Opinion

TRENDS in Parasitology

Vol.23 No.3

Terminology for Blastocystis

subtypes a consensus
C. Rune Stensvold1, G. Kumar Suresh2, Kevin S.W. Tan3, R.C. Andrew Thompson4,
Rebecca J. Traub5, Eric Viscogliosi6, Hisao Yoshikawa7 and C. Graham Clark8
1

Laboratory of Parasitology, Department of Bacteriology, Mycology and Parasitology, Statens Serum Institut, Artillerivej 5,
DK-2300 Copenhagen S, Denmark
2
Department of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia
3
Laboratory of Molecular and Cellular Parasitology, Department of Microbiology, National University of Singapore, 5 Science Drive
2, Singapore 117597
4
WHO Collaborating Centre for the Molecular Epidemiology of Parasitic Infections and the State Agricultural Biotechnology Centre,
School of Veterinary and Biomedical Sciences, Murdoch University, South Street, WA 6150, Australia
5
School of Veterinary Science, University of Queensland, St Lucia, QLD 4072, Australia
6
Institut Pasteur, Inserm U547, 1 Rue du Professeur Calmette, BP 245, 59019 Lille cedex, France
7
Department of Biological Sciences, Faculty of Science, Nara Womens University, Kitauoya-Nishimachi, Nara 630-8506, Japan
8
Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK

Blastocystis is a ubiquitous enteric protistan parasite that

has extensive genetic diversity and infects humans and
many other animals. Distinct molecular methodologies
developed to detect variation and obtain information
about transmission patterns and clinical importance have
resulted in a confusing array of terminologies for the
identification and designation of Blastocystis subtypes.
In this article, we propose a standardization of Blastocystis terminology to improve communication and correlate research results. Based primarily on published smallsubunit ribosomal RNA gene analyses, we propose that
all mammalian and avian isolates should be designated
Blastocystis sp. and assigned to one of nine subtypes.
Multiple Blastocystis subtype terminologies
Blastocystis is a common protistan intestinal parasite that
is found in a wide range of animals, including humans
[13]. Blastocystis is genetically diverse [47] and several
molecular methodologies and tools have been developed to
detect and classify the genetic heterogeneity of this organism (Table 1). There is agreement that at least seven major
clades of isolates exist in mammals and birds [5,7]; however, varying terminologies have been used in the past to
designate the subsets of Blastocystis isolates detected in a
particular study, which makes corroboration, comparison
or criticism of published studies difficult.
The aim of this article is to: (i) summarize the molecular
methodologies that are applied to Blastocystis subtyping;
(ii) provide a key for comparing and correlating previous
studies, as far as is possible; and (iii) propose a standard
terminology for the designation of Blastocystis subtypes.
Molecular methodologies used in Blastocystis research
Small-subunit ribosomal RNA gene (SSU-rDNA) analyses
were useful for establishing that Blastocystis is a stramenopile protist [8]. This taxonomic placement was
Corresponding author: Clark, C.G. (graham.clark@lshtm.ac.uk).
Available online 22 January 2007.
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subsequently confirmed by analysis of other genes [9].

PCR amplification of partial or complete SSU-rDNA has
been combined with either restriction fragment length
polymorphism (RFLP) analysis [4,1019] or dideoxysequencing [68,10,18,2026] to detect genetic diversity,
which was also found in analysis of other genes [9,27].
Comparative analysis of Blastocystis isolates using
elongation factor-1a and SSU-rDNA sequences showed
that phylogenetic trees based on the two genes were
entirely congruent [7]. Arbitrarily primed PCR (AP-PCR)
has also been used to detect variation [28,29]. Subtypespecific sequence-tagged-site (STS) primers have been
developed [30] from products of the initial AP-PCR amplification studies [28] and used in studies by Abe et al. [1517],
Yan et al. [31] and Yoshikawa et al. [12,13,26,30,32,33].
Correlation of data from various studies
The Blastocystis subtype, (sub)group, clade, cluster or
ribodeme designations used in various studies are shown
in Table 2. Some of these correlations have been reported
previously but not in a comprehensive manner.
PCRRFLP analysis of SSU-rDNA (riboprinting) is a
popular, fast and inexpensive tool [34]; however, comparisons between studies are difficult when different numbers
and types of restriction endonuclease are used or when the
PCR products are amplified using different primer pairs.
The assignment of isolates to ribodemes is problematic
when the RFLP banding patterns are generated using only
two or three enzymes because the same patterns can result
from cutting distinct sequences [20,26]. Likewise, when
distinct enzymes are used [11,35], no cross-comparison is
possible unless the isolate representation overlaps between
studies. Comparisons can be made, however, when both
PCRRFLP and dideoxysequencing of the same isolates
are performed. Arisue et al. [20] used in silico RFLP analysis
to correlate results obtained by sequence analysis of SSUrDNA with ribodemes obtained by Clark [4] using PCR
RFLP. Moreover, sequences for three of the isolates used in

1471-4922/$ see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.pt.2007.01.004

Opinion

94

TRENDS in Parasitology Vol.23 No.3

Table 1. Methods used for the detection of subtypes and classification of Blastocystis sp.
Method
PCRRFLP

Gene target
SSU-rDNA (riboprinting)
Elongation factor-1a
(elfaprinting)
SSU-rDNA

Principle
Digestion of PCR products with restriction
endonucleases to yield variable gel banding patterns

Refs
[4,1019]
[27]

PCR amplification followed by dideoxysequencing of

PCR product, with or without cloning

PCRSTS

Unknown

PCR fingerprinting
and AP-PCR
Multilocus sequence
typing

Unknown

PCR amplification with a set of subtype-specific

primers to obtain a specific amplification profile
PCR amplification with arbitrary primers to obtain
specific DNA profiles
Examination of multiple genes by PCR and
dideoxysequencing

[6
8,10,18,20
26]
[12,13,15
17,26,3033]
[28,29]

PCR and
dideoxysequencing

Elongation factor-1a,
cytosolic-type 70-kDa heat
shock protein, SSU-rDNA,
non-catalytic B subunit of
vacuolar ATPase

the riboprinting study are available, so it is possible to

correlate those three ribodemes with the sequence-based
clades [7]. Links between PCRSTS and PCRRFLP results
were made by Yoshikawa et al. [12] and, subsequently, some
of these SSU-rDNAs were sequenced, again enabling crosscorrelation of the classifications. In many cases, however,
such links cannot be made easily.
In contrast to partial RFLP profiling, partial sequencing
enables the unambiguous cross-correlation of subtypes.
Bohm-Gloning et al. [10] produced short SSU-rDNA
sequences from fragments representing the different subgroups they found by PCRRFLP analysis. Likewise, 310bp SSU-rDNA sequences were produced by Stensvold et al.
[25] and 600-bp barcode sequences were reported by
Scicluna et al. [24]. In all three cases, the sequences
enabled the clear and unambiguous assignment of isolates
to the groups recovered from analyses of complete SSUrDNA sequences by Noel et al. [7].
Standardization of terminology
Transmission of Blastocystis is believed to occur by
animal-to-animal, human-to-human, animal-to-human

[9]

and, possibly, human-to-animal routes [7]. Among

the reported SSU-rDNA groups, no exclusively human
clade seems to exist because sequences from human isolates are present in all clades [7,23,24]. Blastocystis isolates from humans have, until now, been designated
Blastocystis hominis, whereas Blastocystis isolates from
other animals have usually been named Blastocystis sp.,
although a small number of specific names have been
published for isolates from certain hosts. The extensive
genetic diversity of this organism, even among isolates
from one host, makes the host-specific naming of species
misleading. In bacteria and certain eukaryotic groups such
as Naegleria [36], the degree of genetic divergence between
the major clades seen in Blastocystis would be considered
sufficient on its own to justify separate species names for
each. However, we believe that a more appropriate terminology is one in which all mammalian and avian isolates
are designated Blastocystis sp. and assigned to a subtype
from 1 to 9 (Table 2, Figure 1). The reason for using
Blastocystis sp., rather than Blastocystis hominis, is that
some reptilian and amphibian species seem to fall within
the range of variation covered by the mammalian and

Table 2. Correlation of Blastocystis subtype designations and suggestion for consensus terminology
Clade a

Subtype b

I
II

III
IV

V
VI

VII

I
II
X
III
IV
IVa
V
VI
VIa
VII
VII

Group and
subtype c
I/1
II/5
I+II/1+5 outlier
III/3
IV/7
IV/7 outliers
V/6
VI/4
VI/4 outliers
VII/2
VII/2 outliers

Subtype d

Ribodemee,f

Subgroup g

Cluster h

Subtype i

Consensus

1
5

3
7

6
4

1, 8 j
6

2, 7, 4?l, 5?
3

9j

10

III
V

I, II
IV

VI m

E
C, D

A
B

1, 1 variant
k

Blastocystis sp. subtype

Blastocystis sp. subtype
Chimaeric sequence
Blastocystis sp. subtype
Blastocystis sp. subtype
Blastocystis sp. subtype
Blastocystis sp. subtype
Blastocystis sp. subtype
Blastocystis sp. subtype
Blastocystis sp. subtype
Blastocystis sp. subtype

Clades described by Arisue et al. [20] and Yoshikawa et al. [26].

Subtypes described by Scicluna et al. [24].
c
Groups and subtypes described by Noel et al. [7].
d
Subtypes described by Yoshikawa et al. [12,30].
e
Ribodemes are groups that share the same SSU-rDNA PCRRFLP patterns and are described by Clark [4] and Yoshikawa et al. [12].
f
Ribodemes in bold are those originally described by Clark [26].
g
Subgroups described by Bohm-Gloning et al. [10] on the basis of PCRRFLP analysis and partial SSU-rDNA sequences.
h
Clusters described by Stensvold et al. [25] on the basis of PCR and sequencing analysis of partial SSU-rDNA sequences.
i
Subtypes described by Yoshikawa et al. [12] using PCRSTS.
j
Ribodemes 8 and 9 described by Yoshikawa et al. [12] differ from those described by Kaneda et al. [14].
k
symbols indicate no equivalent described.
l
Question mark indicates that the subtype equivalence is probable but not proven.
m
Subgroup VI described by Thathaisong et al. [18] equals ribodeme 10 described by Yoshikawa et al. [12] (K.S.W. Tan, unpublished).
b

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Opinion

TRENDS in Parasitology

Vol.23 No.3

95

disease, particularly if not all subtypes have the same

effect on the host. Only a few epidemiological surveys of
Blastocystis in humans have been undertaken using molecular tools, and those that have been published are difficult to compare because of their different methodologies
and terminologies. This standardization of the Blastocystis
nomenclature, coupled with recent methods to simplify the
assignment of isolates to SSU-rDNA subtypes [24,42],
should make the future investigation of the epidemiology
and clinical importance of Blastocystis much easier.
References

Figure 1. Relationships among Blastocystis sp. subtypes. The phylogenetic

analysis of Noel et al. [7] has been redrawn to show the strongly supported
clades with their new subtype designations proposed here. The Bayesian posterior
probabilities are shown. Proteromonas lacertae was used as the outgroup (not
shown).

avian clades [5,7]. If the reptilian or amphibian isolates are

distinct species, the nine clades cannot all be Blastocystis
hominis. This would make it a paraphyletic species
because not all descendents of the common ancestor would
be included. Until the species nomenclature is resolved, it
is proposed that the designation Blastocystis sp. be used.
Noel et al. [7] identified several discrete lineages consisting of one or two isolates that were of uncertain subtype
classification. One of these outliers, AF538348, seems to
be a chimaera [24]. However, two others (designated IVa
and VIa by Scicluna et al. [24]) are elevated here to full
subtype status. The final Noel et al. [7] outliers are on a
long branch within clade VII/2 rather than emerging at the
base. There is 12% SSU-rDNA sequence variation within
most subtypes. Nevertheless, we believe that there is no
need, at present, for the subtypes to be subdivided further.
Concluding remarks
Blastocystis is probably the most prevalent unicellular
parasite in human faecal specimens [2,37]. There are conflicting views as to whether it causes disease in humans,
although it has recently been linked to irritable bowel
syndrome [3841]. The genetic diversity of this organism
might contribute to the uncertainty regarding its role in
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