Infections of Blastocystis hominis and microsporidia in cancer patients: are they opportunistic?

Chemotherapy can cause immunosuppression, which may trigger latent intestinal parasitic infections in stools to emerge. This study investigated whether intestinal parasites can emerge as opportunistic infections in breast and colorectal cancer patients (n=46 and n=15, respectively) undergoing chemotherapy treatment. Breast cancer patients were receiving a 5-fluorouracil/epirubicin/cyclophosphamide (FEC) regimen (6 chemotherapy cycles), and colorectal cancer patients were receiving either an oxaliplatin/5-fluorouracil/folinic acid (FOLFOX) regimen (12 cycles) or a 5-fluorouracil/folinic acid (Mayo) regimen (6 cycles). Patients had Blastocystis hominis and microsporidia infections that were only present during the intermediate chemotherapy cycles. Thus, cancer patients undergoing chemotherapy should be screened repeatedly for intestinal parasites, namely B. hominis and microsporidia, as they may reduce the efficacy of chemotherapy treatments.

The potential use of 29 kDa protein as a marker of pathogenicity and diagnosis of symptomatic infections with Blastocystis hominis.

The present study was performed to characterize the protein profiles of Blastocystis hominis isolates from symptomatic and asymptomatic individuals by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using sera from symptomatic and asymptomatic patients. The presence of immunogenic bands associated with pathogenicity or of diagnostic potentials was also evaluated. The study comprised 80 individuals classified into four groups, 20 each: symptomatic blastocystosis (G1), asymptomatic blastocystosis (G2), other parasitic infections (G3), and healthy control subjects (G4). SDS-PAGE analysis of individual antigens form symptomatic and asymptomatic B. hominis isolates revealed similar and distinctive antigenic bands with significant differences in two high (123.5 and 112.3 kDa) and few low molecular weight bands (48.5, 38, 42.3, and 35.5 kDa). Immunoblotting was performed using symptomatic and asymptomatic antigen pools with sera of the four studied groups. It was found that anti-B. hominis IgG reacted with nine protein bands ranging from 100 to 18 kDa of the symptomatic antigen pool. There was a significant difference between G1 and G2 in the recognition of 64, 56, 38, and 29 kDa antigen bands. Also, anti-B. hominis IgG reacted with five protein bands ranging from 56 to 12 kDa of asymptomatic antigen pool. There was a significant difference between G1 and G2 in the recognition of 29 kDa antigen band. These findings suggest the potential use of the 29-kDa antigen as marker of pathogenicity and implicate its use in the diagnosis and differentiation between symptomatic and asymptomatic blastocystosis.

Encystation--survival of Blastocystis hominis in immunocompetent mice abdomen cavity.

Human Blastocystis hominis were isolated from diarrhea patients' feces and cultured in vitro. Then the cultures were inoculated intraperitoneally to laboratory mice. The B. hominis in living mice were collected and inoculated again to healthy mice. The B. hominis showed dose-dependent pathogenicity in the primary inoculation. No pathogenicity was observed in the secondary inoculation. The protozoan existed in the living mice abdomen cavity for more than 6 months and the cyst was the only form. These results showed that encystation enable the parasite to avoid the immune attack in competent host and simultaneously decrease the pathogenicity to host. Intraperitoneal inoculation to laboratory mice is a good method to maintain and propagate B. hominis. This is also a good model to study the interaction of B. hominis and immune system.

Kinetic analysis of antibody responses to Blastocystis hominis in sera and intestinal secretions of orally infected mice.

The kinetics of antibody production against Blastocystis hominis, an emerging infectious protozoan parasite which causes intestinal disorder in humans and animals, was studied. Sera and intestinal secretions were collected from B. hominis-immunized Balb/C mice for 8 weeks. Flow cytometry was used to monitor the levels of immunoglobulins A (IgA), G (IgG), and M (IgM) from both types of biological samples. The kinetic profile derived from flow cytometry analysis revealed that IgM led the early immune action against B. hominis infection in immune sera while IgA was the predominant antibody isotype in intestinal secretions. Western blotting revealed an array of antigens recognized by both serum and intestinal secretion antibodies. Immunoreactive B. hominis soluble proteins with molecular weights ranging from 28.2 to 77.6 kDa were detected by serum antibodies and 15.1 to 117.5 kDa by secretory antibodies. These antigens may be cytoplasmic or membrane-bound as determined through indirect fluorescent antibody test. Moreover, two immunogens (39.8 and 77.6 kDa) were commonly recognized by serum antibodies, one (70.8 kDa) by secretory antibodies and two (55.0 and 56.2 kDa) by both serum and secretory antibodies, suggesting a possible target in the further understanding of B. hominis pathogenicity, discovery of virulence factors, and development of immunology-based diagnostic protocols and alternative modes of treatment.

[Dot enzyme-linked immunosorbent assay for detection of serum antibody to Blastocystis hominis in humans].

Serum and stool samples were collected from 322 undergraduate students in medical school. Using stool in vitro cultivation as golden standard, 178 cases were found Blastocystis hominis positive and 144 were negative. Dot-ELISA was used to examine the serum samples with a sensitivity of 92.1% (164/178) and specificity of 97.1% (141/144). This revealed that dot-ELISA can be used for antibody detection against Blastocystis hominis.

Secretory and humoral antibody responses to Blastocystis hominis in symptomatic and asymptomatic human infections.

The study included 3 groups of individuals, in the first 2 groups they had positive stool microscopic examinations only for B. hominis indicating blastocystosis, with and without gastrointestinal symptoms, respectively, while the last group included apparently healthy individuals with no parasites in stool. Stool and serum samples of these individuals were subjected to detection of anti-B. hominis fecal and serum IgA and serum IgG antibodies by indirect ELISA, and detection of B. hominis fecal and serum antigens by double sandwich ELISA. In symptomatic B. hominis infections with positive stool microscopy the study recorded first: specific secretory IgA and humoral IgA and IgG antibody responses at a prevalence of 100%, 83.3% and 86.6%, respectively, with an increased significant difference (P<0.001) of each from healthy controls, together with an increase in level of secretory IgA than that of humoral IgA antibody (P<0.001), and second: the presence of specific antigens in stool and serum at a prevalence of 96.6% and 90%, respectively. With an increased significant difference (P<0.001) of each from healthy controls together with the former at a higher level than the latter (P<0.05). In asymptomatic B. hominis infections with positive stool microscopy the study recorded first; absence of each of the studied specific secretory and humoral antibody responses with no significant difference (P>0.05) of each from healthy controls, and second; absence of specific antigens in stool and serum with no significant difference (P>0.05) of each from healthy controls nor from each other. The explanations and implications of these results are discussed.

Programmed cell death in a human intestinal parasite, Blastocystis hominis.

Although programmed cell death (PCD) has been associated with multicellular organisms, there have been more reports of its presence in some protozoans. Our study shows the existence of PCD in an intestinal protozoan, Blastocystis hominis. Light and electron microscopy, biochemical and flow cytometry studies showed apoptosis-like death in B. hominis cells exposed to a cytotoxic monoclonal antibody (MAb 1D5). B. hominis cells displayed key morphological and biochemical features of apoptosis, namely, nuclear condensation and in situ fragmentation, reduced cytoplasmic volume, some externalization of phosphatidylserine and maintenance of plasma membrane integrity. No oligonucleosomal DNA laddering was observed in gel electrophoresis. This study supports earlier observations that the cellular machinery that is required to carry out PCD may have existed before the advent of multicellularity. Our study also ascribes a novel function for the B. hominis central vacuole in apoptosis; it acts as a repository where apoptotic bodies are stored before being released into the extracellular space.

Blastocystis hominis modulates immune responses and cytokine release in colonic epithelial cells.

An experimental in vitro model has been developed in order to determine whether Blastocystis hominis is able to trigger inflammatory cytokine response in colonic epithelial cells. After 24 h incubation of B. hominis with the cell lines HT-29 and T-84, B. hominis cells were not able to cause cytopathic effects, but significant increases in the release of the cytokines IL-8 and GM-CSF could be observed. However, after the first 6 h of co-incubation, the production of IL-8 was not increased in HT-29 cells, and even reduced when Escherichia coli (bacteria or lipopolysaccharide) was present during co-incubation. Similar effects were observed using supernatants of B. hominis culture. These data indicate that B. hominis induces as well as modulates the immune response in intestinal epithelial cells, and we conclude that different pathophysiological events may occur during B. hominis infection.

Serologic response to Blastocystis hominis infection in asymptomatic individuals.

The pathogenic potential of Blastocystis hominis in the human intestine is subject to controversy because the organism has been found in both symptomatic and asymptomatic individuals. To help clarify this issue, we monitored the serologic response to the organism in B. hominis-infected individuals free of gastroenterologic disorders. 1) Serum antibodies to B. hominis were detected in 70% of infected asymptomatic individuals by an indirect immunofluorescence (IFA) test. 2) IFA and immunoelectron microscopy revealed that the antibody response was directed against a surface antigen(s) of the organism. 3) Analysis by immunoblotting implicated a 12 kDa protein of B. hominis. 4) The strongest positive reaction was obtained in an individual chronically infected for more than 2 years. It may be that long exposure to the parasite is necessary for a serologic response.

Soluble-protein and antigenic heterogeneity in axenic Blastocystis hominis isolates: pathogenic implications.

The protein profile and the antigenic cross-reactivity of 18 axenic isolates of Blastocystis hominis obtained from symptomatic patients with chronic diarrhea (14 isolates) showing no evidence of parasitic etiology and from patients with acute diarrhea attributable in 2 cases to Salmonella spp. were analyzed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of soluble proteins showed the existence of a common profile composed of 31 bands, with molecular weights ranging between 24 and >200 kDa, and minor differences in the proteins of 149, 118, 106, 50, 48, 47, and 30 kDa. These differences allowed us to classify the strains into three related patterns (I-III). In an indirect immunofluorescence assay, all strains were serologically identical, but two related antigenic groups (1 and 2) were found in double-immunodiffusion and Western-blot studies. The isolates of protein patterns I and II belonging to antigenic group 1 were isolated from patients with chronic diarrhea, whereas the four isolates from patients with acute diarrhea were clustered in protein pattern III and in antigenic group 2. These results confirm the protein and antigenic heterogeneity of B. hominis and the existence of demes with different pathogenic roles.

Blastocystis hominis in animals: incidence of four serogroups.

In toto, 520 faecal samples from mammals, birds, reptiles, amphibians, fish, and snails were investigated (see Table 1). 91 strains of Blastocystis hominis could be isolated by culture. However, only 48 of them were suitable for axenisation. 96 percent of samples belonged to four serogroups detected in humans but two strains, one from a pig and another from a duck, could not be classified, suggesting the existence of one or two further serogroups. While humans showed mainly serogroups I and II, pigs harboured serogroups III and IV. Four serogroups were isolated from monkeys. The question whether the genus Blastocystis consists of one or more species is discussed.

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.

Significantly increased IgG2 subclass antibody levels to Blastocystis hominis in patients with irritable bowel syndrome.

Blastocystis hominis is a common intestinal parasite of humans in the tropics whose pathogenic role is in dispute. Its presence has been reported in a variety of intestinal disorders resembling irritable bowel syndrome (IBS) such as diarrhea, anorexia, and flatulence. We have therefore investigated a possible link between IBS and blastocystosis by determining IgG antibody levels to B. hominis in patients with IBS. Levels of IgG antibodies were significantly elevated in patients with IBS compared with asymptomatic controls (P < 0.0001, by Student's t-test) in both B. hominis stool culture-positive and stool culture-negative IBS patients. When IgG antibodies were divided into their respective subclasses, only IgG2 levels were significantly increased in IBS patients compared with asymptomatic controls, indicating that the predominant response in these patients may be directed to carbohydrate antigens. The diagnostic usefulness of this test in IBS patients remains to be established because these data are only suggestive of a possible link between B. hominis and IBS. However, we hope that this antibody test will help in elucidating the controversy that surrounds the role of B. hominis as a pathogen at present.

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.

Enzyme-linked immunosorbent assay for detection of serum antibody to Blastocystis hominis in symptomatic infections.

An enzyme-linked immunosorbent assay was devised in order to search for antibodies against Blastocystis hominis in infected humans. Reaction proteins were obtained from washed, axenic B. hominis cells, as sonicate. Sonicate was diluted to provide 17 and 34 micrograms of protein per well. Dilutions of patients' sera were applied, followed by phosphatase-conjugated goat anti-human serum and phosphatase substrate. Color was measured at 405 microns wavelength. Immunoglobulin G antibodies to high titers were found. Of 30 sera tested from 28 patients, 3 were negative at the 1/50 threshold dilution, 8 were positive at 1/50, 3 at 1/100, 2 at 1/200, 3 at 1/400, 6 at 1/800, and 5 at 1/1,600. Normal sera (42 blood bank sera) were all negative at 1/50. Each serum was subjected to multiple testing. Duplicate tests were included for each run, and runs were made from 4 to 6 for each serum. Blastocystis hominis is increasingly recognized to be a cause of human enteric disease, with symptoms often like those in giardiasis. Demonstration of strong antibody response is consistent with this view.

Four serologically different groups within the species Blastocystis hominis.

61 strains of Blastocystis hominis were isolated from 340 stool specimens coming from 140 patients with intestinal symptoms and from 200 healthy persons. The overall female to male ratio was 1.9 to 1. Classification of the 61 isolates of B. hominis was determined with the aid of the immunodiffusion assay. Four serologically different groups could be identified. Their frequency ratio in the population studied was 39.5%:39.5%:18%:3%. Group 3 was found more often than the other three groups in stool samples of patients. However, within the statistical deviation, none of the four immunological different groups was found to be correlated with the disease or the sex of the hosts.

Urticaria by Blastocystis hominis. Successful treatment with paromomycin.

Urticaria and angioedema are easily recognized disorders, but in at least 70 percent of individuals, chronic episodes of urticaria are of unknown causes. We present 10 cases of chronic urticaria associated parasitation by blastocystis hominis. This parasite has not been previously related with urticaria. Both intestinal parasitation as well as urticaria responded successfully to paromomycin sulfate.