Effect of Blastocystis sp. in dengue patients-Increase in the treatment cost and exacerbation of symptoms.

Increasing incidences of dengue have become a global health threat with major clinical manifestation including high fever and gastrointestinal symptoms. These symptoms were also expressed among Blastocystis sp. infected individuals, a parasite commonly seen in human stools. This parasite has been previously reported to replicate faster upon exposure to high temperature. The present study is a hospitalized-based cross-sectional study involved the collection of faecal sample from dengue patients. Stool examination was done by in vitro cultivation to isolate Blastocystis sp. Growth pattern of all the positive isolates were analyzed to identify the multiplication rate of Blastocystis sp. isolated from dengue patients. Distribution of Blastocystis sp. among dengue patients was 23.6%. Dengue patients who were positive for Blastocystis sp. infection denoted a significantly higher fever rate reaching 38.73°C (p<0.05) compared to the non-Blastocystis sp. infected patients (38.44°C). It was also found that Blastocystis sp. infected patients complained of frequenting the toilet more than five times a day (p<0.05) compared to those who were non-Blastocystis sp. infected. At the same time, the duration of hospitalization was significantly longer (p<0.05) for Blastocystis sp. infected dengue patients compared to the non-Blastocystis sp. infected patients. Besides, Blastocystis sp. isolated from dengue patients (in vivo thermal stress) showed a higher growth rate compared to the non-dengue isolated which was exposed to high temperature (in vitro thermal stress). Our findings suggest that presence of Blastocystis sp. during dengue infection could trigger the increase of temperature which could be due to highly elevated pro inflammatory cytokines by both parasitic and virus infection. This could justify why the temperature in Blastocystis sp. infected dengue patients is higher compared to the non-Blastocystis sp. infected patients. Higher temperature could have triggered a greater parasite multiplication rate that contributed to the aggravation of the gastrointestinal symptoms.

Proteogenomic Insights into the Intestinal Parasite Blastocystis sp. Subtype 4 Isolate WR1.

Blastocystis sp. is known for years as a highly prevalent anaerobic eukaryotic parasite of humans and animals. Several monophyletic clades have been delineated based on molecular data, and the occurrence of each subtype in humans and/or animal hosts has been documented. The genome of several representatives has been sequenced revealing specific traits such as an intriguing 3'-end processing of primary transcripts. Here, a first high-throughput proteomics dataset acquired on this difficult-to-cultivate parasite is presented for the zoonotic subtype T4 isolate WR1. Amongst the 2766 detected proteins, we highlighted the role of a small ADP ribosylation factor GTP-binding protein involved in intracellular traffic as major regulator of vesicle biogenesis and a voltage-dependent anion-selective channel protein because both were unexpectedly highly abundant. We show how these data may be used for gaining proteogenomics insights into Blastocystis sp. specific molecular mechanisms. We evidenced for the first time by proteogenomics a functional termination codon derived from transcript polyadenylation for seven different key cellular components.

Blastocystis Isolate B Exhibits Multiple Modes of Resistance against Antimicrobial Peptide LL-37.

Blastocystis is one of the most common eukaryotic organisms found in humans and many types of animals. Several reports have identified its role in gastrointestinal disorders, although its pathogenicity is yet to be clarified. Blastocystis is transmitted via the fecal-to-oral route and colonizes the large intestines. Epithelial cells lining the intestine secrete antimicrobial peptides (AMPs), including beta-defensins and cathelicidin, as a response to infection. This study explores the effects of host colonic antimicrobial peptides, particularly LL-37, a fragment of cathelicidin, on different Blastocystis subtypes. Blastocystis is composed of several subtypes that have genetic, metabolic, and biological differences. These subtypes also have various outcomes in terms of drug treatment and immune response. In this study, Blastocystis isolates from three different subtypes were found to induce intestinal epithelial cells to secrete LL-37. We also show that among the antimicrobial peptides tested, only LL-37 has broad activity on all the subtypes. LL-37 causes membrane disruption and causes Blastocystis to change shape. Blastocystis subtype 7 (ST7), however, showed relative resistance to LL-37. An isolate, ST7 isolate B (ST7-B), from this subtype releases proteases that can degrade the peptide. It also makes the environment acidic, which causes attenuation of LL-37 activity. The Blastocystis ST7-B isolate was also observed to have a thicker surface coat, which may protect the parasite from direct killing by LL-37. This study determined the effects of LL-37 on different Blastocystis isolates and indicates that AMPs have significant roles in Blastocystis infections.

Differential regulation of proinflammatory cytokine expression by mitogen-activated protein kinases in macrophages in response to intestinal parasite infection.

Blastocystis is a common enteric protistan parasite that can cause acute, as well as chronic, infection and is associated with irritable bowel syndrome (IBS). However, the pathogenic status of Blastocystis infection remains unclear. In this study, we found that Blastocystis antigens induced abundant expression of proinflammatory cytokines, including interleukin 1ß (IL-1ß), IL-6, and tumor necrosis factor alpha (TNF-α), in mouse intestinal explants, in mouse colitis colon, and in macrophages. Further investigation utilizing RAW264.7 murine macrophages showed that Blastocystis treatment in RAW264.7 macrophages induced the activation of ERK, JNK, and p38, the three major groups of mammalian mitogen-activated protein (MAP) kinases that play essential roles in the expression of proinflammatory cytokines. ERK inhibition in macrophages significantly suppressed both mRNA and protein expression of IL-6 and TNF-α and mRNA expression of IL-1ß. On the other hand, JNK inhibition resulted in reductions in both c-Jun and ERK activation and significant suppression of all three proinflammatory cytokines at both the mRNA and protein levels. Inhibition of p38 suppressed only IL-6 protein expression with no effect on the expression of IL-1ß and TNF-α. Furthermore, we found that serine proteases produced by Blastocystis play an important role in the induction of ERK activation and proinflammatory cytokine expression by macrophages. Our study thus demonstrated for the first time that Blastocystis could induce the expression of various proinflammatory cytokines via the activation of MAP kinases and that infection with Blastocystis may contribute to the pathogenesis of inflammatory intestinal diseases through the activation of inflammatory pathways in host immune cells, such as macrophages.

Cytokine changes in colonic mucosa associated with Blastocystis spp. subtypes 1 and 3 in diarrhoea-predominant irritable bowel syndrome.

We determined cytokines (e.g. interleukin-8, 10, 12 and TNF-α) expression by peripheral blood mononuclear cells (PBMCs) and in rectal mucosa in diarrhoea-predominant irritable bowel syndrome (D-IBS) with Blastocystis spp. Eighty patients with D-IBS and Blastocystis spp. infection were classified as 'cases' and 80 with D-IBS without Blastocystis spp. infection were classified as 'control'. Cases were subdivided into D-IBS and Blastocystis sp. defined type 1 (subtype-specific primer SB83) and type 3 (SB227). Stool microscopy and culture were performed. Rectal biopsies were obtained for histology and cytokines by real-time PCR for mRNA expression of cytokines. PBMCs IL-8 was similar in different groups but in type 1, IL-8mRNA was increased compared with type 3 (P = 0·001) and control (P = 0·001). In type 1, IL-10 by PBMCs had a low mean value (14·5±1·6) compared with (16·7±1·5) type 3 and (16±2·3) in controls (P<0·001 and P<0·001, respectively). In Blastocystis sp. type 1, low IL-10 was associated with lymphocyte and plasma cell infiltration (P = 0·015 and P = 0·002, respectively). In Blastocystis sp. type 1 and type 3, IL-12 was associated with goblet cell depletion 23 (85%) (P<0·001) and 8 (29%) (P = 0·037), respectively. In Blastocystis sp. type 1, low IL-10 was associated with a proinflammatory response characterized by IL-8.

[Expressions and significance of IL-17 and IL-23 in intestinal mucosa of mice infected with Blastocystis hominis].

OBJECTIVE: To study the expressions of interleukin-17 (IL-17) and interleukin-23 (IL-23) in the intestinal mucosa of BABL/C mice infected with Blastocystis hominis. METHODS: A total of 30 BABL/C mice were randomly divided into different groups: an experimental group, an immunosuppressant group and a normal group. Each mouse of the experimental group and immunosuppressant group was administered intraperieneally with dexamethasone (2 mg, gd, for 5 days) and one of the control group was given physiological saline (0.2 ml). In the experimental group, each mouse was infected with Blastocystis hominis (107 parasites per 0.5 ml) by the intragastric infusion method; in the immunosuppressant group and normal group, the mice were fed with equal physiological saline. On the fifth day post-infection, the duodenum, jejunum, ileum and colon of the mice of the 3 groups were taken out for the tissue section. The pathological changes of bowel mucosa were determined by HE staining, and the expressions of IL-17 and IL-23 in different parts of bowel mucosa were determined by immunohistochemistry assay. RESULTS: The pathological examinations showed intestinal mucosa had various degrees of inflammatory changes. The expressions of IL-17 and IL-23 in the intestinal mucosa of the mice in the experimental group was significantly higher than those in the immunosuppressant group or normal group (both P < 0.05). The expressions of IL-17 and IL-23 in the intestinal mucosa of the mice in the immunosuppressant group were similar to those in the normal group (P > 0.05). The expression of IL-17 in the duodenum or jejunum or colon of the mice was significantly higher than that in the ileum in the experimental group (P < 0.05). The expression of IL-23 in the duodenum or jejunum of the mice was significantly higher that that in the ileum or colon in the experimental group (P < 0.05). CONCLUSIONS: IL-17 and IL-23 are highly expressed in the intestinal mucosa of the mice infected with Blastocystis hominis. IL-23 may also be involved in the immunomodulatory effects of Blastocystis hominis infection, which plays a mutual regulatory role with IL-17.

High levels of oxidative stress in rats infected with Blastocystis hominis.

OBJECTIVE: Numerous studies have revealed the presence of oxidative stress in parasitic infections. However, such studies were lacking in the Malaysian population. Previously, we have provided evidence that oxidative stress is elevated in Malaysians infected with intestinal parasites. Stool examinations revealed that about 47.5% of them were infected with the polymorphic protozoa, Blastocystis hominis. However, they were found to have mixed infection with other intestinal parasites. METHODOLOGY: Therefore, in order to investigate the role of B. hominis alone in affecting oxidative stress status, here we compared the levels of oxidative stress biomarkers in urine and blood samples between uninfected and B. hominis-infected rats. RESULTS: Infected rats exhibited elevated levels of oxidative indices namely advanced oxidative protein products (AOPP), hydrogen peroxide (H2O2) and lipid hydroperoxide (LHP) indicating that their overall oxidative damage level was higher. Ferric reducing antioxidant power (FRAP) was elevated at the initial stage of infection but decreased significantly during the last week of study duration suggesting that the antioxidant status of the host may be overwhelmed by oxidative damage. CONCLUSION: To date, this is the first comprehensive in vivo study to provide evidence for Blastocystis infection to correlate with significant oxidative burst leading to oxidative stress.

Observations on the ultrastructure and viability of the cystic stage of Blastocystis hominis from human feces.

This report describes the ultrastructure and viability of cysts of Blastocystis hominis from feces of infected patients. The cysts were round to ovoid, measured 2-5 microns in size, and contained a condensed cytoplasm that had vacuoles of varying sizes, four nuclei, and as many as six cristate mitochondria. The cell wall was rather electron-lucent. Surprisingly, chromatoid-like structures were found in the cytoplasm and nucleus of some of the cysts. These have not previously been reported in Blastocystis. The cysts can survive in water for up to 19 days at normal temperatures but are fragile at extreme temperatures and in common disinfectants.

Blastocystis hominis infection and intestinal injury.

Blastocystis hominis is an enteric protozoan associated with clinical illness. To determine the prevalence of intestinal injury in patients with B. hominis infection, the authors prospectively evaluated 18 patients with B. hominis infection by endoscopy and a test of intestinal permeability. Seventeen patients had gastrointestinal symptoms. Colonic mucosa appeared normal by lower endoscopy in 12 of 13 patients, and was friable slightly in 1. Duodenal mucosa was normal by upper endoscopy in nine patients. Pathologic examination of mucosal biopsy specimens did not demonstrate evidence of mucosal invasion. 51Cr-edetic acid (51Cr-EDTA) was given to the 18 patients with stools positive for B. hominis and to 32 healthy control subjects. Approximately 100 uCi of 51Cr-EDTA was given orally after an overnight fast, and urine was collected for the following 24 hours. Mean 24-hour urinary excretion of 51Cr-EDTA, calculated as a percent of the administered dose, was 1.31% (0.34-2.76%) in patients with B. hominis infection and 1.99% (0.59-3.48%) in the control subjects. The intestinal permeability to 51Cr-EDTA in blastocystis-infected individuals was not increased, but was decreased significantly compared with healthy subjects (p < 0.005). Therefore, in a group of symptomatic patients with B. hominis infection, endoscopy typically did not show evidence of significant intestinal inflammation, and results of intestinal permeability testing with 51Cr-EDTA did not suggest impaired barrier function of the intestinal mucosa. The clinical literature on B. hominis infection and intestinal injury is reviewed.