Genomic DNA of Blastocystis isolates released into 0.1% Triton X-100 was suitable for amplification and yielded similar results as the genomic DNA extracted with standard kit. The specific B. hominis primers (BH1: GCT TAT CTG GTT GAT CCT GCC AGT and BH2: TGA TCC TTC CGC AGG TTC ACC TAC A) successfully produced the PCR product of about 1,770 bp with all the 7 Blastocystis isolates tested. The restriction fragment length polymorphism (RFLP) patterns yielded by 13 out of 25 restriction endonucleases showed that the 7 isolates could be grouped into 4 subgroups: subgroup-1 consisted of isolate C; subgroup-2 of isolates H4 and H7; subgroup-3 of isolates KP1, Y51 and M12; and subgroup-4 of isolate 27805. The differences between subgroups manifested as clear-cut RFLP patterns. A common band of 230 bp was revealed by Eco R1 in all the Blastocystis isolates tested. The band of about 180 bp was revealed by Alu I, differentiated symptomatic from asymptomatic isolates of this parasite, and might indicate the pathogenicity of this parasite.
Blastocystis hominis has long been described as a non pathogenic protozoan parasite until recently when claims have been made that it can result in pathogenic conditions. Of the 729 stool samples (614 from survey and 115 from pediatric wards) examined, 18.1% of them were found to be positive for one or more intestinal protozoan cyst. The commonest was Giardia intestinalis (8.4%) Followed by Entamoeba coli (7.1%) and Entamoeba histolytica (5.1%) in the normal children without symptoms of diarrhea. When diarrheic stools were examined, the commonest parasite encountered was Giardia (20.4%), followed by E. coli (15.9%) and E. histolytica (9.7%). Blastocystis was observed in 4.4% of the children who had diarrhea and 1.1% among the children taken from the normal population in the rural areas.
The objective of this study was to characterize the polypeptides associated with cysts of Blastocystis hominis. This form is believed to be infective and plays a role in parasite resistance to anti-B. hominis drugs currently used for treatment of Blastocystis associated diarrhea. Cysts were induced through in vitro culture of the parasite in complete medium supplemented with bacterial extract with trypticase, metronidazole or doxycycline. SDS-PAGE analysis showed almost similar polypeptide patterns of parasite extracts obtained from in vitro cultured parasites before and after exposure with the three supplements. Polypeptide bands at 76, 58.5, 48, 45, 40, 38, 32, 25 and 22 kDa were constantly seen in all antigenic preparations and no specific cyst-associated polypeptide was present. However, on immunoblot analysis, 3 out of 16 blastocystosis human sera identified a cyst-associated polypeptide at 60 kDa in all parasite extracts prepared from cultures with the three supplements. In addition, there were associated morphological changes detected in these parasites stained with acridine orange and observed under fluorescence microscopy. Metronidazole induced cyst forms (reddish cells) as early as 12 hours post-exposure; more cyst production (with stronger immunoblot bands) occurred after 24 hours exposure. However, cysts rupture with release and destruction of B. hominis daughters cells occurred after 48 hours exposure. Doxycycline induced less cyst-like forms at 24 hours (weaker 60 kDa band) and less destruction of the cysts (60 kDa band still present at 72 hours post exposure). Bacterial extract and trypticase also induced cysts at 12 hours with increasing numbers up to 72 hours exposure (corresponding increase in intensity of 60 kDa band from samples harvested at 12 to 72 hours post exposure) without any sign of deleterious effect on the parasite.
Previous studies have shown that apoptosis-like features are observed in Blastocystis spp., an intestinal protozoan parasite, when exposed to the cytotoxic drug metronidazole (MTZ). This study reports that among the four subtypes of Blastocystis spp. investigated for rate of apoptosis when treated with MTZ, subtype 3 showed the highest significant increase after 72h of in vitro culture when treated with MTZ at 0.1mg/ml (79%; p<0.01) and 0.0001mg/ml (89%; p<0.001). The close correlation between viable cells and apoptotic cells for both dosages implies that the pathogenic potential of these isolates has been enhanced when treated with MTZ. This suggests that there is a mechanism in Blastocystis spp. that actually regulates the apoptotic process to produce higher number of viable cells when treated. Apoptosis may not just be programmed cell death but instead a mechanism to increase the number of viable cells to ensure survival during stressed conditions. The findings of the present study have an important contribution to influence chemotherapeutic approaches when developing drugs against the emerging Blastocystis spp. infections.
Blastocystis is one of the most common parasites inhabiting the intestinal tract of human and animals. Currently, human Blastocystis isolates are classified into nine subtypes (STs) based on the phylogeny of their small subunit ribosomal RNA (SSU rRNA) gene. Although its pathogenicity remains controversial, the possibility of zoonotic transmission was recognized since eight of the nine STs (except for ST9) have been reported in both humans and animals. A cross-sectional study was conducted to determine the prevalence and subtype distribution of Blastocystis isolated from humans and associated animals in an indigenous community with poor hygiene in Malaysia, where the risk of parasitic infection is high. A total of 275 stool samples were collected, subjected to DNA extraction and amplified by PCR assay. The Blastocystis-positive amplicons were then purified and sequenced. Phylogenetic tree of positive isolates, reference strains and outgroup were constructed using maximum likelihood method based on Hasegawa-KishinoYano+G+I model. The prevalence of Blastocystis infection among humans and domestic animals by PCR assay were 18.5% (45/243) and 6.3% (2/32), respectively. Through molecular phylogeny, 47 isolates were separated into five clusters containing isolates from both hosts. Among human isolates, ST3 (53.3%) was the predominant subtype, followed by ST1 (31.1%) and ST2 (15.6%). Chicken and cattle had lower proportions of ST6 (50%) and ST10 (50%), that were barely seen in humans. The distinct distributions of the most important STs among the host animals as well as humans examined demonstrate that there is various host-specific subtypes in the lifecycle of Blastocystis.
Blastocystis species (spp.) is an emerging pathogen. There are several unsolved issues linked to this parasite ranging from its nomenclature, commensal status, standardization of laboratory diagnostic methods, genotypes and treatment. Recently, there has been an increase in reports of Blastocystis spp. from symptomatic cases which provide enough evidence of its pathogenic potential. A range of signs and symptoms, from gastro-intestinal to cutaneous manifestations have been attributed to Blastocystis infection. Few reports have established an association between intestinal infection with Blastocystis spp. and skin manifestations in form of urticaria, palmoplantar pruritus and allergy with complete resolution of cutaneous lesions with eradication of the parasite. In this report, we describe a case of Steven Johnson's syndrome (SJS) in a 6 years old girl along with infection with Blastocystis spp. marked by diarrhea and abdominal pain. Stool examination revealed the presence of all forms of the parasite with subsequent decrease in parasite burden and diarrhea over a period of time. Interestingly, the clearance of Blastocystis spp. from stool was followed by recovery from skin lesions and other symptoms. In this case, the course of SJS was clearly associated with Blastocystis infection. Though skin manifestation with Blastocystis infection has been previously reported, this is the first report of its association with SJS. This report indicates newer insights of the parasite that are less well studied.
Blastocystis sp. is an enteric protozoan parasite of humans and many animals. Blastocystis sp. subtype 3 (ST3) proves to be the highest frequency case in most populations around the world and it is further distinguished into symptomatic and asymptomatic isolates based on the clinical symptoms exhibited by infected individuals. Phenotypic and genotypic studies implicate the distinctiveness of this parasite which may describe its pathogenesis. However, the antigenic distinctiveness which describes the antibody mediated cell lysis of this parasite has not been explored. This study was aimed to identify the cross-reactivity and cytotoxicity effect between three isolates of symptomatic and asymptomatic Blastocystis sp. ST3 respectively. Antigen specificity and diversity of this parasite was performed by coculturing sera (10-fold dilution) obtained from mice immunised with Blastocystis sp. symptomatic and asymptomatic antigens and the respective Blastocystis sp. ST3 live cells through complement dependant cell cytotoxicity (CDC) assay. The results obtained has shown that, the sera (at 10-fold diluted concentration) from symptomatic and asymptomatic solubilised antigen immunised mice were able to specifically lyse the respective live parasites with an average percentage of 82% and 86% respectively. There were almost 50% crossreactivity observed between the three isolates of Blastocystis sp. ST3 from symptomatic and asymptomatic group proving high antigen diversity or rather low antigen specificity within the same group. However, there was only 17% cross-reactivity observed between the mice sera and parasitic cells of different groups (symptomatic vs asymptomatic isolates) suggesting high specificity between these two groups. We, for the first time have proven that through CDC analysis there were epitopes dissimilarities between Blastocystis sp. ST3 symptomatic and asymptomatic isolates which may allow the parasite to set up diverse immune modulations such as imbalanced Th1/Th2 responses in an infected host.
Blastocystis sp. is a common enteric protozoan parasite found in humans and various type of animal worldwide. Recently, genotypic distribution of Blastocystis sp. was revealed in insects, rodents, avian and mammals, which exposed its potential of transmiting the infections to human. However, very little information on current level of Blastocystis sp. infection were reported in cattle from Malaysia. Herein, a total of 120 stool samples of cattles were collected. While the potential risk of infection such as age, gender, body score, diarrheic condition of the cattle were noted, the management of the farms was also recorded. All stool sample were cultured, but 80 samples were selected for PCR sequencing analysis. The cultivation and microscopic examination revealed only 25% of the cattle (30/120) were infected with Blastocystis sp.. But, 43.8% of the cattle (35/80) were found positive upon PCR sequencing. The study also found that age, body score condition, diarrheic condition and certain farm were associated with the infection (p<0.05). Six subtypes (STs) that were discovered during the study were ST10 (21.3%;17/35), ST5 (8.8%;7/35), ST3 (7.5%;6/35), ST1 (2.5%;2/35), ST4 (2.5%;2/35) and ST14 (1.3%;1/35). Thus, moderate infections of Blastocystis sp. and variants in the genotypic distributions of the cattle suggest its potential for zoonotic transmission. Therefore, this findings could be helpful for further understanding the parasite, which assist studies of its pathogenicity.
Isolates of Blastocystis hominis from infected immigrant workers from Indonesia, Bangladesh and infected individuals from Singapore and Malaysia were assessed for growth pattern and degree of resistance to different concentrations of metronidazole. Viability of the cells was assessed using eosin-brillian cresyl blue which stained viable cells green and nonviable cells red. The Bangladeshi and Singaporean isolates were nonviable even at the lowest concentration of 0.01 mg/ml, whereas 40% of the initial inoculum of parasites from the Indonesian isolate at day one were still viable in cultures with 1.0 mg/ml metronidazole. The study shows that isolates of B. hominis of different geographical origin have different levels of resistance to metronidazole. The search for more effective drugs to eliminate th parasite appears inevitable, especially since surviving parasites from metronidazole cultures show greater ability to multiply in subcultures than controls.