What happens when a common antiparasitic drug, a diabetes medicine, and onion extract meet Blastocystis? This study finds that all three can suppress the parasite, but they do not shift its biochemical environment in the same way. Blastocystis sp.

is a highly prevalent anaerobic intestinal protozoan increasingly associated with gastrointestinal disorders. Its survival within the gut microenvironment depends on metabolic pathways such as glycolysis, where glyceraldehyde-3-phosphate dehydrogenase, or GAPDH, plays a central role.

That survival also involves interactions with nitric oxide, or NO, derived from host defenses and surrounding microbiota. The emergence of metronidazole resistance necessitates exploration of alternative therapeutic strategies. The objective was to evaluate the in vitro effects of metronidazole, metformin, and Allium cepa, or onion, extract on Blastocystis sp.

growth. The study also focused on GAPDH activity and NO levels within the xenic culture medium. Blastocystis sp. can reach prevalence rates of up to 50 percent in developing countries, while its underlying pathogenic mechanisms remain poorly understood.

Because it lacks classical mitochondria, the organism relies primarily on glycolysis for energy production, supported by mitochondrion-like organelles adapted to anaerobic conditions. GAPDH catalyzes a critical step in glycolysis and also has roles in host-parasite interactions, immune evasion, and resistance to oxidative stress.

The dependence of parasites on glycolysis makes them more vulnerable to GAPDH inhibitors than their human hosts, promoting GAPDH as a valuable antiparasitic drug target. Nitric oxide can inhibit GAPDH through S-nitrosylation, while NO also has cytotoxic effects against pathogens, including Blastocystis sp.

Metronidazole remains the most used antiparasitic agent against Blastocystis, but increasing reports of resistance and treatment failure have raised concerns regarding its efficacy. That concern has prompted interest in alternative or adjunctive therapeutic approaches.

Metformin has demonstrated antimicrobial and immunomodulatory effects, including modulation of nitric oxide pathways. Allium cepa contains bioactive constituents with documented antiparasitic and antioxidant properties. The study evaluated metronidazole, metformin, and Allium cepa extract in vitro, focusing on parasite viability, GAPDH activity, and NO levels within a xenic culture milieu.

By assessing parasitological and biochemical changes, the work provides preliminary insights into drug-induced Blastocystis-culture dynamics in the presence of co-existing intestinal microbiota. Those findings were intended to be validated by future mechanistic studies using axenic cultures or parasite-specific enzymatic assays.

Twenty-one Blastocystis-positive stool samples were collected from the Diagnostic and Research Unit of Parasitology at Cairo University’s Faculty of Medicine. The stool specimens came from outpatients presenting with gastrointestinal manifestations, including abdominal pain, diarrhea, nausea, and flatulence, through convenience sampling.

A purposive sampling strategy then included only twenty-one positive samples. Parasite detection was confirmed using direct wet-mount microscopy and the formalin-ethyl acetate concentration technique. Metronidazole was tested at three concentrations: one hundred, five hundred, and one thousand micrograms per milliliter.

The one-hundred-microgram concentration came from one prior study, the one-thousand-microgram concentration was adapted from another, and five hundred micrograms per milliliter bridged the two doses. Choosing doses for metformin and Allium cepa was challenging because satisfactory literature reports on their in vitro effects against Blastocystis sp.

were lacking. Following preliminary viability trials on Blastocystis, onion-extract concentrations of five hundred, one thousand, and two thousand micrograms per milliliter were selected. Blastocystis culture samples were harvested by centrifugation at two thousand times g for ten minutes at four degrees Celsius, washed three times with ice-cold PBS, and lysed in chilled extraction buffer.

The extraction buffer contained fifty millimolar Tris-HCl, one millimolar EDTA, and one percent protease inhibitor cocktail. Cell lysates were cleared by centrifugation at twelve thousand times g for fifteen minutes at four degrees Celsius, and soluble protein concentrations were quantified with the Bradford assay.

NO accumulation in culture supernatants was indirectly evaluated through chemical trapping, using clarified culture supernatant and PTIO. The resulting stable NO-PTIO adducts were extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated under a nitrogen stream, and prepared for gas chromatography–mass spectrometry injection.

Because the data violated normality assumptions, the Kruskal-Wallis test was used for quantitative analysis. Post hoc pairwise comparisons used the Dwass–Steel–Critchlow–Fligner test, and correlations were evaluated with Spearman’s correlation coefficient.

Statistical significance was set at P less than zero point zero five. The mean Blastocystis count in untreated cell culture was sixty-six point zero plus or minus forty-four point five times ten to the fourth cells per milliliter. Every therapeutic agent except the onion five-hundred-microgram preparation significantly decreased parasite count at different concentrations compared with the control group.

Higher drug concentrations were generally more efficient at decreasing the count, with some exceptions. Metronidazole was most efficient in decreasing Blastocystis count, followed by metformin and onion preparations. In the gray untreated-control row, the box and whiskers span most of the horizontal scale, whereas the pink metronidazole rows are tightly clustered near the origin.

The letter labels also show distinct groups, with the control marked “a” and the highest-concentration metronidazole group marked “f,” indicating statistically significant separation. The highest NO level was observed in the untreated control group, at ninety-one point one plus or minus zero point ninety-four units per milligram protein.

All therapeutic agents significantly reduced NO levels, with the lowest value in the metronidazole five-hundred group, at sixty-three point four plus or minus twelve point fourteen units per milligram protein. The highest GAPDH activity was detected in the untreated control group, at zero point one seven plus or minus zero point zero zero six.

All tested agents significantly reduced GAPDH activity compared with the untreated control group, and the lowest activity was observed in the metformin one-thousand group, at zero point zero nine plus or minus zero point zero one zero. The black squares and median lines show that the untreated-control box is tightly clustered near the top, while the treatment boxes are generally lower and more variable.

In the boxplot, the untreated-control box sits distinctly above the treatment boxes, while metformin one-thousand occupies the lowest region. The letter labels reinforce this separation: the control is marked “a,” most treatment groups “b,” and metformin one-thousand “c,” indicating statistically distinct groupings.

Correlations between Blastocystis count, NO levels, and GAPDH activity were analyzed using the Spearman correlation test. NO levels and GAPDH activity were significantly negatively correlated in the metronidazole five-hundred group, but significantly positively correlated in the metformin one-thousand group; both had P less than zero point zero zero one.

A significantly negative correlation was also found between Blastocystis count and the onion one-thousand group, with P equal to zero point zero three four. A key limitation was reliance on xenic culture conditions without a parallel, Blastocystis-free treated stool control.

Xenic culture media maintain a realistic polymicrobial environment of the human gut, reflecting a collective microenvironmental response rather than isolated, parasite-specific mechanisms. Therefore, the observed metabolic and nitrosative suppression cannot be attributed to an isolated effect on Blastocystis viability and may also reflect changes in the microbial milieu.

The study was preliminary and exploratory, and doses were determined from pilot trials and previous in vitro applications because literature reports on metformin and onion extracts against Blastocystis were scarce. The treatments reduced Blastocystis counts and also lowered nitric oxide and GAPDH activity, with metronidazole strongest against parasite count, metformin strongest against GAPDH, and the xenic design leaving the exact parasite-specific mechanism unresolved.