Blastocystis in ulcerative colitis patients： Genetic diversity and analysis of laboratory findings

Adil Coskun， Erdogan Malatyali， Hatice Ertabaklar， Mustafa B Yasar， Ali O Karaoglu， Sema Ertug

1Department of Gastroenterology， Faculty of Medicine， Adnan Menderes University， Aydin， Turkey

2Department of Parasitology， Faculty of Medicine， Adnan Menderes University， Aydin， Turkey

Blastocystis in ulcerative colitis patients： Genetic diversity and analysis of laboratory findings

Adil Coskun1， Erdogan Malatyali2?， Hatice Ertabaklar2， Mustafa B Yasar1， Ali O Karaoglu1， Sema Ertug2

1Department of Gastroenterology， Faculty of Medicine， Adnan Menderes University， Aydin， Turkey

2Department of Parasitology， Faculty of Medicine， Adnan Menderes University， Aydin， Turkey

ARTICLE INFO

Article history:

Received 15 May 2016

Received in revised form 16 June 2016

Accepted 1 July 2016

Available online 20 September 2016

Blastocystis

Objective: To determine Blastocystis frequency and subtypes （ST） in ulcerative colitis （UC）patients and analyse some laboratory fi ndings between Blastocystis positive and negative cases. Methods: Faecal samples from 150 UC patients in Adnan Menderes University， Training and Research Hospital were examined by direct microscopy and cultivated in Jones medium. Blastocystis positive cultures were subjected to DNA isolation and subtypes were identif i ed by sequencing of barcode region. A retrospective analysis was conducted on C reactive protein（CRP）， leukocyte counts （WBC）， neutrophil counts， and sedimentation rates. Results: The overall positive rate of Blastocystis was 8% （12 patients） and the most abundant subtype was ST3 （eight isolates， 66.7%）， followed by ST1， ST2 and ST7. Laboratory findings between Blastocystis infected and non-infected UC patients were not signif i cantly dif f erent. Blastocystis frequency was 3.8% among the patients in active stage， while it was 11.8% among the patients in remission stage. Conclusions: The present study conf i rms previous fi ndings that have indicated the predominance of Blastocystis ST3 in humans and contributes additional evidence that suggests the low colonization of Blastocystis infection in ulcerative colitis patients during active stage.

1. Introduction

Blastocystis is a common intestinal protozoon parasite， found in humans. Recent researches have shown contradictory findings about the pathogenesis of Blastocystis. In a great majority of human cases， Blastocystis infection is asymptomatic； however， nonspecif i c gastrointestinal and urticarial symptoms may be observed［1，2］. It was reported that Blastocystis has many different subtypes （ST）based on its small sub-unit ribosomal RNA （ssRNA）［3］. These subtypes can be detected by molecular methods such as sequencing，restriction fragment length polymorphism or sequence-tagged site-PCR［4］. There have been many attempts that offered a possible subtype related pathogenicity， nevertheless much uncertainty still continues about the relationship［5］. Ulcerative colitis （UC） is a chronic inf l ammatory disease of large intestine and is increasing in frequency throughout the world. There has been increasing evidence that suggests a possible link between gut microbiota and development of UC either by causing inflammation directly or indirectly through an altered immune system［6，7］. Despite being a common inhabitant of human intestinal tract， the role of Blastocystis in UC still needs to be investigated.

The primary aim of the present study was to determine Blastocystis subtypes in UC patients. Additionally， this study attempted to analyse C reactive protein （CRP）， leukocyte counts （WBC），neutrophil counts， and sedimentation rates between Blastocystis positive and negative cases.

2. Materials and methods

2.1. Samples

In the present study， faecal samples from 150 UC patients in Adnan Menderes University， Training and Research Hospital were collected from June 2013 to the end of February 2015.

2.2. Direct microscopy and culture

Faecal samples were examined by direct microscopy of native（0.9% serum physiological） and Lugol’s iodine preparations as a part of routine parasitological examination. An approximate 50 mg of samples were inoculated in 3 mL of Jones’ medium supplemented with 10% foetal calf serum. The cultures were checked for the presence of Blastocystis on third day of inoculation by direct microscopy.

2.3. PCR and sequencing

Prior to DNA isolation the cultures were pelleted by centrifugation at 12 000 g for one minute. Genomic DNA was isolated only from positive cultures by using a commercially available kit DNAzol（Invitrogen） according to manufacturer’s instructions. A single PCR reaction was set with the primers RD5 and BhRDr as described by Scicluna et al.［4］ for amplification of ‘barcode region’， an approximately 600 bp of SSU rRNA gene. The reaction was set in a 30-μL volume containing: 1-2 μL of template DNA， 0.4 pmol of each of the primers， 0.3 U of Taq DNA polymerase （Fermentas），0.2 mM of each dNTP （Fermentas）， 1×Taq buffer with （NH4）2SO4（Fermentas）. PCR amplicons were purified and sequenced by a commercial facility （MedSanTek， Istanbul） by using Applied Biosystems 377 DNA Sequencer.

2.4. Determination of subtypes

Subtypes were determined according to closest or exact match at GenBank nucleotide database using BLAST tool at the National Center for Biotechnology Information website［8］. Moreover， the sequences were queried against the Blastocystis Sequence Typing website database （http://pubmlst.org/Blastocystis/）， curated by Stensvold and sited at the University of Oxford［9］.

2.5. Analysis of laboratory findings

A retrospective analysis was conducted on the following laboratory findings: CRP， WBC， neutrophil counts， and sedimentation rates. The patients with endoscopic activity score below four were considered as in remission stage and the patients with equal or over four were considered as in active stage［10］. Data was analysed with non-parametric Mann-Whitney U test by using Statistical Package for the Social Sciences （SPSS）.

3. Results

The overall positive rate of Blastocystis was 8% （12 out of 150）by culture， however the rate was 4.7% （7 out of 150） by direct microscopy of wet mounts. The most common subtype was ST3（eight isolates， 66.7%）， followed by ST1 （two isolates， 16.7%）， ST2（one isolate， 8.3%） and ST7 （one isolate， 8.3%）. The sequences were deposited to Genbank with accession numbers: KU361317-323.

The mean values and standard deviations of laboratory fi ndings between Blastocystis infected and non-infected UC patients were given in the Table 1. The mean age of Blastocystis infected and non-infected cases were 54.5 ± 12.0 and 46.5 ± 14.0， respectively. Blastocystis frequency was 3.8% （2 out of 52） among the patients in active stage， while it was 11.8% （8 out of 68） among the patients in remission stage， data was available for 120 UC patients. The difference was not significantly significant （χ2=2.41， P＞0.05）. Moreover， the infection rate was 7.8% （eight out of the 102） among male and 8.3% （four out of 48） among female patients （χ2=0.11，P＞0.05）.

4. Discussion

In the present study， ST3 was found as the most common subtype in UC patients. ST3 is thought as human originated subtype and reported as predominant in a variety of study populations. Dogruman-Al et al.［11］， found that ST3 was the most common among IBS， UC and patients with chronic diarrhoea. Additionally， ST1-3 are the usually reported subtypes from Turkey. Another subtype from Turkey is avian ST7 which rarely infects humans［12］. The use of cultures was suggested in many studies for the detection of Blastocystis both in epidemiological studies and routine laboratories，because of the low sensitivity of direct microscopy［13，14］. In the present study， the rate of positive cases was increased from 4.7% to 8.0% by the use of Jones medium. However， Blastocystis frequency could be higher than we detected， because of the fact that some Blastocystis strains might not grow in xenic culture［15］. In the present study， the rate of Blastocystis infection was not related to gender as previously reported［16］.

Table 1Comparison of Blastocystis infected and non-infected UC patients in terms laboratory fi ndings.

The role of Blastocystis in certain gastrointestinal diseases and relations with symptoms are investigated. Nagler et al.［17］ asserted that Blastocystis had no role in Irritable Bowel Disease （IBD）； but the results were based on limited number of patients （f i ve UC patients）and unable to encompass the entire picture. Mumcuoglu et al.［18］reported that the frequency of Blastocystis was higher among IBS patients and the symptoms declined after treatment of Blastocystis. In another study， Blastocystis positive 99 patients were compared with control group and none of the gastrointestinal symptoms were found to be related with Blastocystis infection［19］. It was noted that despite these controversies， Blastocystis should be screened in UC patients when the symptoms are refractory［20，21］. Tai et al.，［20］interestingly noted that the elimination of Blastocystis was supportive in the recovery of gastrointestinal symptoms and also conf i rmed their fi ndings with colonoscopy. In a more recent study， Krogsgaard et al.［22］ compared the frequency of Dientamoeba fragilis and Blastocystis between asymptomatic population and IBS patients in Denmark. The authors reported that the positive rate of Blastocystis was greater in asymptomatic population than in IBS patients （22% vs. 15%） thus indicating that these parasites are not likely to have a direct role in the pathogenesis of IBS.

The laboratory fi ndings: WBC， Neutrophil counts， sedimentation rates and CRP between Blastocystis infected and non-infected cases was not signif i cantly dif f erent in the present study. CRP is an important marker for the disease activity in UC patients； in active stage the level is high and in remission it is usually low［23］. In a previous study， haematological values were investigated between Blastocystis infected and control group and CRP and sedimentation rates were found to be significantly high in infected group［24］. Despite being statistically not signif i cant， in our study the rate of Blastocystis was higher in patients who were in active stage than the patients in remission （11.8% versus 3.8%）. This fi nding was in accordance with the previous study［25］. Additionally， Rossen et al. compared patients with active UC and healthy controls in a cohort，they reported that Blastocystis was signif i cantly less frequent in UC patients （13.3%） as compared to healthy controls［26］. Nishikawa et al. compared mucosa-associated microbiota of patients with active UC and non-IBD controls using terminal restriction fragment length polymorphism （T-RFLP） analysis； they showed that diversity of microbial composition was signif i cantly fewer in active stage［27］. In the present study， data from 120 out of 150 UC patients was available for the activity of diseases. It may be concluded that this inconsistency may be due to the number of patients. Furthermore， a study dealing with the CRP level of Blastocystis infected UC patients in remission stage would be worthwhile. Our study population was limited to 150 patients； increasing the number of patients in future studies would give more brief conclusions.

A limitation of this study is that data has not been supplemented with direct PCR of faecal samples which possibly could increase the positive rate and make comparisons of patient groups more valid. Additionally， the small number of Blastocystis positive UC patients（12 patients） prevented making parametric statistics between groups. Roberts et al. compared dif f erent methods and found PCR as the most sensitive at detecting Blastocystis［28］. Additionally， in another study， the positive rate of Blastocystis was 41.0% by culture and it was 44.6% by PCR［29］.

In conclusion， the present study conf i rms previous fi ndings that indicate the predominance of Blastocystis ST3 in humans and provides additional evidence that suggests low colonisation of Blastocystis infection in UC patients during active stage. Additionally，further studies investigating some other laboratory findings in Blastocystis infected UC patients will be worthwhile.

Conflict of interest statement

We declare that we have no conf l ict of interest.

［1］ Casero RD， Mongi F， Sanchez A， Ramirez JD. Blastocystis and urticaria: Examination of subtypes and morphotypes in an unusual clinical manifestation. Acta Trop 2015； 148: 156-161.

［2］ Nagel R ， Cuttell L ， Stensvold CR ， Mills PC ， Bielefeldt Ohmann H ，Traub RJ. Blastocystis subtypes in symptomatic and asymptomatic family members and pets and response to therapy. Intern Med J 2011； 42（11）: 1187-1195.

［3］ Clark CG ， van der Giezen M ， Alfellani MA， Stensvold CR. Recent developments in Blastocystis research. Adv Parasitol 2013； 82: 1-32.

［4］ Scicluna SM， Tawari B， Clark CG. DNA barcoding of Blastocystis. Protist 2006； 157（1）: 77-85.

［5］ Stensvold CR. Blastocystis: Genetic diversity and molecular methods for diagnosis and epidemiology. Trop Parasitol 2013； 3（1）: 26-34.

［6］ Nishikawa J， Kudo T， Sakata S ， Benno Y， Sugiyama T. Diversity of mucosa-associated microbiota in active and inactive ulcerative colitis. Scand J Gastroenterol 2009； 44（2）: 180-186.

［7］ Wallace KL， Zheng LB ， Kanazawa Y， Shih DQ. Immunopathology of inf l ammatory bowel disease. World J Gastroenterol 2014； 20（1）: 16-21.

［8］ Altschul SF， Gish W， Miller W， Myers EV， Lipman DJ. Basic local alignment search tool. J Mol Biol 1990； 215（3）: 403-410.

［9］ Jolley KA， Maiden MC. BIGSdb: Scalable analysis of bacterial genome variation at the population level. BMC Bioinformatics 2010； 11: 595.

［10］ Rachmilewitz D. Coated mesalazine （5-aminosalicylic acid ） versus sulphasalazine in the treatment of active ulcerative colitis: a randomised trial. BMJ 1989； 298（6666）: 82-86.

［11］ Dogruman Al F， Kustimur S ， Yoshikawa H ， Tuncer C ， Simsek Z ，Tanyuksel M， et al. Blastocystis subtypes in irritable bowel syndrome and inf l ammatory bowel disease in Ankara， Turkey. Mem Inst Oswaldo Cruz 2009； 104（5）: 724-727.

［12］ Dagci H， Kurt O， Demirel M ， Mandiracioglu A， Aydemir S， Saz U， et al. Epidemiological and diagnostic features of Blastocystis infection in symptomatic patients in Izmir province， Turkey. Iranian J Parasitol 2014；9（4）: 519-529.

［13］ Santos HJ， Rivera WL. Comparison of direct fecal smear microscopy，culture， and polymerase chain reaction for the detection of Blastocystis sp. in human stool samples. Asian Pac J Trop Med 2013； 6（10）: 780-784.

［14］ Tungtrongchitr A， Manatsathit S， Kositchaiwat C ， Ongrotchanakun J，Munkong N， Chinabutr P， et al. Blastocystis hominis infection in irritable bowel syndrome patients. Southeast Asian J Trop Med Public Health 2004；35（3）: 705-710.

［15］ Parkar U， Traub RJ， Kumar S， Mungthin M， Vitali S， Leelayoova S， et al. Direct characterization of Blastocystis from faeces by PCR and evidence of zoonotic potential. Parasitology 2007； 134（3）: 359-367.

［16］ Inceboz T， Usluca S ， Over L ， Yalcin G ， Tuncay S ， Ozkoc S. The epidemiology research of Blastocystis hominis in the Dokuz Eylul University Medical Faculty Hospital between 2005 and 2009. T Parazitol Derg 2011； 35（2）: 72-76.

［17］ Nagler J， Brown M， Soave R. Blastocystis hominis in inf l ammatory bowel disease. J Clin Gastroenterol 1993； 16（2）: 109-112.

［18］ Mumcuoglu I， Coskun FA ， Aksu N ， Purnak T， Gungor C. Role of Dientamoeba fragilis and Blastocystis spp. in irritable bowel syndrome. Turkiye Parazitol Derg 2013； 37（2）: 73-77.

［19］ Chen TL， Chan CC， Chen HP， Fung CP， Lin CP， Chan WL， et al. Clinical characteristics and endoscopic findings associated with Blastocystis hominis in healthy adults. Am J Trop Med Hyg 2003； 69（2）: 213-216.

［20］ Tai WP， Hu PJ， Wu J， Lin XC. Six ulcerative colitis patients with refractory symptoms co-infective with Blastocystis hominis in China. Parasitol Res 2011； 108（5）: 1207-1210.

［21］ Jeddy TA， Farrington GH. Blastocystis hominis complicating ulcerative colitis. J R Soc Med 1991； 84（10）: 623.

［22］ Krogsgaard LR， Engsbro AL， Stensvold CR， Nielsen HV， Bytzer P. The prevalence of intestinal parasites is not greater among individuals with irritable bowel syndrome: a population-based case-control study. Clin Gastroenterol Hepatol 2015； 13（3）: 507-513.

［23］ Henriksen M， Jahnsen J， Lygren I， Stray N， Sauar J， Vatn MH ， et al. C-reactive protein: a predictive factor and marker of inf l ammation in inf l ammatory bowel disease. Results from a prospective populationbased study. Gut 2008； 57（11）: 1518-1523.

［24］ Javaherizadeh H， Khademvatan S， Soltani S， Torabizadeh M， Yousef i E. Distribution of haematological indices among subjects with Blastocystis hominis infection compared to controls. Prz Gastroenterol 2014； 9（1）: 38-42.

［25］ Petersen AM， Stensvold CR， Mirsepasi H ， Engberg J， Friis M ?ller A ，Porsbo LJ， et al. Active ulcerative colitis associated with low prevalence of Blastocystis and Dientamoeba fragilis infection. Scand J Gastroenterol 2013； 48（5）: 638-639.

［26］ Rossen NG， Bart A， Verhaar N， van Nood E， Kootte R， de Groot PF， et al. Low prevalence of Blastocystis sp. in active ulcerative colitis patients. Eur J Clin Microbiol Infect Dis 2015； 34（5）: 1039-1044.

［27］ Nishikawa J， Kudo T， Sakata S ， Benno Y， Sugiyama T. Diversity of mucosa-associated microbiota in active and inactive ulcerative colitis. Scand J Gastroenterol 2009； 44（2）: 180-186.

［28］ Roberts T， Barratt J， Harkness J， Ellis J， Stark D. Comparison of microscopy， culture， and conventional polymerase chain reaction for detection of Blastocystis sp. in clinical stool samples. Am J Trop Med Hyg 2011； 84（2）: 308-312.

［29］ Eida AM， Eida MM. Identification of Blastocystis hominis in patients with irritable bowel syndrome using microscopy and culture compared to PCR. Parasitol United J 2008； 1（2）: 87-92.

10.1016/j.apjtm.2016.07.018

Adil Coskun， Department of Gastroenterology， Faculty of Medicine，Adnan Menderes University， Aydin， Turkey.

E-mail: adilcoskun@gmail.com

?Corresponding author: Erdogan Malatyali， Department of Parasitology， Faculty of Medicine， Adnan Menderes University， 09100 Aydin， Turkey.

E-mail: erdogan.malatyali@adu.edu.tr

Tel: +90 256 2121850

Fax: +90 256 2120146

Ulcerative colitis

Subtype

Laboratory fi ndings