Water-borne

Food-borne

Zoonotic


Several techniques used for the diagnosis of

Blastocystis-

oMicroscopy

oCulture

oImmunofluorescent stains

oMolecular techniques


Blastocystis can be difficult to diagnose through

microscopy

Can be confused with Dientamoeba fragilis and

yeast

Iodine wet prep Trichrome stain Iron Haematoxylin stain

http://www.dpd.cdc.gov/dpdx/HTML/ImageLibrary/Blastocystis_il.htm


Not done routinely in diagnostic laboratories. Mainly

used for epidemiological studies

Direct PCR analysis from faecal material and culture

systems

Several extraction techniques available but most are

time consuming and labour intensive

12 different subtypes have been identified from

humans and animals in the past 10 years but there are

continuously being more identified


Blastocystis has been identified from both developing

and developed countries around the world

Higher infection rates have been identified in

developing countries. Due to diagnostic technique

used?

Subtype 3 is the predominant subtype isolated around

the world


Still much debate about the pathogenicity of

Blastocystis.

Suggested to be a pathogen, commensal or an

opportunistic organism

Symptoms include diarrhoea, abdominal cramps and

nausea

Many cases shown where Blastocystis is the only

pathogen present to cause the disease

Been suggested that the presence of >5 cells per 40x

magnification should be considered as pathogenic


The aim of this project was to study

the molecular epidemiology of

Blastocystis sp. in the Sydney

population and comment on the

pathogenic potential of this

parasite


Compared 5 different diagnostic techniques on 510

samples

98 (19%) samples were positive for Blastocystis

Diagnostic

Method

No.

Positives for

Blastocystis

Permanent

stain

MBD

culture

TYGM-9

cutlure

PCR 1 PCR 2

47 81 80 65 92


Parasites No.

Blastocystis sp 35

Blastocystis + Endolimax nana 4

Blastocystis + Giardia intestinalis 2

Blastocystis + Dientamoeba fragilis 1

Blastocystis + Entomoeba histolytica/dispar/moshkovskii

complex

Blastocystis + Enteromonas hominis +Chilomastix mesnili 1

Blastocystis + Iodamoeba butschlii + E. nana + E.histo

complex

Blastocystis + E.nana + Entamoeba coli + Entamoeba

hartmanii

Blastocystis+ D.fragilis + E. coli + I. butschlii + E. nana

Total 47

1

1

1

1


PCR 1 PCR 2

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 M 1 2 3 4 5 6 7 8 9 10 11 12 13 14

M= marker

1= positive control

2= negative control

3- 14= samples


91 readable sequences from the 98 PCR positive samples

Subtype 1 2 3 4 6 8

Number of samples 28 6 41 12 3 1

% 31% 7% 45% 13% 3% 1%


Number of patients

40

35

30

25

20

15

10

5

0

Blastocystis positive patient symptoms

Diarrhoea Abdominal pain Vomiting Nausea

Symptoms

Table of Blastocystis positive patients symptoms

5 patients also noted having traveled overseas


68 patients where Blastocystis was the only organism

isolated. Shows the potential of Blastocystis as a

pathogen. Treatment should be considered when only

Blastocystis is isolated in conjunction with symptoms

Subtype 2

All patients had symptoms including diarrhoea and

abdominal pain

2 patients had another pathogen isolated- 1 with

Cryptosporidium and 1 with Giardia intestinalis

The 3 other samples had no other pathogen found

May suggest that subtype 2 is uniformly pathogenic


Subtype 4

98% of patients noted symptoms of diarrhoea,

abdominal pains and nausea

No patients with subtype 4 had any other pathogen

detected

Suggests this subtype may play a role in causing

gastrointestinal disease in humans


Subtype 3

the most abundant subtype found in humans worldwide

50% of subtype 3 noted some form of symptom

Subtype 1

Been suggested that subtype 1 is a pathogenic

strain.

Only 33% of patients in this study noted

gastrointestinal symptoms

Shows that subtype 1 can not be considered a

pathogenic strain from this study alone


This study highlights the low sensitivity of

microscopy for the diagnosis of Blastocystis

Shows the need for further molecular techniques

when studying the epidemiology and pathogenicity

of Blastocystis

This is the first epidemiological study done in

Australia and shows there is a 19% prevalence of

Blastocystis in people from Sydney

Subtype 3 is the most common subtype isolated

from the Sydney population


This research highlights the potential pathogenicity

of Blastocystis in humans and shows that it could be

subtype related

Shows through the different subtypes isolated that

there is potential for zoonotic transmission

Suggests that subtype 3 occurs through humanhuman

transmission


Thank you

Questions??

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