Effect of artemisinin–naphthoquine phosphate on the viability of Schistosoma haematobium adult worms

Over the 72 h, all S. haematobium worms cultured in RPMI-1640 drug-free medium revealed distinctive wavy as well as peristaltic motion, with maintenance of male–female mating. Worms incubated with PZQ showed immediate muscle contraction, without separation of worms, and all worms were dead 48 h after incubation. Interestingly, incubation of worms with the different concentrations (1–40 μg/ml) of CO-ArNp led to dissociation of S. haematobium pairs and their irreversible debilitation within 24 h; also, all schistosomes displayed only minimal activity, with slow convulsions and abnormal contractions, which were more prominent in female worms that died earlier than males, while male worms appeared shrunken and markedly more coiled than females, and all worms were dead after incubation with 20 and 30 μg/ml for 72 and 48 h, respectively (fig. 1). The LC₅₀ and LC₉₀ of CO-ArNp recorded 72 h post incubation were 11.1 μg/ml and 18.5 μg/ml, while those of PZQ were 0.2 μg/ml and 0.3 μg/ml, respectively, as shown in table 1.

Fig. 1. In vitro effect of artemisinin–naphthoquine phosphate at various concentrations on the mortality of adult Schistosoma heamatobium under culture conditions. The data were generated from at least two independent studies. Note that the bars show the range of percentage of dead worms in the experiments, and the intervals on the x-axis are not equal.

Table 1. In vitro schistosomicidal activity of artemisinin–naphthoquine phosphate combination and a reference drug (praziquantel) on adult Schistosoma haematobium worms after three days’ exposure.

In comparison, treatment of S. mansoni worms with 20 and 40 μg/ml of the same drug combination killed all parasites at the same time points, but males were more afflicted and died earlier than females (El-Beshbishi et al., Reference El-Beshbishi, El Bardicy, Tadros, Ayoub and Taman2015). In an in vitro study performed by Eissa et al. (Reference Eissa, El Bardicy and Tadros2011), the authors declared that miltefosine killed all worms after five days of incubation with 10 μg/ml, and the drug was more active on S. haematobium than on S. mansoni adult worms. Indeed, S. haematobium and S. mansoni, two major human schistosomes, have different sensitivities to particular antischistosomal drugs, which may be attributed to specific intrinsic biochemical processes (Camacho & Agnew, Reference Camacho and Agnew1995), and depend on the enzyme(s) involved (Pica-Mattoccia et al., Reference Pica-Mattoccia, Novi and Cioli1997). Schistosoma haematobium adults exposed to 20 μg/ml artemether for 72 h, showed no apparent changes (Xiao et al., Reference Xiao, Chollet, Utzinger, Matile, Mei and Tanner2001). Our results may be attributed to drug–drug interaction between naphthoquine phosphate and artemisinin exerting a more toxic effect on the worms than using artemisinins alone. Artemisinins interact with haemin and other iron systems, producing toxic compounds and free radicals, which cause oxidative damage of the parasite (Xiao et al., Reference Xiao, Chollet, Utzinger, Matile, Mei and Tanner2001). Additionally, it is well known that 4-aminoquinoline derivatives interfere with haemozoin formation, hence the accumulated free haem kills the parasite through oxidative stress (Corrêa Soares et al., Reference Corrêa Soares, Menezes and Vannier-Santos2009).

Ultrastructure analysis of artemisinin–naphthoquine phosphate-induced surface damage in Schistosoma haematobium adult worms

The characteristics of the tegument of untreated worms (fig. 2a, b) were compared with those exposed to CO-ArNp. Male and female worms incubated with 20 μg/ml CO-ArNp for 48 h showed surface membrane ultrastructural damage (figs 3 and 4). Tegumental changes were more prominent and outspread in male than female schistosomes. Generally, all inspected male worms showed extensive oedema of the tegument, with thinning of tegumental ridges. There were also pronounced changes in the tubercles in the form of reduction in size and number, with shortening and loss of spines leaving a smooth surface (fig. 3a). Erosion of the tegument in several areas of the body, cleft formation with exposure of the subtegumental tissue (fig. 3b), exfoliation of the tegument and roughness of the surface (fig. 3c), as well as extreme distortion of the tegumental folds, with concomitant collapse and deformity of the tubercles, were encountered (fig. 3c, d). Moreover, in vitro incubated worms with CO-ArNp showed longitudinal muscle contraction, and the worms were bent, shortened and deformed, with marked corrugation at the anterior end (fig. 3e). Besides, the gynaecophoric canal revealed contraction deformity at the edges and formation of protuberance in the tegument (fig. 3f). Disfigurement of the ventral sucker was also noticed in the form of marked oedema in > 50% of treated worms (fig. 3g), while marked contraction and deformity, with erosion and vesicle formation, were seen in the remaining males, as well as evident shrinkage and wrinkling of the area between the oral and ventral suckers (fig. 3h). Xiao et al. (Reference Xiao, Shen, Chollet, Utzinger and Tanner2000) declared that such damage to the suckers may predispose schistosomes to loss of power to adhere to the vasculature, hence making uptake of nutrients more difficult. In turn, this may affect the worms’ maturity, fecundity and ability to deposit eggs – the major contributory factor to schistosomiasis morbidity.

Fig. 2. Normal surface ultrastructure of Schistosoma haematobium adult worms (controls). (a) Normal dorsal tegument of a male worm (×5000); S: spines, t: tubercles, w: wrinkles. (b) Normal tegument of a female worm (×3000); fi: fissures.

Fig. 3. SEM micrographs of adult Schistosoma haematobium male worms incubated with 20 μg/ml CO-ArNp for 48 h. (a) Tegumentary changes in a male worm, including reduction in size and number of tubercles, and loss of spines (×5000); r: tubercles reduced in size, ls: tubercles showing loss of spines. (b) Tegumental erosion and splitting (×2400); sp: splitting, e: erosion. (c) Tegumental peeling off, with deformity of the tubercles (×3000); p: peeling. (d) Extensive oedema of the tegument, with deep pitting, as well as marked atrophy of the tubercles (×1600); pi: pitting. (e) Male S. haematobium, showing contraction and deformity at the anterior end, with bending at the mid-body region (×270); b: binding. (f) Gynaecophoric canal with small vesicles and contraction deformity at the edges (×1000); v: vesicles. (g) Marked swelling of the ventral sucker and the tegument around it (×1200); sw: swelling. (h) Ventral sucker, showing contraction and deformity, erosion and vesicles, together with shrinkage of the area between suckers (×1600); df: deformity, e: erosion, sh: shrinkage.

Fig. 4. SEM micrographs of adult Schistosoma haematobium female worms incubated with 20 μg/ml CO-ArNp for 48 h. (a) Tegumental changes in the mid-body, including loss of the normally arranged fissures in some parts, with contraction of the tegument as well as furrows and vesicle formation (×3000); f: furrows, v: vesicle. (b) Marked oedema and pitting of the tegument, as well as peeling off (×1000); pi: pitting, p: peeling. (c) Swollen female tegument with deep crake formation, exposing the subtegumental tissue (×3000); c: crake.

Treatment with the same dose of CO-ArNp and for the same time point revealed less extensive tegumental changes in females, as the damage was localized to the mid-body. Tegumental swelling with absence of normal arranged fissures was evident in all female worms examined; besides, constriction and collapse of the tegument with furrow formation and vesiculation (fig. 4a). In addition, the tegumental surface of some worms (< 50%) was distorted, exfoliated (fig. 4b) and eroded, leaving profound fissures with exposure of the subtegumental tissues (fig. 4c).

The tegument has a pivotal role in protection, immunomodulation, sensation, nutrient ingestion and synthesis of some proteins, osmoregulation, and excretion (Skelly & Wilson, Reference Skelly and Wilson2014). It is noteworthy that alterations in the tegument structure deteriorate its functions and lead to worm death (Shaw & Erasmus, Reference Shaw and Erasmus1987). The results of the present study demonstrated the schistosomicidal capability of CO-ArNp, wherein S. haematobium females revealed faster and higher susceptibility to the combined regimen compared to males, although male worms showed the most prominent and extensive tegumental damage. Corroborating our results, Soliman & Ibrahim (Reference Soliman and Ibrahim2005) reported that both atorvastatin alone and its combination with medroxyprogesterone acetate administered to experimentally infected hamsters led to more marked tegumental damage in S. haematobium male worms than females. Similarly, Eraky et al. (Reference Eraky, Aly, Selem, El-Kholy and Rashed2016) reported more pronounced tegumental alterations in male worms incubated with phytol than females exposed to the same dose and time period. Importantly, the differences in the severity and extent of morphological changes, and death rates and time, caused by Co-ArNp may be ascribed to the ability of the drug to attack different targets in both genders, based on the structural, physical and biochemical variations between schistosome males and females (Shaw & Erasmus, Reference Shaw and Erasmus1987). Drugs attacking schistosomes in a sex-dependent pattern could control disease morbidity and limit the spread of infection by decreasing the number of eggs released.

Considering the results of this study, artemisinin–naphthoquine phosphate exerts in vitro effects on S. haematobium adult worms, as it affects the morphology, tegumental surface and viability of the exposed worms. Therefore, this work opens up perspectives for future research on this drug in vivo to disclose its mechanism(s) of action.