Diagnostic perspectives of evaluation of virulent potential Blastocystis sp. strains according to index of percentage of amoeboid forms and protease activity

Abstract

Introduction. Blastocystis is single-celled, anaerobic, eukaryotic protozoa, that colonizes the intestines of many speсies animals and humans and induce pathological changes with a number of nonspecific symptoms - abdominal pain, diarrhea, flatulence, anorexia, nausea, vomiting, anal itching, uncontrolled weight loss. The combination of two criteria - the detection ≥ 5 cells of Blastocystis sp. observed in the field of view under light microscopy with ×400 magnification and the exclusion of the role of other pathogens is today used to diagnose blastocystosis. Recently, the study of the virulence potential has yielded many new results. Methods for determining the number of amoeboid forms and measuring the protease activity of lysates are simple and promising. The goal of this study was experimental reasoning for determining the level of virulent potential Blastocystis sp. strains of using indicators of the percentage amoeboid forms (PAF) formation in vitro culture and the level of protease activity (Pa) lysates. Materials and Methods. 12 strains of Blastocystis sp. isolated from different faecal samples (FS) were used, which were 3 from asymptomatic and 9 from symptomatic patients. The parasitic burden of native 12 FS was determined by light microscopy (×400 magnification, 1% Lugol's solution) of wet smears (mean number cell in 10 view fields). 200 μl native fresh FS was inoculated in 5 ml of liquid media RPMI/IMDMEM with serum and antibiotics. Blastocystis sp. culture growth was carried out under anaerobic conditions at 37 ^oC for 5 days (determination PAF); and 10 sequential passages in the same medium to stabilize cultures (for Pa lysate). The value of the PAF index for each strain was determined by calculating the percentage of amoeboid forms in 300 counted parasite cells in suspensions smears stained by modified Field's stain method. Рa was determined using 4-day stabilized blastocyst cultures. Cells were precipitated three times at 500 g for 5 min and washed in PBS. Histopaque (1.077 g/ml) was used for the final cleanup (5000 g 10 min). The required concentration of 1×10⁶ cell/ml was determined using haemocytometer, adding PBS. We used 15 cycles of freeze and defrosting to outflow  proteases from cells. The biochemical method of azocasein cleavage by colorometric analysis (LisaScan EM analyzer, λ=450 nm) was used to determine Pa lysates of Blastocystis sp. Results & Discussion.  Amoeboid forms were found in 8 out of 12 (66.7%) Blastocystis sp. cultures, which exceeds that of direct coproscopy (p≤0.05). The lowest levels of PAF (1.7-2.3%) were found in strains that had a low parasites concentration in stool samples. Strains from two groups of patients that had a low parasitic load in the faeces had low levels of Ра (<20 AU). Conversely, strains from symptomatic individuals, and moderate to high parasite counts (2.5-3.8 and ≥ 5 per field, respectively) had levels Pa > 30 AU, which is a sign of virulence. Despite the fact that the generation of amoeboid forms correlates with the pathogenicity of parasites, it should be taken into account that the PAF index depends on the type of nutrient media. We noted a strong positive correlation between PAF a values and Pa values in the strains: in cultures with PAF < 3%, the value of Pa < 20 AU, and in parasites with PAF > 10%, the level of Pa > 30 AU. Conclusion. Blastocystis sp. strains from individuals with symptoms and a moderate to severe parasitic load of faecal samples had higher PAF and Pa values (p ≤ 0.05). We recommend doing an assessment of the virulence potential with the determination of PAF in culture under the following conditions: suspicion of blastocystosis, amoeboid forms are absent, but other morphoforms are present in medium concentrations in smears of native faeces. Virulente signs of blastocystis strains should be considered under the condition - PAF > 10% and Pa > 30 AU.

Keywords: Blastocystis sp., protease activity, amoeboid forms, in vitro.