Blastocsytis sp. is a protozoan parasite that has been linked to common gastrointestinal illnesses. Metronidazole, the first line therapy, was reported to show frequent inefficacy. Previously, Blastocystis sp. isolated from different population showed varying metronidazole resistance. However, the effect of metronidazole treatment on pathogenic potentials of Blastocystis sp. isolated from different populations, which is known to have different gut environment, is unclear. This study investigates the in vitro effect of metronidazole on the pathogenic potentials of Blastocystis sp. isolated from urban and orang asli individuals. Blastocystis sp. ST 3 isolated from symptomatic and asymptomatic individuals were treated with a range of metronidazole concentration. The parasites' growth characteristics, apoptotic rate, specific protease activity and the ability to proliferate cancer cells were analyzed upon treatment with 0.001 mg/l metronidazole. The study demonstrates that Blastocystis sp. isolates showed increase in the parasite numbers especially the amoebic forms (only in urban isolates) after treating with metronidazole at the concentration of 0.001 mg/ml. High number of cells in post-treated isolates coincided with increase of apoptosis. There was a significant increase in cysteine protease of Blastocystis sp. isolates upon treatment despite the initial predominance of serine protease in asymptomatic isolates. Metronidazole resistant Blastocystis sp. also showed significant increase in cancer cell proliferation. Resistance to metronidazole did not show significant different influence on the pathogenicity between Blastocystis sp. isolated from urban and orang asli individual. However, an increase in parasite numbers, higher amoebic forms, cysteine protease and ability to proliferate cancer cells implicates a pathogenic role. The study provides evidence for the first time, the effect of metronidazole towards enhancing pathogenic potentials in Blastocystis sp. when isolated from different gut environment. This necessitates the need for reassessment of metronidazole treatment modalities.
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.
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.
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.
Blastocystis sp. is a gastrointestinal (GI) protozoan parasite reported to cause non-specific GI symptoms including diarrhea, flatulence, abdominal pain, and nausea. Complete eradication of Blastocystis sp. is rather challenging even with the drug of choice, i.e., metronidazole. Here, we report on two Blastocystis sp.-infected individuals, who presented increased parasite load and exacerbated symptoms upon treatment with the usual recommended dosage and regime of metronidazole. The two studies uniquely demonstrate for the first time a cyst count as high as fivefold more than the original cyst count before treatment and show an exacerbation of GI symptoms despite treatment. The study provides additional support in recognizing metronidazole resistance in Blastocystis sp. and its consequences towards the pathogenicity of the parasite.
Blastocystis sp., an intestinal organism is known to cause diarrhea with metronidazole regarded as the first line of treatment despite reports of its resistance. The conflicting reports of variation in drug treatment have been ascribed to subtype differences. The present study evaluated in vitro responses due to metronidazole on ST3 isolated from three symptomatic and asymptomatic patients, respectively. Symptomatic isolates were obtained from clinical patients who showed symptoms such as diarrhea and abdominal bloating. Asymptomatic isolates from a stool survey carried out in a rural area. These patients had no other pathogens other than Blastocystis. Ultrastructural studies using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) revealed drug-treated ST3 from symptomatic patients were irregular and amoebic with surface showing high-convoluted folding when treated with metronidazole. These organisms had higher number of mitochondrion-like organelle (MLO) with prominent cristae. However, the drug-treated ST3 from asymptomatic persons remained spherical in shape. Asymptomatic ST3 showed increase in the size of its central body with the MLO located at the periphery.