The slip extrusion test: A novel method to characterise bolus properties.

The role of mastication is to prepare a bolus for safe swallowing. The Swallow Safe model defines deformability, slippiness, and cohesiveness as key properties that influence whether a bolus is safe to swallow. Defining these properties numerically is difficult and current instruments used for bolus analysis have limitations. The slip extrusion test (SET) was developed to objectively measure the swallowability of the bolus through determination of its resistance to deformation and slip. The test measures the force needed to extrude a bolus through a bag as it is pulled through a pair of rollers, imitating the swallowing action of a bolus. Three food model systems were used to evaluate the SET: (a) viscous solutions with varying viscosity, (b) gels with varying hardness, and (c) particulate systems of varying cohesion. The test was applied to peanut boluses produced in vivo to demonstrate its potential in characterizing boluses. The deformation and slip resistance measurements correlated well with the hardness and viscosity measurements of the gels and viscous solutions respectively (correlation coefficient r = .94 between deformation resistance and hardness; r = .85 for slip resistance and hardness in gels; r = .98 for deformation resistance and viscosity; r = .93 for slip resistance and viscosity in solutions). The advantage of the SET is it can evaluate the swallowability of a wide range of foods of different structure and composition. It could potentially be used to investigate the properties of boluses throughout oral processing and help in establishing the criteria for a safe to swallow bolus in a quantitative way. PRACTICAL APPLICATIONS: The test could be used to measure bolus properties from the initial stages of breakdown to the point of swallow for all types of food. The ability to measure the changes in bolus properties through all stages of breakdown using the same instrument is a significant development. The resistance to deformation and slip are quantitative measurements that could potentially be used to further develop the Swallow Safe model by providing numerical limits to the identified properties. This could be of interest to the development of foods for dysphagia sufferers.

Predominance of subtype 3 among Blastocystis isolates from a major hospital in Singapore.

Blastocystis is an enteric protozoan parasite commonly found in humans and animals. Phylogenetic and genotypic analyses have shown that Blastocystis exhibits extreme genetic diversity, and humans are host to a number of zoonotic isolates. In the present study, the prevalence of Blastocystis in 276 stool samples from a hospital in Singapore was examined, and for the first time, riboprinting using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to determine the genetic diversity of the Blastocystis isolated from the Singapore population. The prevalence rate was determined to be 3.3% (9/276), and Blastocystis displaying two main ribotypes were isolated. As a comparison, we performed PCR-RFLP using two different published methodologies, and both methods allowed the isolates to be divided into two distinct groups based on their riboprint patterns. According to a recently proposed classification scheme, 78% (7/9) of the isolates were of subtype 3, while 22% (2/9) were subtype 1. The predominance of subtype 3 in an urbanized city state such as Singapore is in agreement with the idea that subtype 3 is a genotype of human origin.

Exposure of Blastocystis species to a cytotoxic monoclonal antibody.

A recently described cytotoxic monoclonal antibody (mAb 1D5) raised against Blastocystis hominis isolate B, was tested for reactivity with 13 different isolates of Blastocystis. The isolates used were previously isolated from humans, rats and reptiles and were maintained as axenised cultures throughout the course of this study. Five B. hominis isolates (B, C, E, G and H) were found to react with mAb 1D5 in immunoblotting studies and the indirect fluorescence antibody test. The pattern of fluorescence observed for all five isolates was diffuse and patchy. Immunoblotting studies revealed that mAb 1D5 reacted with a 29-30-kDa protein found in all five isolates. Results of a cytotoxic assay showed that the mAb exhibited a complement-independent cytotoxic effect on all the exposed isolates. Microscopic observations showed differences in morphology between the Blastocystis cells exposed and unexposed to mAb. Acridine orange staining performed on both exposed and unexposed cells showed similar internal structures when viewed under fluorescence microscopy.

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.

Colony growth as a step towards axenization of Blastocystis isolates.

Colonies of Blastocystis from non-axenic cultures grown in agar medium were isolated from bacterial colonies. Axenization of human and reptilian isolates of Blastocystis was achieved using antibiotic treatment to lower bacterial numbers, followed by colony growth to isolate pure parasite colonies.

Survival of Blastocystis hominis clones after exposure to a cytotoxic monoclonal antibody.

Our previous studies have shown that monoclonal antibodies (MAbs) to Blastocystis hominis react mainly with carbohydrate epitopes, while 1 MAb (1D5) reacts specifically with a protein of 30.5 kDa. In the present study, 3 monoclonal antibodies (1D5, 1E7 and 4F7) were used in immunogold localization. 1E7 and 4F7 were found to react primarily with the surface coat, while 1D5 was plasma membrane-specific. In the presence of complement, only 1D5 exhibited a cytotoxic effect on B. hominis whereas 1E7 and 4F7 did not, suggesting that the surface coat of B. hominis could serve as an immunological barrier against host antibodies. Using a recently described agar plating method, only 1D5 exhibited significant (P < 0.01) complement-independent cytotoxicity to B. hominis, inhibiting colony growth at low concentrations. Parasites that had been exposed to 1D5 were morphologically smaller than those that were not exposed to this MAb. Colonies that grew in the presence of 1D5 were isolated and grown in liquid medium containing increasing amounts of the cytotoxic MAb. Two clones that grew well in liquid medium containing 1D5 were also able to develop into colonies in soft agar. This study has shown that the 30.5 kDa protein found on the plasma membrane of B. hominis is a functionally important protein and that not all cells within a certain population would be susceptible to the cytotoxic effects of 1D5. These findings suggest that a heterogenous population exists in continuously maintained cultures of B. hominis.

Experimental Blastocystis hominis infection in laboratory mice.

Young (less than 8 weeks old) immunocompetent BALB/c mice became infected with Blastocystis hominis after inoculation of fecal cysts orally and of in vitro axenic-culture forms intracecally. This study confirmed that the fecal cyst was the form responsible for external transmission and that the mode of transmission was by the fecal-oral route. The infection was self-limiting and the infected BALB/c mice appeared normal except that some of them showed weight loss and lethargy. Both vacuolar and granular forms were found in the cecum, but only cyst forms were observed in the colon. Histological examination of the cecum and colon showed intense inflammatory-cell infiltration, edematous lamina propria, and mucosal sloughing. It is apparent that although B. hominis is not invasive, it is capable of causing pathogenesis in BALB/c mice.

Description of a Blastocystis species from Rattus norvegicus.

Two isolates (WR1 and WR2) of Blastocystis from laboratory-bred Wistar rats were axenized by a method of colony growth in soft agar combined with antibiotic treatment. The colonies were cultured in Iscove's modified Dulbecco's medium (IMDM) and Bacto agar mixture supplemented with 10% horse serum in the presence of thioglycollate. The cells from the colonies had an ameboid outline with a central body. Large inclusions were seen in the central body of some cells. Some granular forms were also found. In the axenic culture of isolate WR2, about one-third of the organisms were granular forms. Cysts were found in the axenic culture of both isolates. This is the first report of such cyst formation in in vitro culture. The karyotypic patterns of both isolates of the rat Blastocystis were analyzed by pulsed-field gel electrophoresis (PFGE). A total of 13 chromosomal bands were separated, ranging from 1.86 Mb to 295 kb. The karyotypic patterns of the rat Blastocystis were different from those of B. hominis and reptilian Blastocystis. On the basis of the above-mentioned differences, the rat Blastocystis is assigned as B. ratti sp. nov.

A speckle target adaptive imaging technique in the presence of distributed aberrations.

Acoustic velocity inhomogeneities in tissue result in aberration of ultrasound images. These aberrations can be modeled as a near field thin phase screen or as a distributed aberrator. The effect of a near field thin phase screen is to time shift the received echo at each element, while distributed aberrators result in both pulse distortions and time shifts from element to element. Most current techniques for the correction of distributed aberrators are limited to application on point targets. A new technique is proposed which uses multiple transmits from spatially shifted transmit apertures (the translating transmit aperture algorithm), in conjunction with phase conjugate filters, to correct for distributed aberrations in the presence of speckle targets. The performance of the translating transmit aperture algorithm in improving the correlation between signals received by elements of different spatial separations is measured, and factors affecting the performance of this technique are investigated in simulation and experiment.

In vitro response of Blastocystis hominis against traditional Chinese medicine.

This is the first inn vitro study on the activity of 20 kinds of crude extracts of traditional Chinese medicine (TCM) on the intestinal parasite, Blastocystis hominis using the criteria of living cell count (LCC) and living cell rate (LCR). LCC and LCR were applied as observation indicators, the former as a fixed-quantity and the latter as a fixed-quality method. LCR calculated percentage rate of living cells using eosin-brilliant cresyl blue staining which could differentiate between living cells and dying or dead cells. There were five extracts with no inhibitory activity, thirteen with moderate inhibition and two with high inhibition. The crude extracts of Coptis chinensis (CC) and Brucea javanica (BJ) were found to be most active against B. hominis. The active concentration of CC was 100 micrograms/ml. The active concentration of BJ was 500 micrograms/ml. The active concentration of metronidazole (MD) was 10 micrograms/ml and this was taken as an active standard drug for B. hominis.

Production and characterization of murine monoclonal antibodies to Blastocystis hominis.

Several hybridomas producing antibodies detected by enzyme-linked immunosorbent assay (ELISA) were established by fusions of mouse myeloma P3.X63.Ag8.U1 with spleen cells from BALB/c mice immunized against an isolate of Blastocystis hominis. Five strongly positive hybrids (6B6, 1D5, 1E7, 4F7 and 4G11) were cloned and all were found to secrete IgM monoclonal antibodies. Four MAbs (6B6, 1E7, 4F7 and 4G11) reacted in immunoblots with a number of B. hominis antigens (mol. wt ranging from 25,000 to 220,000) which were likely to be repeating oligosaccharide epitopes located on glycoproteins, as indicated by pronase and periodate treatment. Another MAb (1D5) reacted with a single antigenic band (mol. wt 30,5000). Similar results were obtained in immunoblots using 4 other B. hominis isolates. Indirect fluorescent-antibody assay (IFA) using MAbs showed 3 patterns of reactivity. 1D5 showed patchy fluorescence, 4F7 showed peripheral fluorescence and 6B6, 1E7 and 4G11 showed bright diffuse fluorescence. These patterns were observed for all 5 human Blastocystis isolates. The MAbs exhibited some cross-reactivity with 2 reptilian Blastocystis isolates but not with Giardia intestinalis, Trichomonas vaginalis or Entamoeba histolytica.

Colony formation of Blastocystis hominis in soft agar.

This is the first description of a method for growing axenized Blastocystis hominis as colonies in petri dishes containing soft agar. Blastocystis cells cultured in two types of agar appeared to show different colonial morphologies as well as differing colony yields. Microscopic examination of the colonies revealed many amoeboid and giant cells. Many cells were also shown to possess thin filament-like structures that appeared to stretch across the central vacuole.

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.

Axenic culture of reptilian Blastocystis isolates in monophasic medium and speciation by karyotypic typing.

The growth of axenic reptilian isolates of Blastocystis in Iscove's modified Dulbecco's medium (IMDM) was studied and the morphology of the parasite was examined by phase-contrast microscopy. The chromosomal patterns of these reptilian isolates of Blastocystis were examined by pulsed-field gel electrophoresis (PFGE) and compared with those of B. hominis and B. lapemi, a sea snake Blastocystis. IMDM with 10% horse serum supported excellent growth of the reptilian Blastocystis isolates. The parasites from all the isolates were predominantly vacuolar, but multivacuolar and amoeboid forms were also seen. Amoeboid forms with rather elongate pseudopodia were also observed. There were some differences in size, morphology, and growth characteristics in the different reptilian isolates. The karyotypic patterns of the Blastocystis isolates from tortoise, iguana, and python were distinctly different from one another and from those obtained with B. hominis and B. lapemi. On the basis of the above-mentioned differences in chromosomal patterns, the tortoise, iguana, and python isolates are described as new species, viz., B. geocheloni sp. nov. from Geochelone carbonaria (red-footed tortoise), B. cycluri sp. nov. from Cyclura cornuta (rhino iguana), and B. pythoni sp. nov. from Python reticulatus (reticulated python).

Clonal growth of Blastocystis hominis in soft agar with sodium thioglycollate.

The present report describes a method for establishment of colonies of Blastocystis hominis from single cells in soft agar. The percentage of colony-forming efficiency (% CFE = number of colonies grown/number of cells inoculated x 100) for the cultures was greatly improved by the addition of sodium thioglycollate. Five human Blastocystis isolates chosen for this study showed no apparent variation in colonial morphology. Isolated colonies were also successfully grown in liquid medium, providing a means of obtaining large numbers of B. hominis cells that had arisen from a single clone.

Serum antibodies to Trichomonas vaginalis in invasive cervical cancer patients.

OBJECTIVE: To evaluate, by seroepidemiology, the possible role of the sexually-transmitted flagellate, Trichomonas vaginalis, in invasive cervical cancer. SUBJECTS AND METHOD: Sera from 121 invasive cervical cancer patients and 242 random age-matched female controls. Antibodies to T. vaginalis were detected by the western blot technique. RESULTS: Antibodies to T. vaginalis were detected in the sera of 41.3% (50/121) of invasive cervical cancer patients compared with only 5.0% (12/242) of female controls. All the reactive sera reacted strongly with the immunogenic surface membrane proteins of T. vaginalis of molecular weights of about 92 and 115 kDa, with variable reactivity to other immunogenic proteins of T. vaginalis. CONCLUSION: The significantly increased relative risk, RR = 3.42 (95% CI = 1.73-6.78), is comparable to the RRs derived in seroepidemiological studies of human papillomavirus, suggesting that T. vaginalis may be even more closely associated with invasive cervical cancer than previously realized.

Elucidation of the life cycle of the intestinal protozoan Blastocystis hominis.

This paper elucidates the status of the different morphological forms of Blastocystis and reports the existence of thin- and thick-walled cysts in B. hominis on the basis of current experimental evidence. It is suggested that the thin-walled cysts are autoinfectious, leading to multiplication of the organism in the intestinal tract. The thick-walled cysts are responsible for external transmission via the faecal-oral route. A life cycle for B. hominis is postulated on the basis of these findings.

Tubulovesicular elements in Blastocystis hominis from the caecum of experimentally-infected rats.

By transmission electron microscopy (TEM), tubulovesicular elements were seen in Blastocystis hominis obtained from the caecum of experimentally-infected rats. These appeared to arise from the peripheral cytoplasm and were rounded, oval or elongate in sections. It is suggested that these elements form a network for transfer of nutrients to the periphery during the process of encystation.

Differentiation of the various stages of Blastocystis hominis by acridine orange staining.

Acridine orange staining differentiates the cystic and the central body forms of Blastocystis hominis and offers a very convenient and easy method to observe the internal structure of the parasite. Acridine orange stains the nuclei and the central body of the rounded vacuolar forms of the parasite bright and dull green, respectively. The colour changes to yellow and then to flaming red-orange when the rounded central body forms of the parasite become cystic.

A multiple fission-like mode of asexual reproduction in Blastocystis hominis.

A non-axenic and an axenic isolate of Blastocystis hominis have been induced to form cysts in vitro using an encystation medium. The morphology of the parasite at different time points was observed by scanning electron microscopy. In day-2 cultures the cysts were spherical and had a non-uniform, coarse outer surface around the body. A deep, pore-like opening was seen in some of the parasites. Most of the cysts from day-4 and day-6 cultures ruptured, revealing small, uniformly sized spherical bodies occurring in grape-like clusters. Acridine orange staining confirmed that these bodies were the progeny of Blastocystis hominis. A multiple fission-like reproduction process giving rise to many daughter Blastocystis occurs within the cyst.