Unveiling the role of EVs in anaerobic parasitic protozoa.

The anaerobic or microaerophilic protozoan parasites such as the enteric human pathogens Entamoeba histolytica, Giardia intestinalis, Cryptosporidium parvum, Blastocystis hominis and urogenital tract parasites Trichomonas vaginalis are able to survival in an environment with oxygen deprivation. Despite living in hostile environments these pathogens adopted different strategies to survive within the hosts. Among them, the release of extracellular vesicles (EVs) has become an active endeavor in the study of pathogenesis for these parasites. EVs are heterogenous, membrane-limited structures that have played important roles in cellular communication, transferring information through cargo and modulating the immune system of the host. In this review, we described several aspects of the recently characterized EVs of the anaerobic protozoa, including their role in adhesion, modulation of the immune response and omics analysis to understand the potential of these EVs in the pathogenesis of these diseases caused by anaerobic parasites.

In vitro Effect of Silver Nanoparticles on Blastocystis hominis.

INTRODUCTION: This study aims to assess the efficacy of silver nanoparticles (Ag Nps) alone and combined with metronidazole (Ag Nps + MTZ) as potential alternative therapeutic agents for Blastocystis hominis. METHODS: The parasites were challenged with Ag Nps, Ag Nps + MTZ and MTZ. To assess the efficacy of drugs, counting of viable parasites was done after 1, 2, and 3 hours of adding the drugs. RESULTS: Blastocystis hominis count was reduced by 20.72%, 28.23%, and 18.92% after one hour of adding Ag Nps, Ag Nps + MTZ, and MTZ, respectively. Cysts count was further reduced by 51.49%, 61.61%, and 40.78% after 2 hours and by 71.69%, 79.67%, and 62.65% after 3 hours of adding the drugs in the same order, respectively. CONCLUSION: There was a statistically significant difference (P<0.05) in the in vitro growth inhibition of the parasite over the different time intervals when using the tested drugs against the control drug.

Characteristics of Factors of Protozoa Blastocystis hominis Persistence.

Persistence activity manifested in the expression of anti-lysozyme, anti-lactoferrin, and antihistone factors promoting inactivation of natural anti-infection resistance factors in the body was revealed in Blastocystis hominis protozoa. Activities of these factors were ranged. The frequency of these factors in clinical isolates of blastocyst decreased in the following order: anti-lactoferrin activity (84.5±3.7%)→anti-lysozyme activity (64.8±5.7%)→anti-histone activity (48.1±2.3%). In healthy humans, the corresponding parameters were 7.3±1.3, 5.3±0.9, and 3.3±0.4%, respectively (p<0.05). It was shown that the studied activities in highly virulent blastocysts were higher than in groups of medium-, low-, and avirulent protozoa.

[Observation on growth status of Blastocystis hominis in three media].

OBJECTIVE: To observe the growth situation of Blastocystis hominis in vitro and select the optimal method for cultivation of B. hominis in different media. METHODS: Ten positive stools with B. hominis were inoculated in three different media for cultivating, namely 1640, Jone's medium and vitro medium. And the stools with good growth status and high quantities of B. hominis were chosen to inoculate in the three media with equal amount after subcultivation, and the number of B. hominis was counted every 24 h for ten days, and the morphological changes and growth status were also observed. RESULTS: The densities of B. hominis in the 1640 and Jone's medium were higher than that in the vitro medium 48 h after the inoculation. The same stool sample was inoculated to the three different media and observed for ten days, and the results indicated that the growth of B. hominis presented regular changes in the three media, the growth peaks were on the third, sixth and ninth day post inoculation; and the density of B. hominis was the highest in the Jone's medium. The morphology of B. hominis was the clearest and most dynamic in the vitro medium, while various reproductive forms were observed in the Jone's medium. CONCLUSION: Jone's medium is suitable for the growth of B. hominis and can be the first choice for the cultivation of B. hominis in vitro, and vitro medium is the best medium for observing the growth situation of B. hominis.

In vitro culture of blastocystis hominis in three liquid media and its usefulness in the diagnosis of blastocystosis.

BACKGROUND: Blastocystis hominis is a common protozoan in the human intestinal tract and can cause the so-called blastocystosis characterized by diarrhea. To date, its identification has depended on the discovery of vacuolar, granular, amoebic, or cystic forms in stool samples using wet mount smears, iodine staining, trichrome staining, or iron hematoxylin staining. The permanent staining methods provide more positive findings. However, mercuric chloride (HgCl(2))-based polyvinyl alcohol (PVA) and Schaudinn fixative are potentially toxic and dangerous to laboratory personnel and, as hazardous chemicals, present problems with disposal. METHODS: To determine whether in vitro culture could be an environmentally safe alternative, the culture growth of B. hominis in three commercially available liquid media (RPMI 1640, 199 Medium, and Dulbecco's modified Eagle's medium (DMEM)) were observed and compared. The sensitivity and specificity of these culture methods to identify B. hominis were compared with those of existing methods used clinically. RESULTS: Conditions for the anaerobic culture of B. hominis in these media were determined as follows: total inoculum sizes no less than 10(5) cells; pH values ranging from 7.0 to 7.5; concentrations of calf or horse serum ranging from 10% to 30% (vol/vol); basic antibiotics added; peaking times at days 3, 6, and 9 (pH 7.5) or days 4 and 8 (or 9) (pH 7.0) at 37°C. No significant differences were noted in multiplication or generation times for the cultivation of B. hominis (p>0.05). In 56 of 398 positive cases, the short-term in vitro culture method achieved the best performance with regard to sensitivity and specificity of the five studied methods. CONCLUSIONS: With the advantages of environmental safety, convenience in preparation and storage, facility in morphologic discrimination, and outstanding performance in terms of sensitivity and specificity, the in vitro culture method could be applied to identify B. hominis for both clinical diagnosis and field study purposes.

In vitro sensitivity of Blastocystis hominis to garlic, ginger, white cumin, and black pepper used in diet.

To determine the growth pattern and in vitro susceptibility of Blastocystis hominis to metronidazole (MTZ), garlic, ginger, white cumin, and black pepper. Stool specimens were collected from 16 irritable bowel syndrome (IBS) and 10 controls between July-November 2010. Stool microscopy and culture for B. hominis was performed. Drug susceptibility assays was done using 0.01 and 0.1 mg/ml of MTZ, garlic, ginger, white cumin, and black pepper. Effect was assessed on B. hominis culture after 48 h. Stool DNA was extracted using stool DNA extraction kit (Qiagen) and polymerase chain reaction (PCR) done using subtype-specific sequence-tagged-site primers. B. hominis genotype 3 and coinfection of 1 and 3 tended to grow well in culture compared to isolated type 1 infection. Exposed to MTZ at a concentration of 0.01 mg/ml, 38% (6/16) B. hominis from IBS did not grow in culture compared to 100% (10/10) of B. hominis from control (p = 0.001). When they were exposed to MTZ at 0.1 mg/ml, 56% (9/16) B. hominis from IBS did not grow in cultures compared to 100% (10/10) from control (p = 0.01). Forty-four percent (7/16) B. hominis from IBS did not grow in culture compared to 100% (10/10) B. hominis from control when exposed to garlic at a concentration of 0.01 mg/ml (p = 0.003) and following exposure to garlic at 0.1 mg/ml, 38% (6/16) B. hominis from IBS did not grow in cultures compared to 100% (10/10) from control (p = 0.001). B. hominis isolates from IBS had a cell count of 6,625 at a MTZ concentration of 0.01 mg/ml that reduced to 1,250 as MTZ concentration was increased to 0.1 mg/ml (p = 0.08). B. hominis from IBS with a mean cell count of 3 × 10(5) at baseline decreased to 1 × 10(4) when exposed to garlic at 0.01 mg/ml (p < 0.001) and to 1 × 10(3) (p < 0.001) when garlic was 0.1 mg/ml. B. hominis from IBS cell count decreased to 1 × 10(5) when exposed to white cumin at 0.01 mg/ml (p = 0.01) and to 1 × 10(5) (p < 0.001) when white cumin was 0.1 mg/ml. Exposed to black pepper at 0.1 mg/ml, cell count of B. hominis from IBS decreased to 1 × 10(5) (p = 0.01). B. hominis from IBS decreased to 1.3 × 10(5) exposed to ginger at 0.01 mg/ml (p = 0.001). B. hominis isolates were mostly genotypes 3, type 1 and 3 coinfection, and non-typeable B. hominis isolates. B. hominis isolates from IBS mostly genotype 1 demonstrated an increased sensitivity to garlic at 0.01 mg/ml with a B. hominis cell count of 3,714 compared to 6,142 when exposed to 0.01 mg/ml of MTZ. However, this sensitivity did not increase as garlic concentration was increased to 0.1 mg/ml, for B. hominis cell count was 6,000 compared to 1,428 as MTZ was increased to 0.1 mg/ml.

Study of the dynamics of Blastocystis hominis reproduction in vitro.

The dynamics of Blastocystis hominis reproduction in vitro in Pavlova's and Nelson-Jones media was studied. The time of generation in these media was 21.5 and 16.7 h, respectively. The duration of the lag phase was 1 day, of log phase 2 days, and of the stationary phase 6 days in both cases. The cell count in the logarithmic phase increased at the expense of the vacuolar forms proliferation. During the stationary phase, the granular forms quantitatively predominated over vacuolar forms, the share of the granular forms reaching almost 100% at late stages of the subculture development.

In vivo encystation of Blastocystis hominis.

Blastocystis from infected stools of a person who showed chronic symptoms of abdominal discomfort and diarrhea were examined over a 6-month period, using transmission electron microscopy, for the ultrastructural changes from vacuolar to cystic stage. The study confirms the irregular shedding phenomenon of the organism previously reported, and for the first time, records sequential changes in encystation in stools collected over a time period. The study also confirms the existence of a precystic stage which has an immature cell wall consisting of a layer of a homogenous electron-dense mass surrounding the cell which acts as a intermediatory stage between the vacuolar and cystic stage.

Evaluation of the in vitro effect of Nigella sativa aqueous extract on Blastocystis hominis isolates.

The effect of Nigella sativa aqueous extract was evaluated against the in vitro growth of 2 different isolates of the intestinal protozoan parasite Blastocystis hominis. Different concentrations (10, 100, 500 microg/ml) of Nigella aqueous extract and metronidazole, an active standard drug for B. hominis, were incubated with B. hominis isolates in culture media at 37 degrees C. Their possible effect on B. hominnis living cell count (LCC) was assessed on Day 1, 3 & 6. The aqueous extract of N. sativa at concentrations of 100 and 500 microg/ml showed a potent lethal effect on both B. homninis isolates, but with different extent. There is no significant difference between the inhibitory effect of N. sativa and metronidazole on the LCC on the 6th day. On assessment of living cell rate (LCR) which calculate percentage rate of living cell, N. sativa at 500 microg/ml concentration has a significant inhibitory effect on both isolates. So, it is considered as the most active concentration of Nigella aqueous extract. These results prove that N. sativa aqueous extract could be useful in the treatment of B. hominis.

[Vitro culture of blastocystis hominis in medium DMEM].

OBJECTIVE: To establish the vitro culture of Blastocystis hominis (B. h) in medium DMEM for the further research on diagnosis, life cycle and pathogenicity of this intestinal protoza. METHODS: The growth, reproduction and relevant factors of B . h under different culture conditions including sorts and concentrations of serum, pHs and number of inoculation were compared. RESULTS: Conditions for the continuously anaerobic culture of B. h in medium DMEM were as follows: the number of inoculation were no less than 10(5) cells per tube, pHs ranged 7.0 - 8.0, concentrations of calf serum (or human serum and horse serum) ranged 10% - 30% , antibiotics and Amphotericin B should be added, subculturing could be choose at the each peaking-day 3,6 or 5 at 37 degrees C. CONCLUSION: The medium DMEM could be used in diagnosis and continuously vitro culture for B. h.

Degradation of human secretory immunoglobulin A by Blastocystis.

Microbial immunoglobulin A (IgA) proteases cleave human secretory IgA, promoting the mucosal adhesion of pathogens. To investigate if the enteric protozoan Blastocystis degrades human secretory IgA, cell lysate and conditioned medium from two species were exposed to immunoglobulin A. Secretory IgA was cleaved by both cell lysate and conditioned medium with mainly cysteine proteinase activity in B. hominis B isolate and aspartic proteinase activity in B. ratii WR1 isolate. These findings suggest that Blastocystis proteases may play a role in parasite survival in vivo.

Fecal-oral transmission of the cyst form of Blastocystis hominis in rats.

The infectivity of two Blastocystis hominis strains, RN94-9 and NIH:1295:1, was examined in 3-week-old SPF Wistar rats. The NIH:1295:1 strain, originally isolated from a guinea pig, was only able to infect rats via intracecal inoculation of the cultured organisms, while the RN94-9 strain, originally isolated from a laboratory rat, was able to infect rats by oral inoculation of the cultures due to the presence of a cystic form in the in vitro culture. Since many cysts were discharged in the feces of the infected rats, the infectivity of the concentrated cysts was compared between the two strains. Successful oral infection was observed in rats inoculated with 1 x 10(2)-1 x 10(6) cysts of the RN94-9 and NIH:1295:1 strains. The infectivity of the ten cysts varied in the three experiments of ten rats, being 20-100% and 30-100% in the RN94-9 and NIH:1295:1 strains, respectively. When an uninfected normal rat was housed with five experimentally inoculated rats, the normal rat became infected, demonstrating the fecal-oral transmission of the cyst form of this parasite. These results show that the Wistar rat is an ideal host for the propagation of strains RN94-9 and NIH:1295:1 of B. hominis, and demonstrate that the cyst form is the only transmissible form of this parasite.

The usefulness of short-term in vitro cultivation for the detection and molecular study of Blastocystis hominis in stool specimens.

When in vitro cultivation was used as the 'gold standard' for the detection of Blastocystis hominis in stool specimens, simple smear and trichrome staining showed sensitivities of 16.7% and 40.2% and specificities of 94% and 80.4%, respectively. In vitro cultivation also enhanced PCR amplification for the detection of B. hominis in stool specimens. Our data show the usefulness of in vitro cultivation for the detection and molecular study of B. hominis in stool specimens.

In vitro susceptibility of Blastocystis hominis isolated from patients with irritable bowel syndrome.

This study aims to determine the growth pattern and in vitro susceptibility of clinical isolates of Blastocystis hominis to different concentrations of metronidazole, furazolidone and ciprofloxacin. Stool specimens from 25 consecutive patients with irritable bowel syndrome presenting to the gastroenterology department of Aga Khan University Hospital between January and May 2003 are examined by microscopy and cultured for B. hominis. Drug susceptibility assays are performed for metronidazole, furazolidone, and ciprofloxacin using final concentrations of 0.01 mg/mL and 0.1 mg/mL. The effect of the drugs is assessed after B. hominis culture for 48 h. With furazolidone and metronidazole, 68% (17/25) and 60% (15/25) of B. hominis isolates, respectively, failed to grow at drug concentrations of both 0.01 mg/mL and 0.1 mg/mL. However, ciprofloxacin failed to suppress growth completely at both concentrations. B. hominis resistance to furazolidone, metronidazole and ciprofloxacin at 0.01 mg/mL was 32% (8/25), 40% (10/25) and 100% (25/25), respectively. B. hominis isolates varied in their degree of susceptibility to the three drugs studied, being greater with furazolidone than with metronidazole, and complete resistance with ciprofloxacin.

Comparison of methods for detecting Blastocystis hominis.

In order to determine the comparative sensitivity of two methods of detecting Blastocystis hominis and to investigate the seasonality of infection with this enteric protozoan parasite, the present study was conducted. In each of two 3-month periods representing winter, spring (February-April) and summer (July-September), 500 routine stool submissions were examined for B. hominis using microscopy following either formol-ether concentration or in vitro culture using Jones' medium. The organism was detected in 39 of the 1,000 samples investigated using the in vitro culture technique and in none of the samples using the formol-ether concentration technique. In 82% of the B. hominis-positive samples, no concurrent bacterial or parasitic pathogens were found, and diarrhoea was the most commonly recorded symptom among patients. Infection was more prevalent in summer than in winter/spring, occurring primarily in the 71-80-year age group. Cysts were detected in 20.5% of positive samples, but only following Ficoll-Paque concentration of formol-ether concentrates. Cyst excretion was more prevalent in summer than in winter/spring.

Irritable bowel syndrome: in search of an etiology: role of Blastocystis hominis.

This study was designed to examine stool specimens of irritable bowel syndrome (IBS) patients for Blastocystis hominis, a common intestinal parasite. One hundred fifty patients were enrolled, 95 IBS cases and 55 controls. These patients provided a medical history, and underwent physical and laboratory evaluations that included stool microscopy and culture for B. hominis and colonoscopy. The 95 cases (51 males and 44 females) had a mean +/- SD age of 37.8 +/- 13.2 years. Stool microscopy was positive for B. hominis in 32% (30 of 95) of the cases and 7% (4 of 55) of the controls (P = 0.001). Stool culture was positive in 46% (44 of 95) of the cases and 7% (4 of 55) of the controls (P < 0.001). Stool culture for B. hominis in IBS was more sensitive than microscopy (P < 0.001). Blastocystis hominis was frequently demonstrated in the stool samples of IBS patients; however, its significance in IBS still needs to be investigated. Stool culture has a higher positive yield for B. hominis than stool microscopy.

[Emphasizing of Blastocystis hominis on culture media]. / Evidentierea microorganismului Blastocystis hominis pe medii de cultura.

The aim of our study was to determine the frequency of Blastocystis hominis in the stool specimens sent to the Parasitological Laboratory of the Mures County Hospital between October 2003 and May 2004. The 124 inoculated specimens showed positive values in 41.93%. After an incubation in anaerobic conditions at 37 degrees C for 24, 72 and 96 hours in liquid and biphasic media, the last proved to be better (69,24%) at an incubation time of 72 hours.

In-vitro cultivation: a sensitive method for detecting Blastocystis hominis.

Currently, the detection of human infection with Blastocystis hominis is usually based on the examination under a light microscope of faecal samples, either directly, as 'simple smears', or after some form of concentration. Whether short-term, in-vitro cultivation would increase the sensitivity of such detection remains a matter of controversy. Over 900 fresh stool specimens, from soldiers in the Royal Thai Army, were each checked for the parasite using three methods: simple smears; formalin-ethyl-acetate concentration; and cultivation in Jones' medium. Although 334 of the samples were found to be culture-positive, the parasites were only detected in 142 of the simple smears, and faecal concentration led to an even lower sensitivity (64 positive samples). In-vitro cultivation does seem worthwhile in the detection of B. hominis carriage in field studies.

Blastocystis hominis: A simplified, high-efficiency method for clonal growth on solid agar.

Colony growth of protozoan parasites in agar can be useful for axenization, cloning, and viability studies. This is usually achieved with the pour plate method, for which the parasite colonies are situated within the agar. This technique has been described for Giardia intestinalis, Trichomonas vaginalis, and Entamoeba and Blastocystis species. Extracting such colonies can be laborious. It would be especially useful if parasites could be grown on agar as colonies. These colonies, being exposed on the agar surface, could be conveniently isolated for further investigation. In this study, we report the successful culture of B. hominis cells as colonies on solid agar. Colonies were enumerated and the efficiency of plating was determined. It was observed that B. hominis could be easily cultured on agar as clones. The colonies were dome-shaped and mucoid and could grow to 3 mm in diameter. Flow cytometric analyses revealed that parasite colonies remained viable for up to 2 weeks. Viable colonies were conveniently expanded in liquid or solid media. Scanning electron microscopy revealed that each colony consists of two regions; a dome-shaped, central core region and a flattened, peripheral region. Older colonies possessed numerous strand-like surface coat projections. This study provides the first report of clonal growth of B. hominis on agar and a simple, effective method for cloning and expansion of B. hominis cells.

Blastocystis hominis: neutral red supravital staining and its application to in vitro drug sensitivity testing.

A simple in vitro drug sensitivity testing system for Blastocystis hominis clinical isolates was developed. Application of supravital staining by neutral red allowed quantitative viability assessment. Four xenic cultures, isolated from human sources, were grown in modified monophasic Robinson's medium and tested for sensitivities to nine available drugs. Assessment was done using the cell-count method from air-dried preparations after supravital staining with neutral red. Also, the light absorbence method was evaluated. Trimethoprim, metronidazole, quinacrine, tetracycline, paromomycin, and two new antiprotozoal drugs, nitazoxanide and deacetyl-nitazoxanide, showed cytostatic or cytocidal effects on all or some Blastocystis isolates. Chloroquine and sulphamethoxazole did not demonstrate any effect at the concentrations studied.