INTRODUCTION
Blastocystis sp. is an anaerobic intestinal parasite belonging to stramenopiles, and considered to be the most commonly isolated parasite worldwide (0.8%–61.8%).[1] It infects both humans and animals with a probable fecal-oral mode of transmission. Blastocystis sp. is also one of the main causes of traveler’s acquired diarrhea.[2]
More than 1 billion people have been estimated to be infected by Blastocystis sp. with a higher prevalence in developing than developed countries. This can be explained by poor hygiene and consumption of contaminated water and food.[3] Egypt has a high rate of infection worldwide.[4]
One explanation of the very high prevalence of Blastocystis in stool samples is the reluctance of physicians to treat the infection. Symptoms are self-limiting, and many patients harboring the infection are asymptomatic and healthy. This is one reason why Blastocystis sp. was regarded by some physicians as a commensal microorganism rather than an infectious one.[5]
In symptomatic patients with Blastocystis infection, clinical manifestations include abdominal pain, diarrhea, flatulence, anorexia and nausea, and manifestations commonly associated with irritable bowel syndrome (IBD).[6]Blastocystis was also regarded as an opportunistic pathogen, in immunocompromised patients.[7] This has led different studies to cite Blastocystis as an emerging pathogen.[3] A possible factor of pathogenicity might be the recently discovered 29-kDa parasite protein and a parasite-associated protease.[8]
Whether Blastocystis is pathogenic or not and to what extent remains controversial. This controversy might be related to the number of other associated parasitic infections, host genetics, whether the infection is acute or chronic, and to the fact that Blastocystis has several subtypes, some of which might cause pathogenicity while others do not.[9] For years, microscopic examination has been the standard tool to assess the presence of Blastocystis infection. However, new molecular diagnostic tools emerged, hence increasing the sensitivity and specificity, and allowing molecular genotyping.[9] Based on sequence analysis, and gene analysis of small subunit ribosomal RNA (SSU-rRNA) of Blastocystis sp., 26 subtypes or species of Blastocystis sp. have been characterized, at least 9 of which have been reported in humans.[9,10]
ST1–ST8 were found both in humans and animals;[11] ST9 was exclusively identified in humans, and ST10–ST17 were identified only in animals.[2,12] Furthermore, the most frequently isolated subtypes from humans were demonstrated to be ST1–ST4, with ST3 being the most common subtype of human infections worldwide.[1] The variation of clinical symptoms in people harboring Blastocystis sp. might be attributed to this genetic variation.[10] ST3 subtype is proposed to be the most pathogenic as it invades the intestine by releasing cysteine protease, which in turn increases the production of interleukin-8 (IL-8) in human intestinal epithelial cells.[13]
Diagnosis of Blastocystis is usually done by microscopic examination; however, detection of parasite-specific DNA from stool samples is suggested to be a much more sensitive technique and can provide a suitable tool for subtyping of the parasite.[14]
Thus, the aim of the current work is to study the prevalence of Blastocystis parasite detected by routine microscopic examination and quantitative polymerase chain reaction (qPCR) in patients with gastrointestinal manifestations referred for colonoscopy. In addition, possible correlation between Blastocystis sp. subtypes and clinical, colonoscopic, and histopathological findings is assessed.
METHODOLOGY
Subjects
One hundred patients suffering from various gastrointestinal manifestations attending the Gastroenterology Department in the Medical Research Institute and referred for colonoscopic examination were enrolled in the present study. Approval of the Committee of Ethics was given (IRB number: 00007555). Informed consent was obtained from all patients enrolled in this study.
Collection and processing of stool samples
Stool samples were collected from all patients in clean plastic containers. Stool samples were homogenized by thorough mixing immediately after delivery to the laboratory. A portion of each sample was kept in a labeled test tube without preservative and stored at –20°C for polymerase chain reaction (PCR) analysis. Another portion was preserved in 10% formalin for concentration and microscopic examination.
Microscopic examination of stool samples
Stool samples were prepared for wet mount examination after formal ethyl acetate concentration as described by Garcia 2016.[14] Preparations were examined systematically at low magnification (×10 objective). Any forms suspected of being protozoa or parasitic ova were examined at high magnification (×40 objective).
DNA extraction for molecular analysis of stool samples
DNA was extracted from fecal samples of all patients using the Bioline fecal isolate DNA (Bioline, UK, catalog number BIO – 52082) according to the manufacturer’s protocol.
Quantitative polymerase chain reaction analysis
Blastocystis was detected using primers selected in conserved regions flanking a nonconserved region of the SSU-rRNA gene amplifying a DNA fragment of 320–342 bp, depending on the subtype. Primer sequence was BL18SPPF1 (5_-AGTAGTCATACGCTC GTCTCAAA-3) for forward primer and BL18SR2PP (5_-TCTTCGTTACCCGTTACTGC-3) for reverse primer.[15] Positive controls from samples positive for Blastocystis by microscopy and negative controls (DNA nuclease-free water) were included.
The setup of real-time PCR was as follows: one 3-min cycle 95°C, followed by 40 cycles of 5 s 95°C, 10 s 65°C, and 20 s for extension 72°C. The criteria used to define a positive reaction of subtypes were a melting temperature of 78°C to 85°C. Primary subtyping was done using the melting temperature of different subtypes as described by Poirier et al.[15]
Sequencing
Purified PCR products were sent for confirmation using sequencing by ABI 3730 xl DNA sequencer using forward and reverse primers of each of the SSU-rRNA PCR products. Accuracy was confirmed by two-directional sequencing. The sequences obtained from the present study were compared to the reference sequences in the NCBI GenBank using the BLASTn software.[16]
Colonoscopy
Colonoscopy and biopsy taking were done for all patients, and diagnosis of underlying colonic diseases was reported using routine histopathological examination. Results were correlated with parasitological and qPCR findings. All infected cases with Blastocystis and other parasites were treated with appropriate drugs.
Statistical analysis
Data were coded and analyzed using the Statistical Package for the Social Sciences (SPSS version 20 Chicago, IL, USA). Agreement between two qualitative variables was done by Cohen’s Kappa agreement test.[17]
RESULTS
The present work enrolled 100 patients suffering from various colonic manifestations attending the Gastroenterology Department in the Medical Research Institute and was referred for colonoscopic examination. Stool samples were collected from all patients and prepared for stool examination to diagnose any intestinal parasite, DNA extraction, real-time PCR amplification, and sequencing to detect the presence of Blastocystis different subtypes.
Intestinal parasitic infections diagnosed by microscopic stool examination
The total parasite infection rate by microscopic examination was 46%. Blastocystis showed the highest percentage of infection with 31 cases (31%) [Figure 1]. Other parasites found: Entamoebacoli 5 cases (5%), Entamoeba histolytica 5 cases (5%), Giardialamblia 2 cases (2%), Enterobiusvermicularis 2 cases (2%), and only one case of Dientamoebafragilis (1%).
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Figure 1: Blastocystis sp. wet mount (×400)
Positive Blastocystis sp. cases detected by quantitative polymerase chain reaction
qPCR diagnosed 58 cases of Blastocystis among 100 patients examined. Differentiation of Blastocystis sp. was done according to different subtypes melting curves (between 78°C and 85°C).
Agreement study between microscopy and quantitative polymerase chain reaction analysis for detection of Blastocystis sp.
Comparing the results of the 100 patients examined by microscope and qPCR, it was found that 23 cases gave positive concordant results and 4 gave negative concordant results. By analysis of the discordant results, 8 cases positive microscopically were negative by qPCR while 35 cases diagnosed positive by qPCR were negative by microscope examination. The percent agreement was only 38.5% between the two methods. Statistical analysis showed a k index = −0.143, indicating slight agreement between both techniques in diagnosing Blastocystis infection.
Subtyping of Blastocystis sp. by quantitative polymerase chain reaction and sequencing
Subtyping of Blastocystis sp. was done by studying the melting curves of the positive samples, as described by Poirier et al.[15] [Figure 2] and confirmed by sequencing of positive qPCR samples [Figures 3 and 4].
Figure 2: Melting curves of positive result for Blastocystis sp. by quantitative polymerase chain reaction with temperature between 78°C and 85°C
Figure 3: Sequence analysis of a Blastocystis sp. Subtype 3 sample, chromatogram
Figure 4: Sequence alignment on GenBank BLASTn of a Blastocystis sp. Subtype 3 sample
Among the 58 samples, 4 subtypes have been detected: Subtype 1 (2 cases, 3.4%), Subtype 2 (19 cases, 32.8%), Subtype 3 (29 cases, 50%), and Subtype 4 (8 cases, 13.8%).
Prevalence of Blastocystis sp. subtypes in patients with different gastrointestinal manifestations
Table 1 shows the various gastrointestinal manifestations among patients infected with different Blastocystis subtypes. The most common gastro-intestinal tract (GIT) symptom correlated with Blastocystis infection was found to be abdominal pain, followed by dyspepsia. The least common symptom was constipation
Table 1: Prevalence of Blastocystis sp. subtypes in patients with different gastrointestinal manifestations
Subtype 3 was found to be the most common subtype associated with GIT symptoms while subtype 1 was detected in only 2 symptomatic patients [Table 1].
Prevalence of Blastocystis sp. subtypes in patients with different colonoscopic findings
Table 2 shows the various colonoscopic findings among patients infected with different Blastocystis subtypes. Normal GIT mucosa was found in 13/58 (22.4%) cases positive for Blastocystis followed by colitis 18/58 (31%). The least common symptom was ileitis, only one case.
Subtype 3 was found to be the most common subtype associated with abnormal colonoscopic findings (associated with 53.3% of the total abnormal findings), while Subtype 1 was detected in only 4.4% of abnormal findings [Table 2].
Table 2: Prevalence of Blastocystis sp. subtypes in patients with different colonoscopic findings
Prevalence of Blastocystis sp. subtypes in patients with different histopathological findings
Table 3 shows histopathological studies after colonoscopy of the positive Blastocystis sp. patients who participated in this study. The main histopathological findings other than normal mucosa were colitis and colon cancer. Ileitis was detected in only two cases.
Table 3: Prevalence of Blastocystis sp. subtypes in patients with different histopathological findings
Subtype 3 was found to be the most common subtype associated with abnormal histopathological findings (associated with 52.7% of the total abnormal findings), while Subtype 1 was detected in only 2.7% of abnormal findings [Table 3].
DISCUSSION
Blastocystis sp. is considered the most prevalent intestinal parasite. This high prevalence drove the assumption of some physicians that Blastocystis should be regarded as a normal commensal in humans. However, it has been noted that not all isolated Blastocystis infections are asymptomatic. In many occasions, Blastocystis infections are associated with clinical symptoms, mainly gastrointestinal.[5]
What makes some Blastocystis sp. infections symptomatic, while others asymptomatic, remains a question to be answered. A hypothesis states that the difference in the pathogenicity of Blastocystis infection might be attributed to different subtypes of the parasite. Blastocystis sp. was reported to have 26 subtypes, infecting humans or animals or both.[10]
In the current study, 100 patients suffering from gastrointestinal manifestations and scheduled for colonoscopy were enrolled. Assessment of the presence of Blastocystis was tested both microscopically and by qPCR. As expected, the sensitivity of detection of Blastocystis by qPCR far exceeded that of microscope (58 positive samples by qPCR versus 31 positive samples by microscope, with agreement of 38.5%). This coincides with several other studies indicating that simple microscopy can miss several cases of Blastocystis sp., and thus, if the symptoms are due to this parasitic infection that goes undetected by microscopy, this might result in inefficient treatment.[10,18] Furthermore, it may be possible that microscopy detects only cases with a relatively high intensity while qPCR being a sensitive technique can detect cases with a very low intensity of infection.[15]
Another advantage of diagnosis by qPCR is the ability to subtype Blastocystis sp. Not all subtypes are detected in humans. In addition, not all subtypes seem to be of the same pathogenicity. In our study, only Subtypes 1, 2, 3, and 4 were detected in stool samples, with the most common detected strain being Subtype 3 and the least common being Subtype 1. This goes hand in hand with several other studies.[3,10,11] Others reported Subtypes 1 and 2 to be the most commonly detected among symptomatic patients.[19,20] It has been reported that the prevalence of different Blastocystis sp. subtypes is related to geographical distribution, with high prevalence of ST1 and ST2 in America, ST1 and ST3 in Europe, Australia, and South Eastern Asia, and ST4 in Europe.[9,21]
Regarding clinical features of Blastocystis infection, literature reported several symptoms, such as abdominal pain, anorexia, flatulence, nausea, diarrhea, in addition to association with IBD.[4,6]Blastocystis has also been regarded as an opportunistic pathogen in immunocompromised patients.[7] On the other hand, other studies proposed Blastocystis sp. to be a commensal nonpathogenic parasite.[1,22]
Conflicting observations about pathogenicity of Blastocystis may be related to the parasitic load, whether the infection is acute or chronic, genetic factors of the host, or to different subtypes of infecting Blastocystis sp.[9,10]
In our study, all patients were presented to the colonoscopy clinic because they suffered from gastrointestinal manifestations. Most of those patients were diagnosed positively with Blastocystis and it has been noticed that abdominal pain (55 patients) and dyspepsia (42 patients) were the most frequently encountered symptoms. Other symptoms such as blood in stools, diarrhea, and constipation occurred at a much less rate. Subtype 3 was the most commonly detected subtype associated with various symptoms followed by Subtype 2, except for constipation in which Subtype 2 was the prevalent subtype detected. Subtype 1, on the other hand, was the least frequently encountered subtype, with only 1 case of diarrhea and 1 case of abdominal pain harboring this subtype.
Abnormality for any reason in the gastrointestinal tract has been proposed as a reason paving suitable venue for Blastocystis. Most of the detected clinical symptoms might be a part of IBD, which is functional gastrointestinal disease characterized by vomiting, nausea, and diarrhea, frequent defecation, diffuse abdominal pain, distention, and alternation in bowel habits in absence of a specific organic pathology.[23,24] Several studies observed the association of Blastocystis with IBS. In some studies, the most predominant type was Subtype 1 followed by 3,[20,25] unlike our study in which the most prevalent subtype was Subtype 3 followed by 2, which coincided with other studies.[26] Those symptoms might be attributed to some protease enzymes secreted by Blastocystis sp. causing mucosal disruption and dysbiosis.[6]
It was suggested that subtyping of Blastocystis sp. might help in determining the pathogenicity of Blastocystis sp.[4] Regarding pathogenicity, we tried to associate the different detected subtypes of Blastocystis sp. to abnormal colonoscopic and histopathological findings.
In spite that all enrolled cases were symptomatic, colonoscopic examination showed that normal mucosa was found in 22.4% of cases associated with Blastocystis infection, with the most prevalent Blastocystis sp. Subtype 2 (53.8% of cases with normal mucosa).
The most common abnormal colonoscopic finding was colitis, followed by colon cancer and inflammatory ulcer, and the least common finding was ileitis. Interestingly, although many studies reported Blastocystis to be commonly associated with IBD, only 3 cases in this study out of 58 had IBD associated with Blastocystis. In our study, Subtype 3 was the most frequently encountered subtype in abnormal colonoscopic findings (representing 53.3%).
Regarding histopathological examination, normal mucosa was the most prevalent finding. Abnormal findings included, in order of cases detected, colitis, colon cancer, inflammatory ulcer, and ileitis. Furthermore, Subtype 3 was the most frequently encountered subtype in abnormal histopathological findings (52.7%).
As mentioned above, the most common abnormal finding in this study was inflammation as assessed by colonoscopy and colitis as detected by histopathological examination. Previous studies showed elevated levels of pro-inflammatory IL-6 and IL-8 cytokines, tumor necrosis factor-alpha, and nuclear factor-kappa B in the serum of Blastocystis sp.-infected rats and cell culture, which highlighted the pathogenic potential of Blastocystis sp.[27]
As for association with colorectal cancer, some papers associated the presence of Blastocystis sp. to colorectal cancer.[10,19,28] Some studies proposed this association was due to the immune compression of cancer patients paving the way to opportunistic infections such as Blastocystis.[10,29] It was also suggested that the Blastocystis antigen has the ability to proliferate HCT116 cells through downregulation of p53 and proapoptotic genes, and upregulation of IL-6, thus helping in the growth of colorectal cancer.[28]
In addition, pathogenicity of Blastocystis in general might be attributed to increased oxidative damage and invasive activity previously demonstrated in rats inoculated with human Blastocystis.[27]
CONCLUSIONS
This study confirmed the importance of qPCR in diagnosis of Blastocystis as its sensitivity far exceeds microscopy. In this study, most patients with gastrointestinal manifestations were infected with Blastocystis. Subtype 3 was found to be the most commonly associated with abnormal clinical, colonoscopic, and histopathological findings, except for constipation and ulcers. This necessitates further studies to assess the mechanism of pathogenicity.
Ethical clearance
The study protocol was approved by the Institutional Committee of Ethics in Medical Research Institute, Alexandria University, Egypt. Approval number: IORG0008812.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
Acknowledgment
We would like to thank nurses of the Experimental and Clinical Internal Medicine Department and Mrs. Naglaa Hassanein and Mrs. Somia Elsebaeey from the Parasitology Department in Medical Research Institute, University of Alexandria, Egypt, for their technical assistance and continuous support. We would like to sincerely thank all participants in the studied group for their cooperation during the different procedures.
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