In vitro susceptibility of human Blastocystis subtypes to simeprevir

Abstract

Introduction and aim: Blastocystis is a common enteric parasite, having a worldwide distribution. Many antimicrobial agents are effective against it, yet side effects and drug resistance have been reported. Thus, ongoing trials are being conducted for exploring anti-Blastocystis alternatives. Proteases are attractive anti-protozoal drug targets, having documented roles in Blastocystis. Serine proteases are present in both hepatitis C virus and Blastocystis. Since drug repositioning is quite trendy, the in vitro efficacy of simeprevir (SMV), an anti-hepatitis serine protease inhibitor, against Blastocystis was investigated in the current study.

Methods: Stool samples were collected from patients, Alexandria, Egypt. Concentrated stools were screened using direct smears, trichrome, and modified Ziehl-Neelsen stains to exclude parasitic co-infections. Positive stool isolates were cultivated, molecularly subtyped for assessing the efficacy of three SMV doses (100,150, and 200 μg/ml) along 72 hours (h), on the most common subtype, through monitoring parasite growth, viability, re-culture, and also via ultrastructure verification. The most efficient dose and duration were later tested on other subtypes.

Results: Results revealed that Blastocystis was detected in 54.17% of examined samples. Molecularly, ST3 predominated (62%), followed by ST1 (8.6%) and ST2 (3.4%). Ascending concentrations of SMV progressively inhibited growth, viability, and re-culture of treated Blastocystis, with a non-statistically significant difference when compared to the therapeutic control metronidazole (MTZ). The most efficient dose and duration against ST3 was 150 µg/ml for 72 h. This dose inhibited the growth of ST3, ST1, and ST2 with percentages of 95.19%, 94.83%, and 94.74%, successively and viability with percentages of 98.30%, 98.09%, and 97.96%, successively. This dose abolished Blastocystis upon re-culturing. Ultra-structurally, SMV induced rupture of Blastocystis cell membrane leading to necrotic death, versus the reported apoptotic death caused by MTZ. In conclusion, 150 µg/ml SMV for 72 h proved its efficacy against ST1, ST2, and ST3 Blastocystis, thus sparing the need for pre-treatment molecular subtyping in developing countries.

Keywords: Blastocystis subtypes; CV, central vacuole; DMSO, Dimethyl Sulfoxide; IBS, irritable bowel syndrome; In vitro; MLO, Mitochondrion-like organelle; MTZ, Metronidazole; PCR, Polymerase chain reaction; Re-culture; SEM, Scanning electron microscopy; SMV, Simeprevir; ST, subtypes; Simeprevir; TEM, Transmission electron microscopy; Ultrastructure; Viability.

Graphical abstract

Fig. 1

Agarose gel analysis of PCR-amplified products of Blastocystis.1a: Amplification with SB227 primer (526 bp) (ST3), 1b: Amplification with SB83 primer (351 bp) (ST1), 1c: Amplification with SB155 primer (650 bp) (ST2).

Fig. 2

Light microscopic findings of Blastocystis forms detected after drug testing by iodine smears and eosin-brilliant cresyl blue dye (×400). 2a: Vacuolar (V) form by iodine smear, 2b: Vacuolar (V) and cystic (C) forms by iodine smear, 2c: Vacuolar (V) and granular (G) forms by iodine smear, 2d: Amoeboid (A) form by iodine smear, 2e: Viable vacuolar (V) form, 2f: Viable cyst (C) form, 2g: Non-viable vacuolar (V) and granular (G) forms, 2h: Non-viable amoeboid (A) form.

Fig. 3

Growth and viability profiles of ST3 Blastocystis along the three-times intervals for the SMV tested doses versus their respective non-treated and MTZ-treated controls. a: Growth profile b: Viability profile. I (non-treated control), III (MTZ-treated control): IIIa (10 µg/ ml), IIIb(100 µg/ml), IIIc(250 µg/ml), IV (SMV-treated): IVa(100 µg/ml), IVb(150 µg/ ml), IVc(200 µg/ml).

Fig. 4

Percentage inhibition of ST3 Blastocystis multiplication and viability by MTZ and SMV versus its respective non-treated control group. 4a: Percentage inhibition of ST3 Blastocystis multiplication, 4b: Percentage inhibition of ST3 Blastocystis viability. I (non-treated control), II (Solvent control), III (MTZ-treated control): IIIa (10 µg/ ml), IIIb(100 µg/ml), IIIc(250 µg/ml), IV (SMV-treated): IVa(100 µg/ml), IVb(150 µg/ ml), IVc(200 µg/ml).

Fig. 5

Re-culturing of ST3 Blastocystis previously inoculated with SMV at different incubation periods versus its respective non-treated and treated controls. I (non-treated control), II (Solvent control), III (MTZ-treated control): IIIa(10 µg/ml), IIIb(100 µg/ml), IIIc (250 µg/ml), IV (SMV-treated): IVa(100 µg/ml), IVb(150 µg/ml), IVc(200 µg/ml).

Fig. 6

Effect of 150 µg/ml SMV for 72 h on the percentage inhibition of multiplication, viability and viability post re-culture of ST1 and ST2 Blastocystis in comparison to ST3.

Fig. 7

(a-f): Scanning electron microscopy of the non-treated Blastocystis and SMV-treated groups. a: Non-treated Blastocystis showing its oval shape (X19000). b: Non-treated Blastocystis showing its round shape and fibrous surface coat attached to cell surfaces of Blastocystis (arrow) (×30000). c: SMV-treated Blastocystis showing remarkable convolution and folding (arrow) (×30000). d & e: SMV-treated Blastocystis showing remarkable convolution and folding and surface membrane pores (arrow) (×30000). f: SMV-treated Blastocystis amoeboid form (×35000).

Fig. 8

(a-e): Transmission electron microscopy of non-treated Blastocystis and SMV-treated groups. a: Non-treated Blastocystis vacuolar form showing central vacuole (CV) and thin rim of cytoplasm with peripheral nuclei (N) (×6,000). b: Non-treated Blastocystis granular form showing central vacuole (CV) almost filled with granules of different electron densities and surrounded with peripheral rim of the cytoplasm (×8,000). c: SMV-treated Blastocystis vacuolar form showing central vacuole (CV) devoid of any electron-dense particles and showing mitochondrion-like organelle (MLO) (×6,000). d: SMV-treated Blastocystis amoeboid form showing central vacuole (CV) devoid of any electron-dense particles and showing mitochondrion-like organelle (MLO) (×6,000). e: SMV-treated Blastocystis showing central vacuole (CV) devoid of any electron-dense particles, and showing mitochondrion-like organelle (MLO) and plasma membrane rupture (arrow) (×6,000).