Terminology for Blastocystis subtypes – a consensus C. Rune Stensvold 1 , G. Kumar Suresh 2 , Kevin S.W. Tan 3 , R.C. Andrew Thompson 4 , Rebecca J. Traub 5 , Eric Viscogliosi 6 , Hisao Yoshikawa 7 and C. Graham Clark 8 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 Women’s 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 Blasto- cystis terminology to improve communication and corre- late research results. Based primarily on published small- subunit 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 [1–3]. Blastocystis is genetically diverse [4–7] and several molecular methodologies and tools have been developed to detect and classify the genetic heterogeneity of this organ- ism (Table 1). There is agreement that at least seven major clades of isolates exist in mammals and birds [5,7]; how- ever, 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 strame- nopile protist [8]. This taxonomic placement was 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 [4,10–19] or dideoxyse- quencing [6–8,10,18,20–26] 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]. Subtype- specific sequence-tagged-site (STS) primers have been developed [30] from products of the initial AP-PCR ampli- fication studies [28] and used in studies by Abe et al. [15–17], 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. PCR–RFLP analysis of SSU-rDNA (riboprinting) is a popular, fast and inexpensive tool [34]; however, compari- sons 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 PCR–RFLP 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 SSU- rDNA with ribodemes obtained by Clark [4] using PCR– RFLP. Moreover, sequences for three of the isolates used in Opinion TRENDS in Parasitology Vol.23 No.3 Corresponding author: Clark, C.G. (graham.clark@lshtm.ac.uk). Available online 22 January 2007. www.sciencedirect.com 1471-4922/$ – see front matter ß 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.pt.2007.01.004