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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 31  |  Issue : 3  |  Page : 786-794

Therapeutic effect of phenyl vinyl sulfone and nitazoxanide on experimentally infected mice with cryptosporidiosis


1 Department of Parasitology, Faculty of Medicine, National Liver Institute, Menoufia University, Menoufia, Egypt
2 Department of Clinical and Molecular Parasitology, National Liver Institute, Menoufia University, Menoufia, Egypt
3 Department of Pathology, Faculty of Medicine, National Liver Institute, Menoufia University, Menoufia, Egypt

Date of Submission22-Oct-2017
Date of Acceptance03-Dec-2017
Date of Web Publication31-Dec-2018

Correspondence Address:
Shaimaa A Farag
Clinical and Molecular Parasitology department, National Liver Institute, Shebin El kom, Menoufia governorate
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_712_17

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  Abstract 


Objective
Evaluation of the therapeutic effect of phenyl vinyl sulfone (PVS) as cysteine protease inhibitors, nitazoxanide (NTZ), and combined therapy on Cryptosporidium parvum infection regarding the parasitological and histopathological parameters.
Background
Cryptosporidium spp. had been identified as the second most important diarrheal pathogen after rotavirus in young children. The immune status of the host plays a critical role. There is no reliable treatment for cryptosporidiosis, as the only approved drug NTZ provides no benefit for immunocompromised patients.
Materials and methods
A total of 180 female laboratory-bred Swiss albino mice were divided into two major divisions, immunocompetent and immunosuppressed, with the following groups for each one, respectively: negative control (I and VI), infected control (II and VII), infected treated with PVS (III and VIII), infected treated with NTZ (IV and IX), and infected treated combined (V and X). Stool examination for oocyst shedding was done at different days postinfection, and mice were killed at 18 and 30 days postinfection (groups A and B, respectively). Histopathological assessment of the ileum was done, and the endogenous developmental stages of the parasite were counted.
Results
Combined therapy in groups V and X resulted in the highest reduction in Cryptosporidium spp. oocysts shedding (P = 0.044 and <0.001, respectively) and mean number of endogenous developmental stages in group X (P < 0.001) than either drugs when used alone. This is followed by NTZ-treated groups and then cysteine protease inhibitors-treated groups, which gave the least reduction.
Conclusion
Combined therapy is more effective than either NTZ or PVS used alone. NTZ is still a better treatment option than PVS.

Keywords: Cryptosporidium spp., cysteine protease inhibitors, Giemsa stain, nitazoxanide, periodic acid–Schiff stain, phenyl vinyl sulfone


How to cite this article:
El Shafei OK, Saad AGE, Harba NM, Sharaf OF, Samaka RM, Farag SA. Therapeutic effect of phenyl vinyl sulfone and nitazoxanide on experimentally infected mice with cryptosporidiosis. Menoufia Med J 2018;31:786-94

How to cite this URL:
El Shafei OK, Saad AGE, Harba NM, Sharaf OF, Samaka RM, Farag SA. Therapeutic effect of phenyl vinyl sulfone and nitazoxanide on experimentally infected mice with cryptosporidiosis. Menoufia Med J [serial online] 2018 [cited 2019 Jun 19];31:786-94. Available from: http://www.mmj.eg.net/text.asp?2018/31/3/786/248760




  Introduction Top


Cryptosporidium species are unicellular apicomplexan protozoan parasites that infect humans and a wide variety of other vertebrates[1]. Water is the most commonly reported way of transmission in Cryptosporidium spp. outbreaks[2].

In young children, Cryptosporidium spp.had beenidentified as the second most important diarrheal pathogen after rotavirus[2],[3]. In developing countries, diarrhea is the third leading cause of deaths, and diarrhea in children is associated with subsequent impairment in growth, physical fitness, and cognitive function[1].

Cryptosporidium spp. has been considered as one of the most common opportunistic parasites. The immune status of the host plays a critical role in determining the severity of cryptosporidiosis. Infection is self-limited in immunocompetent hosts but can be severe and persistent in the immunocompromised such as patients with AIDS or malnourished children[1].

In immunocompetent hosts, the organisms are mainly localized to the distal small intestines, whereas in immunocompromised, the parasites have been detected in the gut, biliary tract, and respiratory tract[4].

There is no reliable treatment for cryptosporidiosis especially in immunocompromised patients. Nitazoxanide (NTZ) is the only Food and Drug Administration-approved drug for the treatment of cryptosporidiosis in immunocompetent patients older than 1 year, but it is not approved for patients with AIDS[5]. Moreover, clinical trials demonstrated that there is no difference in mortality or parasitological responses between the patients who received NTZ and placebo[6]. Therefore, the need for specific and effective therapy remains a goal.

Cysteine proteases (CPs) are enzymes for protein catabolism through the cleavage of peptide bonds. Parasitic CPs are included in two clans (classes): clan CA (lysosomal cathepsins) and clan CD (caspase). CP facilitate cell invasion, nutritive degradation of host proteins, and the modification of parasite proteins during life cycle transitions.

The most commonly used cysteine protease inhibitors (CPIs) are vinyl sulfones (VSs) and histone deacetylases (HDACs)[7]. Small-molecule inhibitors targeting parasite clan CA enzymes have shown much promise for their use in the therapy of parasitic diseases. N-methylpiperazine-phenylalanyl-homophenylalanyl-vinylsulfone-phenyl (K11777) was tested on Cryptosporidium spp.in vitro and in γ-interferon receptor knockout (IFN-γR-KO) mouse model. The infected animals regained weight and appeared to be healthy when they were killed[8]. Moreover, Ndao et al.[9] found that K11777 displays potent anticryptosporidial activity in vitro and in vivo.

Vinyl sulfone compounds had been tested for treatment of Plasmodium falciparum and Plasmodium vivax and resulted in inhibition of CPs. In Fasciola gigantica, it showed potent activity and resulted in immediate death of the adult flukes[7]. To the best of our knowledge, phenyl vinyl sulfone (PVS) is not tested before as a proposed treatment of cryptosporidiosis.

This work aims to evaluate the therapeutic effect of PVS against cryptosporidiosis on immunocompetent and immunosuppressed mice in comparison with NTZ and the effect of their combination regarding the oocyst shedding intensity in stool, mean number of developmental stages in ileum, and the pathological changes.


  Materials and Methods Top


Ethical consideration

All the experiments were carried out in accordance with the recommendations in the guide for the care and use of laboratory animals of the Ethics Committee of Theodor Bilharz Research Institute (TBRI).

Experimental design

This study was conducted on 180 laboratory-bred female albino mice weighing ∼20–25 g. They were purchased from the Schistosome Biological Supply Program in TBRI. The mice were kept on a standard diet and water in the biological unit of TBRI under a temperature of 24°C.

Mice were classified into 10 groups, and each was divided into two subgroups (groups A and B) according to time of scarification [18 and 30 days postinfection (PI), respectively]: group I, uninfected and untreated; group II infected with Cryptosporidium parvum (C. parvum) oocysts and untreated; group III, infected and treated with CPIs; group IV, infected and treated with NTZ; group V, infected and treated combined; group VI, immunosuppressed with dexamethasone (DEX) and uninfected; group VII, immunosuppressed, infected, and untreated; group VIII, immunosuppressed, infected, and treated with CPIs; group IX, immunosuppressed, infected, and treated with NTZ; and group X, immunosuppressed, infected, and treated combined (all groups were composed of 20 mice for each one, except groups I and VI, which were composed of 10 mice).

Immunosuppression was induced by giving DEX (Dexazone 0.5 mg), which was manufactured and provided by Kahira Pharmaceuticals and Chemical Industries Company (Shoubra, Cairo, Egypt), orally at a dose of 0.25 μg/g/day for 14 successive days before infection. The mice were maintained on DEX throughout the experiment[10].

Cryptosporidium spp. oocysts were obtained by collection of scrapings of the ileal mucous membrane and cecal content from naturally infected calves from different slaughter houses[11]. The specimens were examined for Cryptosporidium spp. oocysts by modified Ziehl–Neelsen (Z-N) staining method; a commercially available Z-N stain ready for use from Egyptian diagnostic media was used[12].

Positive calf specimens were preserved by an equal volume of 2.5% potassium dichromate solution (El Gomhouria Company for Drugs, Tanta, Egypt) at 4°C for use as an infecting inoculum[11].

The infective inoculum was prepared in accordance with Reese et al.[13]. For counting Cryptosporidium spp. oocysts in 1 ml of specimen sediments, 50 μl from the sediment was taken and stained by modified Z-N acid-fast stain, and then the mean of three counts of oocysts per high-power field was calculated and multiplied by 20 to get the number of oocysts in 1 ml[14].

All mice in the studied groups except the control groups were infected orally with the prepared inoculums; this occurred on day 15 of DEX taking in the immunosuppressed groups[10]. The animals were inoculated intraesophagally with the prepared inoculums using a tuberculin syringe connected to a polythene tube. The amount given to each mouse was adjusted to contain ∼1 × 105 oocysts[11].

Drugs

Nitazoxanide

NTZ (Nitazode 100 mg) is manufactured by Sigma Pharma (Egypt) as a suspension for pharmaceutical industries, for Al Andalous Medical Company, Cairo, Egypt. Nitazode was administrated orally in a daily dose of 100 mg/kg BW started 10 days PI and lasted for 10 days[10],[15].

Phenyl vinyl sulfone

PVS powder was manufactured and provided by Sigma-Aldrich (USA). A stock solution was prepared as stated by Ndao et al.[9]; the drug was given orally at a dose of 35 mg/kg of BW twice per day started 10 days PI for a period of 10 consecutive days.

The mice were killed by cervical dislocation at 18 and 30 days PI (groups A and B, respectively). Control groups were killed at the same time as the other groups.

Stool examination and counting of oocyst (oocyst shedding)

From the second day of inoculation, fresh fecal pellets were collected from each group daily until the first detection of oocysts in stools was recorded. Then fresh fecal pellets from each mouse in each group were collected separately every 2 days over the experiment, according to the different groups[11].

Each sample was concentrated by formol–ether concentration method and then 1 mg was prepared as a fecal smear and stained by the modified Z–N stain and examined microscopically. The number of Cryptosporidium spp. oocysts was counted in 10 high-power field; the means of oocysts per mg for each animal and then for each group of animals were calculated[11].

Histopathological study

The terminal 2 cm of the ileum of killed mice in all groups were submitted to Pathology Department, Faculty of Medicine, Menoufia University, for routine processing. Serial sections 5 μm in thickness were stained with the following stains: hematoxylin and eosin (H and E), Giemsa, and periodic acid–Schiff (PAS); all these stains were provided by Midco Trade Company (Giza, Egypt).

Assessment of hematoxylin and eosin-stained slides

Pathological changes in the submitted tissues were recorded. Mucosal changes such as crypt villous ratio, blunting of villi, atrophy, degree of atrophy, ulceration, dysplasia, and reactive atypia, and lamina propria changes such as degree of inflammatory infiltrate, status of blood vessels, and edema were recorded.

Moreover, counting the endogenous stages of the parasite in ten villous crypt units, the mean numbers per villous crypt unit for each group of animals were calculated with assessment of the cure rates in treated groups[10].

Assessment of Giemsa-stained and periodic acid–Schiff-stained slides

Giemsa-stained and PAS-stained slides were prepared in group A to highlight Cryptosporidium spp. developmental stages at the brush border of the intestinal villi[16],[17].

Statistical analysis

Data were collected, tabulated, and statistically analyzed by using statistical package for the social sciences program, version 20 (SPSS; SPSS Inc., Chicago, Illinois, USA). Descriptive statistics included mean, SD, and percentage.

Analytical statistics included Fisher's exact test, Student's t-test, and Mann–Whitney test. Statistical significant was considered when P value less than 0.05[18].


  Results Top


Parasitological results (oocyst shedding)

Using modified Z-N stain, oocysts appear in stool as round objects (4–5 μm), with some degree of red staining of the internal structure [Figure 1]a,[Figure 1]b, [Figure 1]c.
Figure 1: (a) Oocyst shedding in stool in immunocompetent groups at different days postinfection. (b) Oocyst shedding in stool in immunosuppressed groups at different days postinfection. (c) Cryptosporidium spp. oocysts in stool stained with modified Ziehl–Neelsen stain (×1000). (d) Sections showed destructed mucosal glands with reactive atypia (circles) (hematoxylin and eosin, ×400). (e and f) Developmental stages at the brush borders of the epithelial cells of intestinal villi appeared as small round or oval basophilic bodies ∼2–5 μm in diameter (hematoxylin and eosin, ×1000); (e) group IIb (arrows); (f) group VIIb (arrows).

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Combined therapy in immunocompetent group V and immunosuppressed group X showed highest reduction in the mean number of oocyst shedding per milligram of stool, followed by treatment with NTZ (groups IV and IX), and then with CPIs (groups III and VIII). In group V at 30 days, mean oocyte shedding PI was 0.778 ± 1.20 (P = 0.044) [Figure 1]a, whereas in group X, the mean was 4.43 ± 0.49 (P < 0.001), in all treated immunosuppressed groups [Figure 1]b.

There were statistical significant differences between infected immunocompetent groups and corresponding immunosuppressed groups regarding oocyst shedding at different days PI, whereas the oocyst shedding was higher in immunosuppressed groups than corresponding immunocompetent groups all over the study (P < 0.001 for all) [Table 1].
Table 1: Comparison between infected immunocompetent and corresponding immunosuppressed groups regarding oocyst shedding at different days PI

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Histopathological results

The endogenous stages of the parasite appeared as small round [Figure 2] or oval bodies ∼2–5 μm in diameter using H and E stain [Figure 1]e and [Figure 1]f, PAS stain [Figure 2]b,[Figure 2]c,[Figure 2]d, and Giemsa stain [Figure 2]e and [Figure 2]f. The parasite was easily demonstrated in PAS-stained sections than Giemsa-stained and H and E-stained sections.
Figure 2: (a) Ileal mucosa showed blunting of villi and moderate degree of chronic inflammatory infiltrates in group IIa (hematoxylin and eosin, ×200). (b) Ileal mucosa in group VIIa, displayed massive atrophy, ulceration, decreased crypt villous ratio, and blunting of villi. Lamina propria showed moderate degree of cellular infiltrates and extensive edema (hematoxylin and eosin, ×100). (c) Ileal mucosa in group IIb showed mild atrophy, ulceration with sloughing of villi, and scarce numbers of lymphocytes (hematoxylin and eosin, ×100). (d) Ileal mucosa in group VIIb showed severe atrophy of mucosa, severe blunting of villi, and severe inflammatory infiltrates (hematoxylin and eosin, ×100). (e) Ileal mucosa in group IXb with moderate degree of atrophy, decreased crypt villous ratio, and blunting of villi. Lamina propria showed extensive degree of edema and mild degree of inflammatory infiltrates (hematoxylin and eosin, ×100). (f) Ileal mucosa in group VIIIb showed massive atrophy, ulceration, decreased crypt villous ratio, and blunting of villi. Lamina propria showed moderate degree of cellular infiltrates and extensive edema (hematoxylin and eosin, ×100).

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Different histopathological parameters were assessed in H and E-stained slides as demonstrated in [Figure 3] and [Figure 1]d.
Figure 3: (a) High-power view demonstrated preserved periodic acid–Schiff positive goblet cell and well-appreciated brush border in group I (periodic acid–Schiff, ×400). Cryptosporidium spp. parasites disrupt the uniform dark pink brush border focally appeared as blue to purple dots (b) group IIa, (c) group VIIa, (d) group Xa (periodic acid–Schiff, ×1000 for b, c, and d). Cryptosporidium spp. parasites demonstrated at the brush border of mucosa; (e) ileal section in group IIa (arrows); and (f) ileal section in group VIIa s (circles) (Giemsa, ×1000 for e and f).

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Developmental stages/villous crypt unit was calculated in hematoxylin and eosin-stained slides

The highest reduction in mean number of endogenous developmental stages of C. parvum/villous unit between immunocompetent groups A was in group V, followed by group IV (P < 0.001 for both) [Table 2].
Table 2: Comparison of mean number of endogenous developmental stages of C. parvum/villous unit among immunocompetent and immunosuppressed subgroups a and b

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There were statistical significant reduction of mean number of endogenous developmental stages between immunosuppressed groups A and B in all groups, except group VIIIa [Table 2].

There were statistical significant differences between immunocompetent subgroups and the corresponding immunosuppressed subgroups regarding the number of endogenous developmental stages as (P < 0.001 for all) (results not showed in table).

Cure rate in different groups B at the end of the study

There was a significant difference between them (P = 0.001) [Table 3].
Table 3: Cure rate in different group B at the end of the study (30 days postinfection)

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Mortality rate in infected groups throughout the study

There was a significant difference between them (P = 0.03). There was no deaths in group II or NTZ-treated group IV, whereas the highest mortality rate was detected in group VII (30%) followed by PVS-treated groups VIII and III (25, 20%, respectively). It was noticed that there was a high mortality rate in PVS-treated groups, whereas there was a reduction in mortality rate in NTZ-treated groups (results not showed in table).


  Discussion Top


In current study, the onset of oocyst shedding in stool was on the second day PI in immunosuppressed mice and the fourth day in the immunocompetent groups; this finding coincides to some extent with Chai et al.[19] who stated that the starting point of oocyst excretion was on fourth day PI in both immunocompetent and immunosuppressed rats.

Lacroix et al.[20] found that the duration of oocyst shedding was ∼3–4 weeks. In this study, the duration of oocyst shedding in immunocompetent mice (group II) was ∼30 days, which was in agreement with Abdou et al.[11]. Although in infected immunosuppressed group VII, it continued to shed oocysts in large numbers till the 30th day. This result was in agreement with Benamrouz et al.[21] who showed that in Dex SCID mice inoculated with low inocula, the oocysts excretion increased reaching a mean of more than 10.000 oocysts/g of feces at 45 days PI. On the contrary, Chai et al.[19] reported a short patent period (about 9 days), and this discrepancy is probably attributable to the different durations of immunosuppression.

In this work, the maximum oocysts shedding was observed in immunocompetent group II at 13 and 15 days PI (mean: 16.05 ± 0.48) and in immunosuppressed group VII from 20th to 22th days PI (mean: 23.81 ± 0.59); this finding is contradictory with Heine et al.[22] who mentioned that the intensity of shedding peaked 1 week PI in both immunocompetent and immunosuppressed animals, and this may be because of the different species of mice used in their study.

In this study, the terminal part of the ileum was chosen to assess the course of infection because it was reported to be the site with the heaviest burden of intestinal cryptosporidiosis in both immunocompetent and immunosuppressed mice, as it has been suggested that the biochemical conditions in the ileum are favorable for the parasite, and also the presence of specific receptors[10],[11].

The endogenous stages of the parasite were specifically seen in microvillous region of the intestinal mucosa and also detected in the lumen of the crypts in immunosuppressed mice; this result supports those of Mead et al.[23].

Giemsa-stained and PAS-stained sections were used to highlight Cryptosporidium spp. developmental stages at the brush border of the intestinal villi[17]. Cryptosporidium spp. organisms are positive for Giemsa stain, as they are stained deep purple[16]. Although they are PAS negative, they appeared as blue to purple dots disrupting the uniform dark PAS-positive pink brush border focally[17].

In the present study, PAS stain gave more demonstration of Cryptosporidium spp.than Giemsa stain. This may be explained by Shams et al.[24] who found that Giemsa staining technique is problematic because of the prolonged staining period, the perilous decolorization stage, and poor color disparity.

It can be noted that the severity of infection (regarding intensity of oocyst shedding in stool and the mean number of endogenous developmental stages of C. parvum/villous unit) and its duration were significantly higher in infected immunosuppressed mice than the corresponding immunocompetent ones throughout the duration of the experiment (P < 0.001) either treated or not whatever the drug used. These findings were in agreement with Certad et al.[25] and could be explained by Rasmussen et al.[26] who mentioned that treatment of rats by DEX depressed both B and T cells responses.

Combined therapy in groups V and X showed the highest reduction in the mean number of oocyst shedding and the mean number of endogenous stages of the parasite in the ileum, followed by NTZ-treated groups (groups IV and IX), with the least reduction seen in CPIs-treated groups III and VIII.

The effects of NTZ on reduction of oocyst shedding in immunocompetent or immunosuppressed animals were to some extent in agreement with Ollivett et al.[27] who revealed that 85% of the NTZ-treated calves stopped oocysts shedding by the end of the observation period. In immunosuppressed animals, similarly a significant reduction in oocysts excretion was obtained in immunosuppressed gerbil at dose of 200 mg/kg for 12 days[28].

On the contrary, Theodos et al.[15] mentioned that no significant difference in oocysts shedding was observed between groups of mice treated with NTZ and placebo control group. In addition, the prophylactic and therapeutic use of NTZ in calves did not show the expected positive effect on Cryptosporidium spp. infection[29]. These discrepancies may be explained by different drug formulations, doses, and different animal models used.

Reduction in the number of endogenous stages was highly significant in group IVa (P2 <0.001) but was not significant in group IVb (P5 =0.16), whereas groups IXa and IXb showed significant reduction in both groups (P8 and P11 were 0.015 and <0.001, respectively). These results were in agreement with Abdou et al.[11].

Results of CPIs were to some extent in agreement with Ndao et al.[9] where treatment of IFN-γR-KO mouse model with K11777 for 10 days caused gradually decrease in oocyst shedding to very low levels by day 35 PI (P < 0.01). Moreover, intestinal sections from treated mice (105 mg/kg BID orally) showed small numbers of endogenous developmental stages.

The percentage of complete cure in NTZ-treated group IV (50%) was in agreement to some extent with Abdou et al.[11] who stated that after NTZ treatment, infected immunocompetent mice stop oocyst shedding in eight mice of the 10.

However, there was no complete cure in treated immunosuppressed groups (VIII, IX, and X) at the end of the study, as proved by the presence of Cryptosporidium spp. oocysts in their stool smears and the endogenous stages of Cryptosporidium spp. in their ileal sections. Amadi et al.[30] recorded cessation of diarrhea in only 56% of patients receiving NTZ compared with 23% of patients receiving placebo, whereas Sadek and El-Aswad[10] stated that NTZ caused 61.53% cure percentage in immunosuppressed rats. The discrepancy in results could be contributed to different models used.

Mortality rate was higher in immunosuppressed groups than in immunocompetent groups. These findings coincided with those of Sadek and El-Aswad[10] and also with Mead et al.[23] who demonstrated that deaths apparently resulted from hepatic dysfunction.

Immunosuppressed group VIII treated with CPIs showed no significant reduction in mortality rates. However, in immunocompetent group III, it was 20%, whereas there was no deaths in control group II. This result was surprising, and causes of deaths in groups treated with PVS (III, VIII, V, and X) should be further investigated to know the reason. These results contradict those of Sajid et al.[8] where K11777 (1 and 2 mg BID)-treated infected mice 48 h PI and continued for 14 days, 90% of mice 9/10 mice survived with 1 mg, and with 2 mg survivors were 60% 6/10. Moreover, Ndao et al.[9] observed that K11777 rescued mice from lethal Cryptosporidium spp. infection, as there was a dose-dependent increase in survival of mice.

Reduction in mortality rate in NTZ-treated group IX in this study was in agreement with Checkley et al.[3] who recorded significant reduction in mortality in malnourished children treated with NTZ.

Pathological changes observed in immunocompetent group II were in agreement with Mahmood et al.[31], where they began to subside even without treatment in group IIb as cryptosporidiosis is self-limiting disease in immunocompetent hosts.

Major histopathological changes were observed at groups VIIa and VIIb. Similar changes were observed by Soufy et al.[29] and explained by that it could be attributed to Cryptosporidium spp. displacing brush borders resulting in shortening and fusing of the villi. Villous atrophy may be explained by toxins secreted by Cryptosporidium spp. that directly damage the epithelial cells.

On comparison between groups IIa and VIIa, it was noted that the degree of inflammatory infiltrate and reactive atypia were more in group IIa. This could be explained that during Cryptosporidium spp. infection, T-lymphocyte, macrophages, and polymorphonuclear cells migrate to lamina propria as a part of the host defense mechanisms against this parasite[32]. Therefore, the signs of inflammation will appear earlier in immunocompetent than immunosuppressed (e.g. at 18 days PI) individuals and will be more severe, whereas later at 30 days PI (group B), inflammation starts to subside in immunocompetent groups while still present and severe in immunosuppressed groups.

Combined therapy in group V showed the best improvement in pathological changes, where the degree of inflammation was mainly mild with significant improvement in the degree of reactive atypia.

NTZ-treated immunosuppressed group IX showed the best improvement in intestinal mucosa and no ulceration especially at 30 days PI. Moreover, group IV showed significant decrease in the degree of inflammation. These results are in agreement with Sadek and El-Aswad[10] where pathological changes were moderate in group treated with NTZ, whereas they were severe in control group.

Treatment with CPIs (groups III and VIII) and also combined therapy in group X did not ameliorate undesired result appearing in intestinal morphology at either groups A or B. This finding contradicts that obtained by Ndao et al.[9] where K11777-treated IFN-γR-KO mouse model [210 mg/kg (body weight) BW/day] showed only minimal inflammation and no epithelial changes. This may be because of the different formula of vinyl sulfone, different mouse model, or different dose of infecting inoculate.

The mechanism behind the additive effects of NTZ and CPIs, observed in the present study, was not known. It may be attributed to different synergistic mechanisms of actions of both compounds. NTZ is a broad-spectrum thiazolide anti-infective agent used for the treatment of gastrointestinal infections. It counteracts inflammation and may thus be applicable in a wide variety of diseases[33],[34].

Moreover, PVS is a selective clan CA inhibitor of CPs. Proteases are critical in cell biology, and they are implicated in the host-parasite interaction[7]. Small-molecule inhibitors of clan CA CPs from parasites were validated as drugs for amelioration, if not cure, of many protozoan[35] and helminthic diseases[7].

There are various CPIs that were experimentally used for treatment of cryptosporidiosis. EDTA, iodoacetic acid, E-64, and phosphoramidon inhibited an azocasein proteinase found on the surface of C. parvum sporozoites[36].

Phenyl-methyl-sulfonyl fluoride combined with E-64 inhibited greater than 95% of the azocasein hydrolysis but did not inhibit oocyst excystation[37]. Other protease inhibitors including antipain, aprotinin, leupeptin, soybean trypsin inhibitor, and phenyl-methyl-sulfonyl fluoride also reduced parasite numbers[38].

CPIs used in this study are not at a stage of drug development sufficient to complete cure. Further studies are needed to detect the bioavailability, toxic hazards, and possible adverse effects of PVS.

More studies to develop other novel promising CPIs for treatment of cryptosporidiosis, either as a complementary or alternate therapeutic line to NTZ, are also recommended, as findings from in vitro and animal studies suggest that drug combinations might have some efficacy[3].

To the best of our knowledge and thorough search in English literature, the combination of NTZ with CPIs was not demonstrated before, so more studies upon this combination must be investigated as it would be useful to explore the feasibility of combining the most widely used anticryptosporidial drugs to find active combinations against cryptosporidiosis.


  Conclusion Top


The best drug efficacy on Cryptosporidium spp. was combined treatment of CPIs and NTZ in either immunocompetent or immunosuppressed mice, regarding reduction of oocyst shedding and the mean number of endogenous developmental stages in the ileum.

Moreover, we could conclude that NTZ is more effective in treatment of cryptosporidiosis than PVS used in this study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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