ABSTRACT
Since the identification of Blastocystis subtypes (ST) in the last decade, much has been learned about the genetic diversity of Blastocystis isolates in different populations, except pregnant women. The objective of this study is to investigate the genetic diversity of Blastocystis in pregnant women and analyse some demographic factors.
The faecal samples from 100 pregnant women were collected at an Obstetrics and Gynecology Department in Muğla, Turkey. Thereafter, Blastocystis positivity was detected by direct microscopy and culture. The positive cultures were subjected to DNA isolation, and the Blastocystis barcode region was amplified with polymerase chain reaction. Next, the sequences were queried against GenBank nucleotide and Blastocystis STs (18S) databases.
Blastocystis was detected in 14% (14 out of 100) of the faecal samples by culture and 10% (10 out of 100) of the samples by direct microscopy. Nine of Blastocystis isolates (64.4%) were ST3, three (21.4%) were ST1 and two (14.2%) were ST2. Neither the demographic features nor the gastrointestinal symptoms were statistically related to Blastocystis infection.
The findings in this study agreed with the most of the previous human studies that found ST3 as the most abundant genotype. This study reported the frequency of Blastocystis in pregnant women and highlighted the importance of comprehensive studies with more cases of Blastocystis during pregnancy.
Keywords: Blastocystis, pregnancy, subtypes, symptoms
INTRODUCTION
Blastocystis is a highly prevalent enteric protozoon in humans and a variety of animals (1). Despite of the controversy about Blastocystis pathogenicity, recent researches has revealed that it may be considered as an opportunistic pathogen in immunocompromised cases (2). Even though there is not a complete immunosuppression, pregnancy is a period with immunomodulation of mother (3). Additionally, iron deficiency anaemia (IDA), protein, zinc, folate deficiency may develop due to the impairment of absorption caused by intestinal parasitic infections in pregnancy. These deficiencies may result in some fatal consequences: increased perinatal mortality rates, low birth weight and intrauterine tension. It was reported that pregnant women infected with Entamoeba histolytica were more susceptible to penetration of intestinal mucosa and the infection increased the risk of preterm delivery two fold (4). Heavy infection with Schistosoma mansoni increased risk of anemia in a cross-sectional survey in Tanzania (5). Soil transmitted helmint infections including Ascaris lumbricoides, Trichuris trichiura and hookworms were soil transmitted helminth infections were significantly associated with anemia in pregnant women (6).
The complex, collective and highly active population of living microorganisms in gastrointestinal tract of humans is defined as gut microbiota and it exerts a marked influence on the host both in homeostasis and diseases. A number of host-related and environmental factors contribute to the formation and change of microbiota from infancy to adolescence. Gut microbiota establish some dramatic changes during pregnancy, primarily as a result of increased energy requirements of the developing foetus. Additionally, recent evidence suggests that the colonisation and immune system of foetus is highly influenced by maternal gut microbiota (7). The position and role of Blastocystis in gut microbiota is a growing area of interest, some authors have asserted that it can be an indicator of a healthy gut microbiota (8).
The genetic heterogeneity of Blastocystis became a major area of interest, recently. Blastocystis isolates have been classified into 17 different genotypes/or subtypes, with small subunit ribosomal RNA (SSU rRNA) gene sequence analysis. The nine of them were isolated from human faecal samples; ST3 is the predominant subtype which is also known as human subtype (9). Subtype distribution of Blastocystis was investigated in a variety of study groups and some factors factors have been found to be related to the Blastocystis infection. However, to the best of our knowledge, no previous study has investigated Blastocystis subtypes during pregnancy. The aims of this study were to determine the genetic diversity of Blastocystis in pregnant women and also analyse gastrointestinal symptoms and demographic factors for Blastocystis infection.
Samples
Faecal samples were collected from 100 pregnant women who admitted to Muğla Sıtkı Kocman University, Training and Research Hospital, Clinic of Obstetrics and Gynaecology in-between November 2014 and April 2015. A semi-constructed questionnaire was used to collect data which included the demographic characteristics: residence, livestock farming, having pet animal, drinking water supply, home ownership, occupation and monthly income. The presence of common gastrointestinal symptoms (abdominal pain, diarrhoea, constipation, flatulence, urticaria and lack of appetite) was also asked to the women in the questionnaire. A single faecal sample was taken from each woman and brought to the laboratory with a plastic container without fixatives.
The study was approved by Muğla Sıtkı Koçman University Clinical Research Ethics Committee (07.11.2014). The informed consents were obtained from pregnant women in the present study.
Direct Microscopy, Culture and Polymerase Chain Reaction
All samples were examined with both direct microscopy and Jones medium for the presence of Blastocystis forms. Positive cultures were centrifuged at 12,000 g for one minute and pellets were used for DNA isolation with a commercial kit (DNAzol, Invitrogen). The barcode region was amplified with a set of conserved polymerase chain reaction primers (RD5 and BhRDr) for the analysis of approximately 600 bp of SSU rRNA coding gene (10). The reaction was set in 30 µL volume: 1-2 µL of template DNA, 0.3 U of Taq DNA polymerase, 0.2 mM of each dNTP, 0.4 pmol of each of the primers and 1× Taq buffer with (NH4)2SO4. The amplicons were purified and sequenced by a commercial facility with 377 DNA Sequencer (Applied Biosystems).
Determination of Subtypes and Phylogenetic Analysis
The barcode sequences of isolates were queried against both Genbank and Blastocystis Subtype (18S) databases. Sequences were aligned with references by using ClustalW algorithm in Molecular Evolutionary Genetics Analysis version 6.0 (MEGA) and a phylogenetic tree was constructed with the Neighbor-Joining method in 1.000 replicates bootstrap test. Additionally, the evolutionary distances of sequences were calculated with the maximum composite likelihood (11-13).
Statistical Analyses
The study did not have defined exclusion criteria. All of the pregnant women who volunteered to participate were included in the study. The qualitative variables including residence, livestock farming, having pet animal, drinking water supply, home ownership, occupation and monthly income were analysed with chi-square test in Statistical Package for Social Sciences (SPSS 19.0) software. The analysis of gastrointestinal symptoms (abdominal pain, diarrhoea, constipation, flatulence, urticaria and lack of appetite) was also performed in the same way. The significance level was set at α=0.05.
RESULTS
The mean age of pregnant women was 27.4±5.3 and the ages were varying from 16 to 44. Blastocystis was found in 14 of 100 (14%) pregnant women by culture; however, the rate was 8% by direct microscopy. The subtype distribution of isolates were as follows: ST3 nine isolates (64.4%), ST1 three isolates (21.4%) and ST2 two isolates (14.2%) (Figure 1). Blastocystis SSU rRNA gene partial sequences were deposited to Genbank with accession numbers: MH349731, MH349744 and MH349753.
Among the Blastocystis positive 14 cases, the most common gastrointestinal symptom was flatulence 57.1% (n=8), followed by abdominal pain (n=6, 42.5%) and constipation (n=4, 28.5%).
Blastocystis negative pregnant women (n=86) were defined as control group and compared to Blastocystis positive pregnant women (n=14). Statistical analysis of demographic factors and symptoms with the presence of Blastocystis was presented in
Table 1. None of these factors were statistically related with Blastocystis positivity.
Additionally, the comparison Blastocystis positivity according to the presence and absence of symptoms was given in Table 2. It was found that Blastocystis positivity was not statistically different between the cases with and without symptoms.
DISCUSSION
Blastocystis is one of the most common protozoa in human faecal samples. Despite the recent development in the understanding of Blastocystis genetic diversity and symptomatology, the role of Blastocystis infection during pregnancy is not a well-studied area. The present study was designed to investigate Blastocystis frequency and subtype distribution in pregnant women. The Blastocystis frequency was 14% in our study, there were limited number of studies in the literature that investigated Blastocystis in pregnant women. It was reported that Blastocystis infection contributed to the development of IDA during pregnancy. They found that Blastocystis frequency was significantly higher in IDA patients (40%) than non-anemic pregnant controls (6.3%). Additionally, the overall frequency was 26.5% among all studied subjects (14). Another study investigated intestinal parasitism among pregnant women who lived in low socio economic residential areas in Bogotá, they reported that Blastocystis spp. frequency was 25% (15). Our study investigated the subtype distribution of Blastocystis in a pregnant population for the first time in the literature. As pathogenicity has been a controversial and much disputed area in Blastocystis researches, a variety of study populations such as cancer, ulcerative colitis, and Irritable Bowel syndrome patients were subjected in similar studies (16-18). Our findings were in accordance with the reported Blastocystis subtype distribution in these studies, all have found Blastocystis ST3 as the predominant genotype.
There were a few studies that investigated the frequency of Blastocystis in Muğla and surrounding cities. The city is located in the south-western of Turkey and the centrum is about 30 km from the seacoast in the Gulf of Gokova. It was reported that 7.4% of school aged children were infected with Blastocystis in Mugla (19). In addition, subtype distribution was found as follows: ST3 (34.2%), ST1 (31.4), ST2 (25.7) and ST7 (2.8%). In a study, Blastocystis subtypes from the culture positive 61 samples were investigated. They found a comparable result in that study: ST3 (38.6%), ST2 (29.5%), ST1 (20.5%), ST1+ST3 (9.1%), and ST1+ST2 (2.3%) (20). Moreover, a study reported that most common Blastocystis subtype was ST3 with a frequency of 52.6, followed by ST2 (22.1%), ST1 (17.9%), ST7 (4.2%), ST2+ST3 (2.1%) and ST1+ST3 (1.1%) in Aydin (21). In the present study we detected three different subtypes (ST1, 2, and 3) that is comparable to the other studies.
In the present study we have compared Blastocystis frequency with respect to residence, livestock farming, having pet animal, drinking water supply, home ownership, occupation and income. None of these parameters were significantly related with Blastocystis presence. In the literature some have reported that Blastocystis infection has been related to the consumption of untreated water, close contact with animals, intra-family infections and mother’s education (22-24).
In the present study, none of the gastrointestinal symptoms were found to be related with presence of Blastocystis. Flatulence, abdominal pain and constipation were the most common clinical findings in Blastocystis infected pregnant women. Blastocystis infections are highly asymptomatic and gastrointestinal symptoms are seen in a small proportion of infected cases (25). It has also been suggested that Blastocystis may be a marker of a healthy intestinal flora (26). However, in a number of studies, non-specific gastrointestinal symptoms such as diarrhea, abdominal pain and bloating, as well as dermatological symptoms have been linked to Blastocystis infection (27). In addition a study reported that fever was common in symptomatic Blastocystis infected individuals (28). The main limitation of such studies, however, was the lack of elimination of other possible factors such as pathogens, underlying diseases and immune status of cases. In brief, the current data are rather controversial, and there is no general agreement about clinical importance of Blastocystis infection. However, recently Blastocystis pathogenesis is reasonable thought to be a multi-factor phenomenon that is highly depended on both Blastocystis and host related factors (29).
CONCLUSION
Although the current study was based on relatively small number of participants, it highlighted the importance of Blastocystis in pregnancy because of the high infection rate. Therefore, parasitological examinations are recommended in gynaecological clinics. Further studies regarding the role of Blastocystis in extended series of pregnant women would be worthwhile.
