Biochemical and Microbiological Interactions of Molecularly Detected Blastocystis hominis: a cross-sectional study.

doi:10.21203/rs.3.rs-2789621/v1

Background: Blastocystis hominis (B. hominis) is a cosmopolitan intestinal protozoan that has been related to several gastrointestinal disturbances simulating irritable bowel syndrome (IBS). However, the underlying pathogenicity of blastocystosis in human studies remains indistinct.

Methods: In a cross-sectional study, 167 stool samples from patients attending internal medicine department, Kafrelsheik university hospital were examined. Polymerase chain reaction (PCR) -based identification using known sequenced-tagged site (STS) primers allowed the isolation of the positive samples and genotyping of the parasite. Reducing sugar and pH were investigated in patients’ stool samples.

Results: Patients who participated in the study were from both sexes where blastocystosis infection was most prevalent in the age group 20 – 29. Of 167 cases, twenty-seven (16.1%) were molecularly confirmed blastocystosis infections. Genotype 3 was solely detected. Of these, 26 (19%) cases presented with diarrhea, and 27 (17.1%) cases suffered abdominal pain. Additionally, 20 (16.8%) cases had increased flatulence, and only two patients manifested vomiting. The seasonal cycle of the parasite was explored being higher in summer and spring. Our results highlight the consistent chemical association of carbohydrate intolerance and acidic fecal pH with genotype-3 of B. hominis that seemed to augment the irritable bowel syndrome (IBS) -like manifestations of the parasitic infection. Specimens positive for B. hominis showed dominant growth of E. coli. Co-culture of B. hominis with E. coli and Candidashowed their eminent growth whereas the parasite was suppressed. B. hominiswith Giardia species co-infections showed a significant rise in lactate dehydrogenase (LDH) enzyme in fecal samples compared with B. hominissolely.

Conclusion: there is an association between Blastocystis hominis infections and carbohydrate intolerance and fecal acidity. B. hominis was observed to be closely related to IBS-like manifestations with the dominatingly isolated genotype-3. Also, B. hominis appeared to have an enhancing effect on the growth of E. coli and C. non-albicans. Blastocystosis seemed to heve damaging effects on the cells of the intestinal brush border especially when co-existing with Giardia sp. thus increasing levels of LDH.

Blastocystis hominis

PCR

pH

reducing sugar

lactate dehydrogenase

E. coli

Blastocystis hominis is a single-celled anaerobic intestinal parasite that had been detected in the small and large intestines of humans [1, 2]. Recently, blastocystosis is related to multiple gastrointestinal and extra-intestinal disorders [3]. Abdominal pain, diarrhoea, nausea, anorexia, abdominal distention, gas production, lactose intolerance, constipation, and loss of weight are all symptoms of B. hominis infection [4] with the noteworthy correlation between the parasite and irritable bowel syndrome [5, 6], ulcerative colitis [7], terminal ileitis [8], Blastocystis -associated enteritis [9].

Accordingly, what are the possible interactions that occur between the parasite and its microenvironment to produce these clinical manifestations? The virulence factors, pathogenicity, and other potential contributing factors to the presentation of the illness remain unknown [4, 10].

The apical plasma membrane of the intestine in mammalians is comprised of abundant microvilli that encounter the brush border enzymes responsible for the terminal stages of digestion and breaking down of the carbohydrates and protein confronting the enterocytes of the small intestine [11]. Since early infancy, β-glucosidases enzymes catalyze the assimilation of lactose present in milk and dairy products into glucose and galactose. Disruption of the intestinal brush border and lactase enzyme leads to lactose intolerance or mal digestion of lactose leading to its fermentation by the enteric microbiota resulting in gas production and abdominal bloating [12].

Several studies have reported the relationship between parasitic infections and lactose intolerance: for instance, Giardia lamblia infections [13]. Additionally, rotavirus damages the epithelial lining of the enteric brush border hence the absorptive surface becomes reduced and numerous digestive enzymes are altered and disrupted. In this accordance, this viral infection is associated with osmotic diarrhoea that occurs predominantly due to carbohydrate malabsorption [14].

In 2002, Dagci et al [15] deduced that Blastocystis hominis can produce damage in the enteric mucosa. In 2008, Hussein et al. [16] determined the deep effect of the genetic subtype in the virulence of Blastocystis species. Studies on the SSU rRNA gene showed that Blastocystis species are 17 genotypes [17, 18], with distinctive host specificity and different clinical manifestations ranging from acute gastroenteritis, chronic gastrointestinal manifestation with a duration exceeds two weeks, or asymptomatic carrier [19]. However, in a prior study IBS was demonstrated to be associated with similar gastrointestinal manifestations for instance carbohydrate intolerance in 21% of the patients besides chronic non-specific diarrhea and bloating were recorded in 15% and 50% of the cases respectively [20].

Several studies reported the inhibitory effect exerted by Blastocystis on beneficial bacteria, for instance, Bifidobacterium and Lactobacillus [21]. On the other hand, some authors deduced that Blastocystis is asymptomatic in the major sector of the colonized subjects and that it triggers the presence of higher diversity of enteric commensals and healthy microbiota [22]. These differences appeared to be related to Blastocystis subtypes [23]. Blastocystis-positive individuals have been recognized to harbor lower abundances of Faecalibacterium prausnitzii compared with healthy individuals, signifying the role of Blastocystis in the alteration of the physiological balance of the enteric microbiota causing dysbiosis. Controversy, Blastocystis (ST1 and ST3) had been manipulated in fecal microbiota transplantation therapy in patients with persistent Clostridium diffcile infection to recur the normal balance of the gut microbiota [24]. Moreover, dysbiosis towards fungal growth had been associated with manifestations like irritable bowel manifestation [25] simulating symptoms of blastocystosis [6].

Another point, the impact of blastocystosis on LDH enzyme is still not recognized. LDH is a stable enzyme present in the cytoplasm of all cells that increases rapidly coincidently with the damage of the plasma membrane [26].

We aimed in the current work to inquire about the biochemical, microbial, and enzymatic interactions of Blastocystis parasites that contribute to its clinical vignette.

This cross-sectional study was conducted on 167 gastrointestinal symptomatic patients attending internal medicine out-patient clinics of Kafrelsheik university hospital after ethical approval by Faculty of Medicine ethical approval committee numbered MKSU 50-11-23. Inclusion criteria were the presence of gastrointestinal manifestations in the associated clinical sheet, patients didn’t receive any anti-parasitic treatment in the last 12 months and consented to grant the team of the study with fecal samples on three alternate days. Patients were classified into age groups according to Salehi et al. [27].

All collected samples were subjected to the following steps.

Physical and microscopic examination [28]:

The collected stool samples were subjected to macroscopic examination to check for the smell and consistency and detect the presence of mucus, blood, or worms.

A direct wet smear (unstained and iodine stained) was performed to screen for Blastocystis sp. and to exclude the presence of other parasites. The intensity of infection in the form of the number of parasites per high-power field (400 ×) was determined [29]. Only high intensity samples were included.

Chemical examination:

Reducing sugar in stool:

Approximately one volume of the fresh fecal sample (within 2-4 hours): two volumes of water were placed into a clean test tube. 2 ml (15 drops) of Benedict’s reagent (CuSO4) was transferred in the test tube. The mixture was then heated using Bunsen burner for 3-5 minutes and observed for alteration in color or precipitate formation. Result was interpreted where green color with no sediment is corresponding to (+) or 0.05-0.15 percent sugar in the solution; green color with sediment denotes (++) or 0.2-0.4, olive-yellow color means (+++) or 0.5 to 0.75 percent sugar; orange color reflects (+++) or 1 to 1.65 percent sugar, while red color is equivalent to (+++++) or ≥ 2.0 percent sugar (Fig. 1). No change in color reflected the absence of sugar in the fecal sample (Tolipova et al. 2018) [30].

Measurement of pH:

The commercial urine test strips (Medi-Test combi 10^RSGL, LOT no. 67111) were utilized to measure pH in the fresh fecal samples. Readings were taken after 30 secs and compared with the color scale supplied with the kit. The test strips contain methyl red (3µg) and bromothymol blue (10µg) which are reacting substances that change in color between pH 5 and pH 9 (grading from orange to green to turquoise) (Fig. 1).

Genotyping of Blastocystis Parasites

Thirty-three stool samples that were confirmed microscopically for isolated blastocystosis were subjected to Genomic DNA extraction using QIAamp® DNA Mini Kit. The extracted DNA was amplified by PCR targeting specific SSU rDNA using the primers RD5 (5'- ATCTGGTTGATCCTGCCAGT-3`) and BhRDr (5'- GAGCTTTTTAACTGCAACAACG-3') to amplify the ~600 bp fragment for confirmation of the presence of Blastocystis parasites [31,32]. The PCR reaction mixture was prepared in 25 µl; 12.5 µl master mix, 1 µl of each primer, 0.1 µl Taq polymerase, 7.4 µl distilled water and 3 µl of the extracted DNA. The cycling conditions were as follows: an initial denaturation for 4 minutes at 94°C followed by 35 cycles of denaturation for 60 sec. at 94°C, annealing for 60 sec. at 55°C and extension for 80 sec. at 72°C, finally final extension for 5 minutes at 72°C. The amplified products were visualized with 1.5% agarose gel electrophoresis after ethidium bromide staining.

To detect subtype of Blastocystis hominis, a PCR reaction was performed using 7 sets of standardized subtype-specific Sequence-Tagged-Site (STS) primers [33]. Each set was used in a separate reaction (Table 1). The PCR reaction mixture was prepared in 25 µl; 12.5 µl master mix, 1 µl of each primer, 0.1 µl Taq polymerase, 8.4 µl distilled water and 2 µl of the extracted DNA. The cycling conditions were as follows: an initial denaturation for 4 minutes at 94°C followed by 40 cycles of denaturation for 30 sec at 94°C, annealing for 30 sec at 58°C and extension for 60 sec at 72°C, finally final extension for 5 minutes at 72°C.

Evaluation of Blastocystis (ST3) positive fecal samples for bacteria and fungi.

To evaluate the predominant microorganisms that overgrown with blastocystosis infection (ST3), stool specimens were cultured in MacConkey agar, Selenite broth, Salmonella -Shigella agar (BBL) for Enterobacteriaceae and Sabouraud’s for fungi. In the case of weighty colony formation, supplementary biochemical reactions and serological tests were implemented.

Assessment of the effect of Enterobacteriaceae (E. coli) and fungi (C. non-albicans) on the growth of Blastocystis (ST3).

Bacteria preparation. To investigate the impact of Blastocystis on the normal microbiota, isolates of E. coli were collected [34] by centrifugation (5525×g, 15 min) from tryptone soya broth after being incubated for 48 hrs. Sediments were washed 3 times with sterile phosphate-buffered saline (PBS) at pH 7. The pellet was suspended in a sterile Jones’ medium [35]. The optical density (O.D. 620) of the E. coli suspensions was accustomed to 1.5±0.04 in Jones’ medium and the aliquots of the E. coli were diluted to 1:10⁴ with PBS. Fifty µL from the dilution was then spread on Tryptone Soy Agar (TSA) plates. Plates were incubated at 37 °C for 48 to 72 hrs and colonies were counted. The concentrations of bacterial suspensions were finally adjusted at 1× 10⁹CFU/ml.
Fungi preparation. To evaluate whether the incidence of gut fungi can affect Blastocystis counts in vitro, Blastocystis (ST3) was individually co-cultured with representative Candida non-albicans (C. non-albicans) isolated from previously cultured gut microbiota. The fungi were gathered from Sabouraud’s broth (pH 5.8) after incubation for 6 days at 24.5 °C. Sabouraud’s broth comprises glucose (20 g/L) and mycological peptone (10 g/L). Isolates of C. non-albicans were centrifuged at 23 × 10²× g × 10 min. Sediments were washed 3 times in PBS. Fungi cells were counted by a hemocytometer and accustomed to 1× 10⁹ CFU/ml [36].
Blastocystis (ST3) was separately co-cultivated with E. coli and C. non-albicans in vitro. Blastocystis (ST3), E. coli, and C. non-albicans were washed 3 times with pre-reduced sterile (PBS). The reduced PBS for co-culture guaranteed the presence of a low oxygen environment essential for the viability of Blastocystis cells rather than a simple PBS formulation that might result in bacterial overgrowth [21].

Next, 1× 10⁹ CFU/ml of each of E. coli and C. non-albicans was co-cultured to 1× 10⁷ cells/ml of Blastocystis in one ml of PBS. Controls comprised merely organisms of Blastocystis (1× 10⁷cells/ml), E. coli (1× 10⁹ CFU/ml), and C. non-albicans (1× 10⁹ CFU/ml) suspended in 1 ml of PBS. After incubation for 24, 48, and 72 hrs. at 37 °C, cells of Blastocystis (ST3) were counted using a hemocytometer. All experimented co-cultures were repetitively performed at least four times.

d. Enzymatic Assessment of fecal LDH (Kinetic UV method)

To evaluate damaging influence of the parasite on intestinal brush border, lactate dehydrogenase enzyme was measured in Blastocystis (ST3) sole infection (14 cases) and Blastocystis (ST3) with Giardia sp. co-infection (14 cases). LDH is determined by measuring the decrease in absorbance at 340 nm. Test composite. Reagent 1 (R1 Buffer): Phosphate buffer (pH 7.5) 50 mmol/L Pyruvate 3.0 mmol/L Sodium Azide 8.0 mmol/L. Reagent 2 (R2 Coenzyme): NADH > 0.18 mmol/L Sodium azide. Fecal specimens were diluted and the ratio of Direction Decrease Sample: Reagent Ratio was 1: 50 (at 37 C^o). Read time was 1- 3 minutes after Zero adjustment against air reagent blank. Read initial absorbance was after 30 seconds. The mean absorbance change/minute (DA/min) was recorded. Reference range was 1.00 - 2.5 AU. [37].

STATISTICAL METHODS:

Data were coded and entered using the statistical package for the Social Sciences (SPSS) version 28 (IBM Corp., Armonk, NY, USA). Data was illustrated using frequencies (number of cases) and relative frequencies (percentages). For comparing categorical data, Chi square (c2) test was performed. Exact test was consumed instead when the expected frequency is less than 5 (Chan, 2003). P-values less than 0.05 were classified as statistically significant.

Clinical, seasonal data, and stool chemical examination results:

From 167 stool samples, Blastocystis sp. was detected microscopically in 33 (19.7%) samples (Fig. 2A). Only 27 (16.1%) out of them were proved to be positive by PCR (Fig. 2).

Among the recorded positive Blastocystis samples, the highest prevalence was among ag group (20-29) years old (9 samples = 21.4%) followed by the age below 20 years old (8 samples = 19.5%) and blastocystosis infection was neither detected in age groups 50-59 years old nor above 60 years old with no statistical significance difference between blastocystosis and age groups (p value = 0.207). Blastocystis infection was more prevalent in females as it was detected in 16 samples (20.3%) while in males it was detected in 11 samples (12.5%) with no statistical significance difference (p value =0.174) (Table 2).

All patients were symptomatized with gastrointestinal manifestations such as diarrhea, abdominal pain, flatulence and vomiting with no significant association observed (P value= 0.051, Chi-square= 0.021; P value= 0.357, Chi-square= 0.169; P value= 0.724, Chi-square= 0.176 and P value= 0.535, Chi-square= 0.175 respectively) (Table 3).

On tracking seasonal variability of Blastocystis infection, 77.8% (21/27) of molecularly detected Blastocystis was in the summer and spring and a significant relationship was observed between the season and blastocystosis (P value= 0.021, Chi-square= 0.001) (Table 4).

On examining stool-reducing sugars, all Blastocystis-positive stool samples ((Fig. 3A) were positive for reducing sugar (Fig. 3B, C) with statistically significant differences if compared with samples without parasitic infection (P value= > 0.001, Chi-square= 0.000). +1 sugar was detected in 80% of cases whereas the remaining 20% were +2 sugar. Similarly, they were all acidic by measuring stool samples’ pH (P value= > 0.001, Chi-square= 0.000) with an average value of 5.5.

Results of growth of E. coli in stool samples positive for Blastocystis (ST3).

In general, Blastocystis (ST3) positive samples showed an associated increase in the values of the enteric microbiota colony-forming unit per milliliter, predominantly for E. coli, compared with Blastocystis-negative controls. No other pathogenic bacteria species were detected. Illustrative images of the E. coli colonies on MacConkey agar plates and positive tests for biochemical reactions are shown (Fig. 4A&B).

Impact of E. coli and fungi (C. non-albicans) when co-cultivated with Blastocystis (ST3) in vitro.

When E. coli was co-cultivated with Blastocystis (ST3), the parasite cells displayed inhibited growth, whereas bacteria attained high counts (P value < 0.03). However, C. non-albicans showed profounder suppression of Blastocystis (ST3) cell counts and increased growth of fungi compared to E. coli (Fig. 4C) (P-value < 0.001).

Impact of Blastocystis (ST3) on LDH.

Overall, mean values of LDH in Blastocystis (ST3) sole infection and Blastocystis (ST3) - Giardia species co-infections were higher when compared with healthy controls (P value= 0.03). Yet, Blastocystis (ST3)-Giardia sp. co-infections exerted higher LDH values (P value= 0.02).

Blastocystis is the most encountered protozoa in the stool of humans and many animals worldwide with no proved data on its pathogenic role in humans yet [38]. In our study, B. hominis was detected in 19.7% and 16.1% by microscopic examination and PCR respectively. This percentage was in accordance with other study in Beni-Suef University Hospital, Egypt

[39] in which the authors documented a prevalence of 19.1% but differs from other studies in Egypt representing higher prevalence rates of 41.7%, 35.7%, 39% by [38, 40, 41] respectively. On the other hand, lower prevalence rate (8.1%) was reported by Salehi et al. [27] in Iran. This difference may be due to different geographical distribution of the study population and may be also due to the use of different diagnostic techniques.

The prevalence of Blastocystis homonis was higher in the age group 20–29 years old and > 20 years old which nearly similar to that reported by Hegazy et al. [38] who stated higher prevalence in the age group 14–35 years old and likewise, El Safadi et al. [42] reported that patients aged 0–14 years were the most affected age group (26.3%) followed by patients aged 15–49 years (22.2%) and patients aged < 50 years were the least affected group (13.6%), but in contrast to our finding, Salehi et al. [27] stated that it was more prevalent in the age group 30–39 years old with no statistical significance.

Analyzing prevalence of this parasite in both genders, it was 20.3% in women and 12.5% in men, with no significant statistical relation between gender and Blastocystis infection. This agrees with other studies that have reported no significant relationship between gender and blastocystosis [38, 43, 44].

Age and gender variances in Blastocystosis rates may be attributed to the accompanying risk factors and environmental circumstances more willingly than individual’s physiological properties [45].

Regarding clinical presentations of the study population, they were variable including diarrhea, abdominal pain, flatulence, and vomiting with no observed significance differences. Likewise, no significance differences were found among various clinical symptoms in blastocystosis infections in a study conducted in Australia [46].

Like many other studies, there is seasonal variability observed in the prevalence of Blastocystis which was higher in the spring and summer compared to other seasons with a significant statistical relationship (P value = 0.021) between its prevalence and the season (27,42,47,48).

All our patients with blastocystosis had chemically confirmed sugar malabsorption, acidic fecal pH and classical semi formed stools. In 2021, Basuony et al. [49] disclosed the hidden relationship between B. hominis and lactose intolerance. A previous study conducted by Ba´lint et al. [50] reported the existence of B. hominis infections in association with lactose intolerance in six percent of their cases where the manifestations of irritable bowel syndrome was recorded in 12%. Interestingly, lactose-free diet was reported to reduce symptoms and parasite numbers in patients with B. hominis infection [51]. Blastocystosis appeared to reduce the activity of lactase enzyme as a part of its immunological paradigm through the induction of TNF-α in the superficial epithelial cells of the lamina propria. Simultaneously, this was associated with increased apoptosis in the enteric epithelial cells and elevated BAX/BLC2 ratio [49]. Blastocystis parasites had been suggested to stimulate T cells, monocytes, macrophages, and natural killer cells through the upregulation of TNF-α, IFN-γ, and IL-12 that might be related to the absorption of Blastocystis-derived antigens via paracellular and transcellular pathways [20, 52]. Mirza et al. [53] reported that both parasites and their lysates can damage the intestinal epithelial cells and degrade the tight junction proteins in the form of occludin and ZO1, thus the intestinal permeability become increased. Furthermore, Blastocystis ratti was found to rearrange F-actin protein, reduce the transepithelial resistance, increase the epithelial permeability in addition to triggering apoptosis in the intestinal epithelial cells [54]. Parker et al. [55] demonstrated increased turnover of the epithelial cells coating the intestinal villi with irreversible reduction in the length of the enteric villi as pathological traits in blastocystosis infection.

In the current study, the predominant frequencies belonged to genotype-3. Interestingly, like our results, a study conducted in Makkah, Saudi Arabia identified the high evidence of genotype 3 followed by genotype1 and genotype 2. All three genotypes were associated with clinical symptoms [56]. Also, in Iran a recent study demonstrated the increased frequencies of genotype 3 (%56.06). However, they identified the existence of mixed infection of genotypes 3 with 4 in %42.88 of their cases [57]. In contrast to our results, a recent study predominantly defined genotype-1 in 87 (65%) followed by type-3 in 49 (37%). The same authors reported IBS in association with genotype 1 in 75 (86%) while in genotype 3 clinical manifestations were present in 23 (47%) [20].

In 2021, a study conducted in northern Egypt demonstrated the close phylogenetic correlation between human and animal in the isolates of genotype-3 hypothesizing the zoonotic transmission of the parasite and thus its epidemiological existence [41]. In 2019, El Saftawy et al. [29] proposed that the virulence of genotype-3 to stand beyond the increased intensity of Blastocystis infections and the associated clinical manifestations.

In the clinical vignette, acute diarrheal disease is chiefly involved among patients with habitually a self-limiting course [58]. However, recent studies have questioned the value of B. hominis in the results of the fecal examination in individuals with diarrheal sickness. In the current study, E. coli yielded positive overgrowth in samples infected with B. hominis (ST3); and interestingly in vitro co-culture showed the inhibitory effect of E. coli on Blastocystis proliferation. Thus, stool cultures in patients infected with B. hominis in many instances might not reveal the presence of pathogenic bacteria while exerting dysbiosis. A finding that can still be significant in some clinical conditions, particularly in immunocompromised subjects [59]. These findings may signify an extra non-beneficial effect of using empirical antibiotics prior to obtaining culture causing additional grief dysbiosis in the gut microbiota in patients infected with blastocystosis.

The current results might be attributable to a competitive relationship between E. coli and Blastocystis on the metabolism of the lactic acid [49]. E. coli are gram negative, facultative anaerobe and lactose fermenting bacteria that produce hydrogen sulfide [60]. Park et al. [61] reported that E. coli synthesizes lactose operon for lactose transportation and α -1,2-Fucosyltransferase for lactose solubility. Another explanation is that E. coli yields the production of endotoxins, for example, lipopolysaccharides which could be phagocytosed by the parasite causing its destruction. Hence, it is worth demonstrating the impact of gut commensals on the proliferation of the parasite [62]. In contrast to our results, Lepczyńska and Dzika [36] demonstrated that the counts of Blastocystis cells significantly increased starting from day 2 of co-incubation after the addition of E. coli in vitro. Also, Ganas et al. [62] reported the beneficial effect of E. coli on the growth of Histomonas meleagridis parasites.

There is scarce research concerning the Candida-protozoa interactions, whereas the main bulk of research demonstrated the interactions between the enteric bacteria and fungi [63]. Our study manipulated the interface between Blastocystis cells (ST3) and Candida non-albicans in vitro to evaluate the susceptibility to blastocytosis in patients colonized by C. non-albicans as one of the natural enteric microbiota. In the current study, C. non-albicans prohibited the growth of Blastocystis cells profoundly. This result might be attributable to the competition between the Blastocystis cells and Candida for nutrition and the colonized enteric space [64]. Regarding the triggered acidity of fecal specimens infected with Blastocystis (ST3), Sherrington et al. [65] demonstrated the high adaptability of Candida to any possible alterations in the enteric pH. Lepczyńska and Dzika [36] assumed that Candida inhibits the growth of pathogenic protozoa but to a small degree and that the toxins produced by Candida do not affect the proliferation of the protozoa despite being destructive to the bacteria and intestinal brush border.

In the current study, Blastocystis exhibited an overall increase in the LDH levels either solely or when co-existing with Giardia species infection. Also, Blastocystis-Giardia coinfections revealed the highest values. Kumar et al. [26] determined that LDH increases with different forms of cellular damage involving apoptosis and necrosis. Basuony et al. [49] reported that blastocystosis trigger apoptosis via TNF-α and increasing apoptotic biomarkers in the intestinal brush borders. Also, Giardia duodenalis was reported to trigger cellular apoptosis through the production of reactive oxygen species, mitochondria-mediated pathway, and caspases [66]. Therefore, there were elevated levels of LDH in blastocystosis that was augmented by Giardia species infection.

To summarize, our data underlined the disregarded association between Blastocystis hominis infections and carbohydrate intolerance and fecal acidity. B. hominis was observed to be closely related to IBS-like manifestations with the dominatingly isolated genotype-3. However, this effect is always overlooked by the apparent association between the carbohydrate dietary component and upsurge of the gastrointestinal manifestations without complete recognition for the associative pathogenic pattern of the parasite. B. hominis appeared to have an enhancing impact on the growth of E. coli and C. non-albicans; yet the same pathogens do not exert the same proliferative effect on B. hominis. Blastocystosis appeared to exert damaging effects on the cells of the intestinal brush border especially when co-existing with Giardia sp. thus increasing levels of LDH.

Ethics approval and consent to participate.

This study was approved by ethical approval committee of Faculty of Medicine with reference numbered MKSU 50-11-23.

Consent for publication

Not applicable

Availability of data and materials

Available

Competing interests

Authors declare that there is no conflict of interest.

Funding

Not applicable

Authors’ contributions

All authors contribute to the practical work of the research and writing of manuscript.

Acknowledgements

Not applicable

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Table 1: sequence and expected PCR product size of standardized subtype-specific Sequence –Tagged-Site (STS) primers.

Sub-type	STS Primers		Sequence	Expected product size(bp)
1	SB83	F	5'- GAAGGACTCTCTGACGZTGA-3`	351bp
		R	5'-GTCCAAATGAAAGGCAGC-3`
2	SB155	F	5'-ATCAGCCTACAATCTCCTC-3`	650bp
		R	5'-ATCGCCACTTCTCCAAT-3`
3	SB227	F	5'-TAGGATTTGGTGTTTGGAGA-3`	526bp
		R	5'-TTAGAAGTGAAGGAGATGGAAG-3`
4	SB332	F	5'-GCATCCAGACTACTATCAACATT-3`	338bp
		R	5'-CCATTTTCAGACAACCACTTA-3`
5	SB340	F	5'-TGTTCTTGTGTCTTCTCAGCTC-3`	704bp
		R	5'-TTCTTTCACACTCCCGTCAT-3`
6	SB336	F	5'-GTGGGTAGAGGAAGGAAAACA-3`	317bp
		R	5'-AGAACAAGTCGATGAAGTGAGAT-3`
7	SB337	F	5'-GTCTTTCCCTGTCTATTCTGCA-3`	487bp
		R	5'-AATTCGGTCTGCTTCTTCTG-3`

Table 2: The rate of Blastosystis in different age groups and in both sexes and its statistical significance

		PCR				P value	Chi-square
		+ve (n= 27)		-ve (n= 140)
		Count	%	Count	%
Age	< 20	8	19.5%	33	80.5%	0.207	0.017
	20-29	9	21.4%	33	78.6%
	30-39	4	10.0%	36	90.0%
	40-49	6	25.0%	18	75.0%
	50-59	0	0.0%	11	100.0%
	>60	0	0.0%	9	100.0%
Gender	Male	11	12.5%	77	87.5%	0.174	0.067
Gender	female	16	20.3%	63	79.7%	0.174	0.067

Table 3: The percentage of clinical data and its statistical significance

		PCR				P value	Chi-square
		+ve (n= 27)		-ve (n= 140)
		Count	%	Count	%
Diarrhea	Yes	26	19.0%	111	81.0%	0.051	0.021
Diarrhea	No	1	3.3%	29	96.7%	0.051	0.021
Abdominal pain	Yes	27	17.1%	131	82.9%	0.357	0.169
Abdominal pain	No	0	0.0%	9	100.0%	0.357	0.169
Flatulence	Yes	20	16.8%	99	83.2%	0.724	0.176
Flatulence	No	7	14.6%	41	85.4%	0.724	0.176
Vomiting	Yes	2	9.1%	20	90.9%	0.535	0.175
Vomiting	No	25	17.2%	120	82.8%	0.535	0.175

Table 4: The seasonal variability of Blastocystis infection and its statistical significance

		PCR				P value	Chi-square
		+ve (n=27)		-ve (n=140)
		Count	%	Count	%
Season	Spring	11	30.6%	25	69.4%	0.021	0.001
	Summer	10	19.2%	42	80.8%
	Autumn	4	8.2%	45	91.8%
	Winter	2	6.7%	28	93.3%

Biochemical and Microbiological Interactions of Molecularly Detected Blastocystis hominis: a cross-sectional study.

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