INTRODUCTION
The high number of samples positive for Blastocystis sp., an organism that was rarely reported in routine parasitological diagnosis until three decades ago, has received increasing attention in recent years. Although, the pathogenic role of Blastocystis sp. is controversial, accumulating evidence from recent studies supports the idea that they constitute an emerging pathogen (Tan, Reference Tan2008; Tan et al. Reference Tan, Mirza, Teo, Wu and Macary2010). This enigmatic organism has also been identified in a wide range of animals. The taxonomic status of Blastocystis sp. has remained elusive until recently; phylogenetic studies based on small-subunit ribosomal DNA (SSU rRNA) have demonstrated that it is a member of phylum Stramenopiles (Silberman et al. Reference Silberman, Sogin, Leipe and Clark1996; Santín et al. Reference Santín, Gómez-Muñoz, Solano Aguilar and Fayer2011).
Blastocystis sp. is usually detected using conventional parasitological methods such as the direct method, concentration method with formalin–ether and stained smears (Stensvold et al. Reference Stensvold, Nielsen, Mølbak and Smith2008; Tan, Reference Tan2008). Culturing methods have been regarded as the gold standard for the detection of Blastocystis sp., because although these methods show high sensitivity (Yoshikawa et al. Reference Yoshikawa, Dogruman-AI, Turk, Kustimur, Balaban and Sultan2011; Zhang et al. Reference Zhang, Qiao, Wu, Da, Zhao and Wei2012). Polymerase chain reaction (PCR) amplification of Blastocystis DNA from cultures or feces is thought to be the most sensitive detection method (Tan, Reference Tan2008). DNA-based methods have been developed to identify genetic variation between Blastocystis sp. forms that are morphologically indistinguishable using a microscope (Alfellani et al. Reference Alfellani, Stensvold, Vidal-Lapiedra, Uche Onuoha, Fagbenro-Beyioku and Clark2013). Molecular studies have focused predominantly on determining the prevalence of Blastocystis subtypes (STs), as well as possible factors associated with pathogenicity (Tan et al. Reference Tan, Mirza, Teo, Wu and Macary2010). Studies using the sequence analysis of Blastocystis sp. small subunit ribosomal RNA genes (SSU rDNA) have demonstrated that there are approximately 17 different lineages, termed subtypes (ST1–ST17), isolates from mammals and birds (Wawrzyniak et al. Reference Wawrzyniak, Poirier, Viscogliosi, Dionigia, Texier, Delbac and El Alaoui2013). Human isolates have been shown to have a higher occurrence of STs 1–4, with limited presence of STs 5–9 (Stensvold et al. Reference Stensvold, Suresh, Tan, Thompson, Traub, Viscogliosi, Yoshikawa and Clark2007; Scanlan, Reference Scanlan2012; Alfellani et al. Reference Alfellani, Stensvold, Vidal-Lapiedra, Uche Onuoha, Fagbenro-Beyioku and Clark2013).
The molecular epidemiology of Blastocystis sp. infections remains unknown in many parts of the world. Recent studies have provided further information regarding ST distribution among human populations (Tan, Reference Tan2008; Alfellani et al. Reference Alfellani, Stensvold, Vidal-Lapiedra, Uche Onuoha, Fagbenro-Beyioku and Clark2013); however, very few studies have been conducted in Latin America (Santín et al. Reference Santín, Gómez-Muñoz, Solano Aguilar and Fayer2011). To date, a small number of studies subtyping Blastocystis (Malheiros et al. Reference Malheiros, Stensvold, Clark, Braga and Shaw2011; David et al. Reference David, Guimarães, Oliveira, Oliveira-Siqueira, Bittencourt, Nardi, Ribolla, Franco, Branco, Tosini, Bella, Pozio and Cacciò2015; Ramírez et al. Reference Ramírez, Sánchez, Hernández, Flórez, Bernal, Giraldo, Reyes, López, García, Cooper, Vicuña, Mongi and Casero2016) have been conducted in Brazil. The aim of this study was to investigate the prevalence of Blastocystis STs in clinical stool samples from the Hospital das Clínicas of the Faculdade de Medicina de São Paulo (HC-FMUSP), Brazil.
MATERIALS AND METHODS
Study population
This study was approved by the local Research Ethics Committee of the Hospital das Clínicas of the Faculdade de Medicina de São Paulo under protocol n°. 488·701. Sixty Blastocystis sp.-positive stool samples were selected based on the positivity in routine parasitological examinations conducted at the Section of Parasitology, Central Laboratory, HC-FMUSP, Brazil. The techniques employed at this laboratory include the Faust method, Lutz method and permanent-stained smears (Garcia, Reference Garcia2001), considering positivity in at least one of the parasitological methods. Fifteen Blastocystis sp.-negative stool samples, determined by the parasitological methods, and 15 stool samples positive for other parasite infections (Ascaris lumbricoides, Entamoeba histolytica, Giardia lamblia, Endolimax nana and Entamoeba coli) were used as controls. Once the parasitological techniques were performed, the stool samples were aliquoted and stored at −20 °C for subsequent DNA isolation.
DNA extraction
Approximately 200 mg of stool sample were washed twice with phosphate-buffered saline (0·01 m L−1, pH 7·2). DNA was extracted from the pellet using the commercial QIAamp® DNA stool MiniKit (QIAGEN, Hilden, Germany), according to the manufacturer's instructions. DNA was eluted in 100 µL of elution buffer and quantified using a NanoDrop ND-1000 UV–VIS spectrophotometer v.3.2.1 (NanoDrop Technologies, Wilmington, DE, USA).
DNA amplification and sequencing
DNA amplification was performed by PCR using the primers RD5 (5′-ATC TGG TTG ATC CTG CCAG T-3′) and BhRDr (5′-GAG CTT TTT AAC TGC AAC AAC G-3′), located on SSU rRNA, as described by Scicluna et al. (Reference Scicluna, Tawari and Clark2006); these primers amplify a ~600 bp fragment. PCR were performed in a 10 µL volume containing ~50 ng µL−1 of DNA, 2·0 µg BSA, 0·2 mm each dNTP, 1·5 mm MgCl2, 2 pm of each primer, 1× PCR buffer and 1·25 U GoTaq ® DNA Polymerase (Promega Corporation, Madison, USA). PCR amplification was conducted with a Master cycler ep gradient S thermocycler (Eppendorf, Hamburg, Germany) with the following conditions: initial denaturation step at 94 °C for 2 min; 30 cycles of 94 °C (denaturation) for 1 min, 61 °C (annealing) for 1 min and 72 °C for 1 min (extension); and a final extension step of 72 °C for 2 min. The PCR products were loaded on a 2% agarose gel containing Syber safe (Invitrogen™, Thermo Fisher Scientific Corporation, Waltham, USA) and subjected to electrophoresis in 1× TAE (Tris-acetate-EDTA) buffer. Positive and negative controls (DNA from positive cultures and PCR mix without DNA template, respectively) were included in each round of amplification.
PCR products were purified using of the ExoSAP enzyme (GE Healthcare, Piscataway, NJ, USA), according to the manufacturer's instructions, and then submitted to a sequencing reaction for incorporation of labelled ddNTPs (dideoxynucleotides) using the ABI PRISM® BigDyeTM Terminator kit (Applied Biosystems, Thermo Fisher Scientific Corporation, Waltham, USA). Direct DNA sequencing was performed using an automated ABI 3500 sequencer (Applied Biosystems).
Positive controls
The positive controls consisted of approximately 200 mg of Blastocystis sp.-positive stool samples cultured as described by Zerpa et al. (Reference Zerpa, Huichol, Náquira and Espinoza2000). The cultures were maintained at 37 °C and the pellets obtained by centrifugation at 12, 400 g for 1 min were examined using Lugol staining smear with optical microscopy (Olympus CX 41) after 24 and 48 h.
For control testing of extracted DNA, all samples without amplification were tested using universal primers (forward: 18SEUDIR 5′-TCTGCCCTAACTACTTTCGATGG-3′ and reverse: 18SEUINV 5′-TAATTTGGCCTGCGCCTG-3′) that amplify a 140-bp region of the eukaryotic 18S ribosomal RNA gene, as described by Wang et al. (Reference Wang, Cuttell, Bielefeldt-Ohmann, Inpankaew, Owen and Traub2013).
Subtyping analysis
Sequence analysis was performed using BioEdit software (Biological Sequence Alignment Editor http://www.mbio.ncsu.edu/bioedit/page2.html). Sequence alignments were conducted using CLUSTAL W and aligned with Blastocystis sequences from GenBank (http://www.ncbi.nlm.nih.gov/GenBank/tbl2asn2). Phylogenetic analyses were performed using MEGA 5 and the neighbour-joining method with 1000 bootstrap replication for phylogenetic tree construction. The Proteromonas lacertae sequence (accession number U37108) was used as the outgroup. Sequences were compared with Blastocystis SSU rDNA sequences available from the National Center for Biotechnology Information (NCBI) using the BLAST program (Basic Local Alignment Search Tool). Additionally, Blastocystis STs were identified by determining the exact match or closest similarity against all known Blastocystis STs using www.pubmlst.org/blastocystis.
RESULTS
Of the 60 clinical stool samples positive for Blastocystis sp. by parasitological methods, 47 were positive by PCR (78% positivity). PCR positivity was higher compared with other parasitological methods: 8% positivity by Faust, 35% by Lutz and lower than permanent-stained smears (87%) (Table 1). The expected DNA fragment (~600 bp) was detected in all PCR-positive samples and no DNA amplification of the target fragment was observed in the negative samples and stool samples positive for other parasite infections.
Comparison of Parasitological methods and conventional PCR (cPCR) for detection of Blastocytis in the stool samples
All PCR positive samples were sequenced and those that obtained good quality (n = 40) were compared with Blastocystis sequences deposited in the GenBank database (BLAST) and exhibited 98–100% similarity. Seven sequences were excluded from the analysis, since they presented low-quality possibly due to poor amplification of DNA.
The phylogenetic analysis was performed and four different STs were identified (Fig. 1). ST1 was found in nine samples (22·5%), ST2 was present in five samples (12·5%), ST3, the most common ST, was found in 24 samples (60%) and ST6 was present in two samples (5%). Based on information available at www.pubmlst.org/blastocystis, three distinct alleles were identified within ST3 (alleles 34, 36 and 37), two in ST2 (alleles 12 and 71) and one in ST1 and ST6, corresponding to alleles 4 and 134, respectively (Table 2).
Phylogenetic analysis of Blastocystis sp. SSU rDNA sequences (~600 bp) generated in this study (identified by triangles) and reference sequences from GenBank (identified by accession number and subtypes). The phylogenetic tree was constructed using the neighbour-joining method. Bootstrap values are based on 1000 replicates. Bootstrap values of <70% are not shown.
Distribution of Blastocystis SSU rDNA alleles retrieved from the samples on each subtytpes
a Corresponds to the number of samples identified on the phylogenetic tree.
DISCUSSION
Blastocystis sp. is the most common intestinal parasites found in human feces and are considered an emerging parasite with a worldwide distribution (Tan, Reference Tan2008). The clinical features associated with blastocystosis range from non-specific intestinal symptoms to cutaneous disorders and the severity of these diseases varies from acute to chronic infections, a possible reason for these differences is genetic diversity (Tan, Reference Tan2008; Tan et al. Reference Tan, Mirza, Teo, Wu and Macary2010; Scanlan, Reference Scanlan2012). Although to date, no specific association has been observed between clinical findings and Blastocystis STs (Özyurt et al. Reference Özyurt, Kurt, Mølbak, Nielsen, Haznedaroglu and Stensvold2008).
A variety of parasitological techniques can be used to detect Blastocystis sp. in clinical samples (Tan, Reference Tan2008). However, this technique has low sensitivity, leading to misdiagnosis or underestimation of the true prevalence of Blastocystis sp. (Scanlan, Reference Scanlan2012; Alfellani et al. Reference Alfellani, Stensvold, Vidal-Lapiedra, Uche Onuoha, Fagbenro-Beyioku and Clark2013). Moreover, this parasite appears in polymorphic forms and variable size in stool samples, further hindering its diagnosis. PCR is an alternative method used to detect Blastocystis sp. in epidemiological studies, offering increased sensitivity and specificity as well as rapid diagnosis (Parkar et al. Reference Parkar, Traub, Kumar, Mungthin, Vitali, Leelayoova, Morris and Thompson2007). The results of the present study demonstrate that PCR was more efficient than the Faust and Lutz methods in the detection of Blastocystis sp. Another advantage of using molecular techniques compared with conventional methods is that they can provide more information in terms of genetic variability and zoonotic relationship (Parkar et al. Reference Parkar, Traub, Kumar, Mungthin, Vitali, Leelayoova, Morris and Thompson2007).
In this study, 60 samples were diagnosed as Blastocystis sp. positive using parasitological technical (considered the Faust method, Lutz method and permanent-stained smears), while the PCR technique confirmed the presence of the DNA parasite in only 47 stool samples. Possible reasons for this difference are PCR inhibition stool samples, degradation of DNA due to extended storage time, or misidentification by light microscopy (Forsell et al. Reference Forsell, Granlund, Stensvold, Clark and Evengard2012; Mattiucci et al. Reference Mattiucci, Crisafi, Gabrielli, Paoletti and Cancrini2016). However, negative PCR results were not caused by inhibition of the amplification by fecal components, because amplification using universal primers was observed in all samples.
The suggested criteria for reporting infection intensity by parasitological methods are the observation of five or more parasites per high-powered field (×400) for wet mounts or under oil immersion (×1000) using permanent-stained smears (Tan, Reference Tan2008). Other studies have included more detailed reports of parasite abundance (Leder et al. Reference Leder, Hellard, Sinclair, Fairley and Wolfe2005; Özyurt et al. Reference Özyurt, Kurt, Mølbak, Nielsen, Haznedaroglu and Stensvold2008) and quantified parasite abundance using definitions such as rare (one to two parasites in every 10 high-power fields), few to moderate (one parasite in every one to five high-power fields), and abundant (five or more parasites per high-power field) (Leder et al. Reference Leder, Hellard, Sinclair, Fairley and Wolfe2005). In the present study, 13 of the 60 Blastocystis sp. positive samples identified using parasitological methods failed to produce Blastocystis-specific bands by PCR, five of which were reported as rare, four as few to moderate and four as suggestive forms by parasitological analyses. Mattiucci et al. (Reference Mattiucci, Crisafi, Gabrielli, Paoletti and Cancrini2016) have suggested that negative PCR results may be due to the amount of Blastocystis DNA being lower than the detection level.
Blastocystis in mammals and birds are subdivided into 17 STs, nine of which (ST1–ST9) have been found in humans (Stensvold and Clark, Reference Stensvold and Clark2016). In 2007, a consensus of Blastocystis terminology was proposed, based in comparison with representative sequences of all known STs (Stensvold et al. Reference Stensvold, Suresh, Tan, Thompson, Traub, Viscogliosi, Yoshikawa and Clark2007). The ST distribution in the present study was quite similar to that found in other countries. Indeed, the studies reported thus far indicate that the majority of human infections with Blastocystis sp. are attributable to ST3 isolates (Özyurt et al. Reference Özyurt, Kurt, Mølbak, Nielsen, Haznedaroglu and Stensvold2008; Meloni et al. Reference Meloni, Sanciu, Poirier, El Alaoui, Chabé, Delhaes, Dei-Cas, Delbac, Luigi Fiori, Di Cave and Viscogliosi2011; Forsell et al. Reference Forsell, Granlund, Stensvold, Clark and Evengard2012; Ramírez et al. Reference Ramírez, Sánchez, Hernández, Flórez, Bernal, Giraldo, Reyes, López, García, Cooper, Vicuña, Mongi and Casero2016), as was the case in the present study (60%). It is suggested that dominance of ST3 may be related to its human origin (Özyurt et al. Reference Özyurt, Kurt, Mølbak, Nielsen, Haznedaroglu and Stensvold2008; Meloni et al. Reference Meloni, Sanciu, Poirier, El Alaoui, Chabé, Delhaes, Dei-Cas, Delbac, Luigi Fiori, Di Cave and Viscogliosi2011; Yoshikawa et al. Reference Yoshikawa, Koyama, Tsuchiya and Takami2016).
An analysis of samples from indigenous regions of Brazil conducted by Malheiros et al. (Reference Malheiros, Stensvold, Clark, Braga and Shaw2011) showed different results from those of the current study; ST1 was the dominant ST identified (41%). It is possible that ST distribution is affected by the ethnic origin of the population and the limited contact between indigenous groups and people in other communities. ST1 and ST2 are also common in different regions in human isolates, while the other STs are found only sporadically (Alfellani et al. Reference Alfellani, Stensvold, Vidal-Lapiedra, Uche Onuoha, Fagbenro-Beyioku and Clark2013). It is important to note the absence of ST4 in our study, similar to the results of other studies conducted in South America (Malheiros et al. Reference Malheiros, Stensvold, Clark, Braga and Shaw2011; David et al. Reference David, Guimarães, Oliveira, Oliveira-Siqueira, Bittencourt, Nardi, Ribolla, Franco, Branco, Tosini, Bella, Pozio and Cacciò2015). In contrast, ST4 is observed with high frequency in continental Europe and the UK (Forsell et al. Reference Forsell, Granlund, Stensvold, Clark and Evengard2012; Alfellani et al. Reference Alfellani, Stensvold, Vidal-Lapiedra, Uche Onuoha, Fagbenro-Beyioku and Clark2013; Mattiucci et al. Reference Mattiucci, Crisafi, Gabrielli, Paoletti and Cancrini2016).
Another important finding of this study is the presence of ST6, rarely detected in human isolates. ST6 is considered as an avian ST, which points to a possible zoonotic origin (Mattiucci et al. Reference Mattiucci, Crisafi, Gabrielli, Paoletti and Cancrini2016). The reported differences in the number of STs identified in human populations as well as their relative abundance might indicate different reservoirs and transmission routes (Noël et al. Reference Noël, Dufernez, Gerald, Edgcomb, Delgado-Viscogliosi, Ho, Singh, Wintjens, Sogin, Capron, Pierce, Zenner and Viscogliosi2005; Meloni et al. Reference Meloni, Sanciu, Poirier, El Alaoui, Chabé, Delhaes, Dei-Cas, Delbac, Luigi Fiori, Di Cave and Viscogliosi2011).
In the present study, alleles 34, 36 and 37 were detected in ST3. This is in agreement with observations reported by David et al. (Reference David, Guimarães, Oliveira, Oliveira-Siqueira, Bittencourt, Nardi, Ribolla, Franco, Branco, Tosini, Bella, Pozio and Cacciò2015) and Mattiucci et al. (Reference Mattiucci, Crisafi, Gabrielli, Paoletti and Cancrini2016). In contrast to our findings, Ramírez et al. (Reference Ramírez, Sánchez, Hernández, Flórez, Bernal, Giraldo, Reyes, López, García, Cooper, Vicuña, Mongi and Casero2016) showed a high diversity of alleles from ST1, ST2 and ST3. It is important to highlight that the comparison SSU rDNA alleles within the same ST can help determining the differences between the strains, which may possibly contribute to the identification of the potential for zoonotic transmission and pathogenic strains.
The present study is one of the few to analyse the incidence of Blastocystis STs in South America. Thus, our results are very important for understanding the geographic distribution of STs in Latin America. Moreover, these findings provide an initial molecular analysis that will enable future examination of unknown epidemiological aspects.
ACKNOWLEDGEMENTS
We would like to thank all the patients who provided the stool samples used in this study. We are very grateful to Ms Magali Orban for her technical assistance in conducting the parasitological analysis of fecal samples.
FINANCIAL SUPPORT
This research was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 2015/18213-6), Brazil.
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