Detection of Blastocystis in Stool Isolates Using Different Diagnostic Methods

Background: Blastocystis is a parasite that inhabits human intestinalis. It is commonly identied in asymptomatic individuals, while its pathogenic role is underestimated. In Syria, studies on this parasite are very few, and it is not documented in the laboratory reports. The present study aimed to evaluate the sensitivity of three different diagnostic methods in the detection of Blastocystis among patients with no specic or clear symptoms. Methods: Stool samples were collected from 70 patients suffering from various gastrointestinal symptoms. All samples were examined microscopically using iodine staining smears, and after in vitro cultivation at 37°C for 48- 72 h using Jones' medium. Molecular detection of Blastocystis was determined by fragment amplication of the SSU rRNA gene using PCR. Results: Blastocystis was identied in: 49 cases (70%) by direct microscopic examination, in 60 isolate (85.7%) by in vitro culture and in 64 (91.4%) of cases using molecular detection. Comparative analysis revealed that the sensitivity of microscopic detection for Blastocystis was 73.4% while it was 90.6% for in vitro culture and 95.9 - 96.7% using PCR detection method. Blastocystis was found alone in 32 (65.3%) of cases, while co-infection was detected in 17 (34.7%) samples. Conclusions: Our ndings highlighted the importance of considering Blastocystis in laboratory diagnosis. Molecular methods are recommended for screening clinical specimens for Blastocystis infection especially among individuals with no common particular symptoms. If not applicable, two different diagnostic techniques are required for accurate diagnose of this parasite.

Blastocystis pathogenicity is controversial; for many years, this parasite was considered with no clinical relevance due to its high prevalence in asymptomatic individuals [9]. However, several studies reported its presence in both asymptomatic and symptomatic patients [10][11][12]. No speci c gastrointestinal symptoms are associated with the presence of this parasite; some individuals show abdominal pain, acute or chronic diarrhea, while others show atulence, bloating, anorexia, and weight loss as well as urticarial lesions [13,14]. Recently, an association has been reported with irritable bowel syndrome (IBS) and in ammatory bowel disease (IBD) [15,16].
Blastocystis has a highly polymorphic appearance, direct microscopic examination of fecal samples, is considered the traditional diagnostic method of human infection with this parasite [9,17]. In Syria, Blastocystis epidemiology, diagnose, infection sources and the way of its transmission are not well studied. Therefore, we aimed in this study to employ a comparative analysis between three different diagnostic methods, especially among patients with no speci c or clear symptoms in order to shed light on this parasite and to show the importance of its both pathogenicity and diagnosing.

Clinical samples
Seventy patients, who presented to the clinics of internal medicine at three major hospitals in the city of Damascus (Al Assad University Hospital, Al-Mouwasat University Hospital and Kids Hospital), participated in this study. All patients signed informed consent and completed simple questionnaire including gender, age, presence of symptoms (i.e. abdominal pain, diarrhea, vomiting, fever, nausea, headache and discomfort) and environmental conditions, such as type of water supply and contact with household animals. Stool samples were collected into sterile containers, then each obtained sample was divided into three parts for use in microscopy, culture, and PCR assays. Samples were collected in the period between February and December 2020. This study has been approved by the ethical committee of Damascus University.

Macroscopic and Microscopic examination
Firstly, stool specimens were examined by naked eye to determine color and presence of blood or mucous. Then, Lugol's iodine stained smears were prepared from each stool sample to detect Blastocystis and other parasite forms using light microscopy at 40× and 100×. To avoid obscurity in microscopic diagnosis of the polymorphic Blastocystis, only the presence of vacuolar form in more than one eld of stool smear was considered as positive.

Culture
Approximately 50-100 mg of stool samples were subjected to culture in Jones' medium (Lio lchem, Italy) supplemented with 10% horse serum and antibiotics (penicillin 100u/ml, streptomycin 100 μg/ml). Samples were incubated at 37°C in OSK incubator (OSK 9639b, Japan). The growth of Blastocystis and the distinct morphological and reproductive stages were con rmed by microscopic observation of culture at 48, and 72 h of incubation using Lugol's iodine staining and light microscopy at 400×.

Genomic DNA extraction and PCR ampli cation
Total genomic DNA was extracted from 200 to 250 mg of each stool sample, using QIAamp DNA stool mini kit (Qiagen, Valencia, CA) as described by Skhal et al. [18]. A pair of diagnostic primers [19] was used to amplify a 310 bp fragment of the small subunit ribosomal RNA gene (SSU-rRNA) in case the presence of Blastocystis.
Each PCR experiment contained a negative control (4 µl of nuclease-free water) for contamination detection. PCR cycling conditions were as follows: initial denaturation at 94•C for 3 min, then 30 cycles of 94•C for 60 s, 58 •C for 60 s and 72•C for 60 s. The nal extension was at 72•C for 5 minutes. The PCR products were electrophoresed in 2% agarose gel stained with ethidium bromide (Sigma-Aldrich, USA) along with a 100 bp DNA ladder (GeneDirex Inc, Taiwan ROC) as a size standard. The nal results were visualized under a UV transilluminator and photographed for documentation 2.5 Statistical study Diagnostic accuracy was calculated as: sensitivity, speci city, positive and negative predictive values.
The macroscopic examination results showed that 23 of the stool samples were noted to have watery appearance, whereas the remaining had normal consistency and color. Also, no pus, blood, and helminth were observed in the morphological evaluation of the specimens.
Out of the 70 samples, 49 samples (70%) were determined positive for the presence of Blastocystis, using direct microscopic examination with Lugol's staining. The vacuolated form of Blastocystis was the most common, in which cellular structures as the central vacuole, band of cytoplasm, nuclei and surface coat were clearly distinguishable (Fig 1: A).
Our in vitro cultured samples results showed the growth of Blastocystis in 60 (85.7%) stool isolates. The most detected forms of Blastocystis in culture media were the vacuolar and granular forms (Fig 1: C), while the cystic and amoeboid forms were present in a smaller proportion.
PCR ampli cation of the 310 bp fragment of the SSUrRNA gene was successfully obtained from 64/70 studied cases (91.4%) (Fig 2). Two samples only showed no ampli cation.
Comparative result analysis revealed that sensitivity and speci city identi ed by microscopy in comparison with PCR assay were 73.4% and 66.7%, respectively. However, the in vitro culture showed 90.6% sensitivity and 66.7% speci city when compared with PCR (Tables 3-4).

Discussion
Blastocystis is an enteric parasite found in animals and humans with a worldwide distribution [20,21]. Many studies have emphasized the importance of its pathogenic role [22][23][24]. In Syria, Blastocystis is considered part of the intestinal ora; it is not yet recognized as a pathogen and it is not speci ed usually, in the results of any laboratory analysis of the fecal sample. Hence, we aimed to focus on the different diagnostic methods and its sensitivity and speci city in the accurate diagnosis of Blastocystis.
Our ndings showed notable difference in the detection of Blastocystis in stool isolates using the three diagnostic methods. The presence of this parasite was recorded in 70% of samples by microscopy, in 85.7% by in vitro culture and in 91.4% by conventional PCR.
Laboratory diagnosis of Blastocystis can be challenging and the prevalence data can be in uenced depending on the method of diagnosis, making choosing the accurate method an important task in diagnosing Blastocystis [25].
Blastocystis is highly polymorphic it has variation in size and shape [26,27]. Our microscopic data showed that the vacuolar and granular forms were mostly detected using microscopic examination. This nding is in agreement with previous results since these forms are easily distinguished from other protozoa [28,29]. On the contrary, other studies indicated the presence of vacuolar, granular, amoeboid and cyst forms of Blastocystis in the microscopic detection [30,31]. Thus, relying on using microscopic examination only in diagnosis is controversial and many studies underestimated it [27,32]. Additionally, the low microscopic sensitivity recorded in our study may be according to some researches due to the Lugol's staining method which shows less sensitive than cultivation in Jones' medium [17,33].
On the other hand, our data showed that in vitro culture failed to detect the parasite in 6 positively proved cases by PCR. This results may be explained either because they were disintegrated prior to culturing or for some conditions that affected its growth and hence detection in culture [11,34].
Despite its high cost, PCR is considered as a gold standard detection assay with no time consuming, in comparison with in vitro culture method that is time consuming, yet microscopy detection needs experience but with low cost. Our diagnosis results are consistent with previous studies that indicated that molecular assays and in vitro culture are superior over the direct microscopic examination in the detection of Blastocystis from human stool isolates [17,35,36]. However, some studies suggested that in vitro culture is superior to direct PCR assay [32,37].
False-negative results using PCR were detected in two positively con rmed isolates by microscopy and culturing techniques. Even though, there is no clear explanation for such results, low concentration of DNA, the presence of PCR inhibitors in some specimens or degradation of parasite material during storage may be the cause [11,38].
Remarkably, the majority of our samples (65.3%) showed the presence of Blastocystis alone, while coinfection with other intestinal parasites was detected in 34.7%. This nding strongly indicates the importance of considering Balastocystis in laboratory diagnosis. It also agrees with that our patients showed enormously different symptoms, making it hard to associate Blastocystis presence with speci c gastrointestinal symptoms and emphasize the importance of recognizing it as a pathogen agent.

Conclusions
This study is the rst in Syria to highlight the importance of Blastocystis diagnosis. We strongly recommend considering this parasite in laboratory reports, diagnosis, and treatment especially when no coinfection is present. Additionally, molecular methods are an excellent tool for the accurate detection and identi cation of Blastocystis in stool samples. But if not feasible for a diagnostic laboratory, it is recommended to use at least two different diagnostic techniques; such as microscopy in parallel with in vitro culture in Jones' medium for accurate diagnose of this parasite.

Declarations Ethics approval and consent to participate
This study has been approved by the ethical committee of Damascus University (date: 26/8/2019, number: 4031). Informed written consent was obtained from the patients or a member of their family in case of children. Con dentiality of the details of the participants was assured. All methods, used in this study, were carried out in accordance with relevant guidelines and regulations.

Consent for publication
Not applicable Availability of data and materials The datasets used in the current study are available from the corresponding author on reasonable request.

Competing interests
No con ict of interest is associated with this publication, and there has been no signi cant nancial support for this work that could have in uenced its outcome.   Figure 1 Iodine stained smears: A. Blastocystis (vacuole forms "arrows") 100×; B: co-infection Entamoeba cysts "red arrow" with Blastocystis "black arrows" 100×; C: Blastocystis in vitro culture in Jones' medium (granular "red arrows"; vacuolar "black arrow"; amoeboid form "green arrow" 40×. Ethidium Bromide-Stained 2% agarose gel electrophoresis of PCR products. Lanes M ; molecular weight marker (100bp). Lanes 1-7 PCR Product ; a single speci c PCR fragment of 310 bp. NTC: negative control for contamination detection.