Short communication Determination of internal transcribed spacer regions (ITS) in Trichomonas vaginalis isolates and differentiation among Trichomonas species Alexandra Ibáñez-Escribano a,b, ⁎, Juan José Nogal-Ruiz a,b , Vicente J. Arán a,c , José Antonio Escario a,b , Alicia Gómez-Barrio a,b , J.F. Alderete d a CEI Campus Moncloa, UCM-UPM and CSIC, Madrid, Spain b Departamento de Parasitología, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza Ramón y Cajal s/n, 28040 Madrid, Spain c Instituto de Química Médica (IQM), Spanish National Research Council (CSIC), C/Juan de la Cierva no 3, 28006 Madrid, Spain d School of Molecular Bioscience, College of Veterinary Medicine, Washington State University, Pullman WA, USA abstract article info Article history: Received 29 April 2013 Received in revised form 22 December 2013 Accepted 23 December 2013 Available online 9 January 2014 Keywords: Trichomonas vaginalis rDNA Internal transcribed spacers (ITS) Protozoa Phylogenetic study Clades The nucleotide sequence of the 5.8S rRNA gene and the flanked internal transcribed spacer (ITS) regions of six Trichomonas vaginalis isolates with different metronidazole sensitivity and geographic origin were genotyped. A multiple sequence alignment was performed with different sequences of other isolates available at the GenBank/EMBL/DDBJ databases, which revealed 5 different sequence patterns. Although a stable mutation in po- sition 66 of the ITS1 (C66T) was observed in 26% (9/34) of the T. vaginalis sequences analyzed, there was 99.7% ITS nucleotide sequence identity among isolates for this sequence. The nucleotide sequence variation among other species of the genus Trichomonas ranged from 3.4% to 9.1%. Surprisingly, the % identity between T. vaginalis and Pentatrichomonas hominis was ~83%. There was N 40% divergence in the ITS sequence between T. vaginalis and Tritrichomonas spp., including Tritrichomonas augusta, Tritrichomonas muris, and Tritrichomonas nonconforma and with Tetratrichomonas prowazeki. Dendrograms grouped the trichomonadid sequences in robust clades ac- cording to their genera. The absence of nucleotide divergence in the hypervariable ITS regions between T. vaginalis isolates suggests the early divergence of the parasite. Importantly, these data show this ITS1-5.8S rRNA-ITS2 region suitable for inter-species differentiation. © 2014 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Trichomonas vaginalis is the causative agent of trichomonosis, the number one, nonviral sexually transmitted infection (STI) worldwide with an annual incidence of more than 170 million cases [1]. Infection by this protist produces a wide range of adverse clinical outcomes, such as cervical [2] and prostate cancer [3], and infertility or atypical pelvic inflammatory disease [4]. Additionally, trichomonosis also in- creases the predisposition to HIV [5,6]. In pregnant women, this STI is significantly associated with premature labor, low-birth-weight infants and premature rupture of the placental membranes [7]. The T. vaginalis isolate G3 has a genome of ~160 Mb with 65% of re- peated and transposable elements as well as different proteins with do- mains homologous with those of bacteria, viruses, and protozoa [8]. The ribosomal genes, in particular the DNA region encoding for the 18S ribo- somal RNA gene (18S rRNA), are considered one of the main genetic markers used for phylogenetic analysis due to its slow evolution and conserved nature. The rRNA genes are essential for protein synthesis and ribosome generation and are more conserved within species than the non-transcribed spacers [9]. Internal transcribed spacers (ITS) are more variable and, therefore, may have utility for species differentiation [10,11] or even for isolate differentiation among some parasites [12,13] as ITS regions are removed via splicing during transcript processing [9]. ITS1 separates 18S rRNA from 5.8S rRNA, while ITS2 separates 5.8S rRNA from 28S rRNA. Many reports have been focused on improving the sen- sitivity of diagnostic methods targeting the 18S rRNA gene of T. vaginalis [14]. Some reports have analyzed the nucleotide sequences of the ITS1- 5.8S rRNA-ITS2 to characterize phylogenetically the parabasalids [14–16]. Nonetheless, few studies have been conducted to determine the utility of the ITS region as a molecular tool for T. vaginalis inter- and intra-species differentiation. There is a paucity of reports studying the ITS1 region to search the intragenomic variation of parabasalid pro- tozoa like T. vaginalis, as evidenced by a report on Dientamoeba fragilis [17]. As there have been few studies overall on the T. vaginalis ITS loci, we felt it to be important to determine whether the ITS1-5.8S rRNA-ITS2 genomic region is a useful tool for differentiation among T. vaginalis isolates themselves and for distinguishing T. vaginalis from other Trichomonas species. We, therefore, conducted a comparative analysis of the nucleotide sequences obtained from T. vaginalis isolates with those of other related species obtained from the GenBank/EMBL/DDBJ Parasitology International 63 (2014) 427–431 ⁎ Corresponding author at: Departamento de Parasitología, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza Ramón y Cajal s/n, 28040 Madrid, Spain. Tel.: +34 91 394 1818; fax: +34 91 394 1815. E-mail address: alexandraibanez@ucm.es (A. Ibáñez-Escribano). 1383-5769/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.parint.2013.12.017 Contents lists available at ScienceDirect Parasitology International journal homepage: www.elsevier.com/locate/parint