Strongyloidiasis hyperinfection syndrome treatment with ivermectin: in-vitro and in-vivo studies

Salwa A Shams Eldin; Nancy M Harba; Rehab M Samka

doi:10.4103/1687-7942.139687

RESEARCH ARTICLE

Year : 2014 | Volume : 7 | Issue : 1 | Page : 27-36

Strongyloidiasis hyperinfection syndrome treatment with ivermectin: in-vitro and in-vivo studies

Salwa A Shams Eldin¹, Nancy M Harba¹, Rehab M Samka²
¹ Department of Medical Parasitology, Faculty of Medicine, Menoufyia University, Menoufyia, Egypt
² Department of Pathology, Faculty of Medicine, Menoufyia University, Menoufyia, Egypt

Date of Submission	06-Oct-2013
Date of Acceptance	12-Dec-2013
Date of Web Publication	25-Sep-2014

Correspondence Address:
Nancy M Harba
MD, Departments of Medical Parasitology, Faculty of Medicine, Menoufyia University, Shebin El-Kom, 23154, Menoufyia
Egypt

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/1687-7942.139687

Abstract

Background
Strongyloides stercoralis is an intestinal nematode that usually causes asymptomatic infection in immunocompetent individuals and reverts to life-threatening hyperinfection in immunocompromised individuals.
Objective
The aim of the study was to evaluate the effect of ivermectin (IVM) treatment on S. stercoralis larvae in-vitro cultured forms. In-vivo evaluation was performed by assessment of parasitological, histopathological, and ultrastructural changes in the lungs of mice with Strongyloides hyperinfection syndrome before and after treatment with IVM.
Materials and methods
S. stercoralis larvae were collected from agar plates cultures of positive stool samples from different areas in Menuofyia Governorate. The in-vitro study involved the examination of S. stercoralis larvae grown in agar plates (APC) after exposure for 2 h to IVM (15 μl/ml) by scanning electron microscope (SEM) and after 24 h for larval motility. In-vivo study involved 96 mice that were divided into four groups (24 mice in each). Group GI was immunosuppressed by dexamethasone and infected with S. stercoralis larvae; GII was immunosuppressed then infected and treated by IVM (0.5 mg/kg); GIII was infected with S. stercoralis larvae; and GIV was infected and treated by IVM. Fecal larval output was carried out on 1 ^st , 6 ^th , 11 ^th , and 13 ^th days postinfection (dpi); histopathological examination of the lung was conducted on 2 ^nd , 7 ^th , 12 ^th , and 14 ^th dpi; and lung ultrastructure study by transmission electron microscopy (TEM) was performed on 7 ^th and 14 ^th dpi.
Results
IVM caused severe destruction of S. stercoralis larvae detected by SEM after 2 h and resulted in their death after 24 h. In-vivo results recorded significant decrease in fecal larval output in the treated groups (GII and GIV) and in GIII in comparison with GI. The latter showed significant continuous increase in fecal larval output throughout the period of study. Histopathological and ultrastructural results showed marked pathological changes in GI that were still evident until the last day of the study. IVM induced mild improvement in lung tissues in GII in comparison with GIV. The latter showed obvious improvement with great preservation of lung tissue. In GIII, mild pathological effect of infection was still evident by the end of study.
Conclusion
Dexamethasone-immunosuppressed mice infected by S. stercoralis showed intestinal and pulmonary hyperinfection. IVM showed significant improvement of all parameters studied mainly in the immune group and was very effective on motility of S. stercoralis larvae in vitro.

Keywords: dexamethasone, ivermectin, pulmonary hyperinfection, Strongyloides stercoralis, scanning electron microscope, transmission electron microscopy

How to cite this article:
Shams Eldin SA, Harba NM, Samka RM. Strongyloidiasis hyperinfection syndrome treatment with ivermectin: in-vitro and in-vivo studies. Parasitol United J 2014;7:27-36

How to cite this URL:
Shams Eldin SA, Harba NM, Samka RM. Strongyloidiasis hyperinfection syndrome treatment with ivermectin: in-vitro and in-vivo studies. Parasitol United J [serial online] 2014 [cited 2023 Nov 29];7:27-36. Available from: http://www.new.puj.eg.net/text.asp?2014/7/1/27/139687

Introduction

Strongyloides stercoralis is an ubiquitous soil-transmitted intestinal nematode of humans. It is endemic in the tropical and subtropical regions with estimated prevalence of 60-100 million individuals worldwide ^[1],[2],[3] . S. stercoralis is unique among intestinal nematodes in its ability to complete its life cycle within the host through an autoinfective cycle, which may result in infection for long decades ^[4],[5] . Besides, S. stercoralis can adapt for both free-living and parasitic cycles ^[6] . Humans are generally infected transcutaneous by filariform larvae transmitted from the soil into the skin ^[2] . After exposure, larvae migrate to the respiratory system by bloodstream. The parasite is then swallowed, and it penetrates the duodenum wall where the female deposits eggs in small bowel mucosa. Hatched rhabditiform larvae develop into a filariform stage that repenetrates the intestinal wall causing autoinfection. Those expelled with feces, when mixed with soil, start an asexual free-living cycle or maturate to infective filariforms that penetrate the skin ^[7],[8] . The female S. stercoralis worm can generate the progeny even without male copulation giving the species a survival advantage ^[9] .

The clinical presentation of human strongyloidiasis varies with the immune status of the host ^[10] . The uncomplicated intestinal form of disease may be asymptomatic or vary from mild diarrhea with nonspecific abdominal pain to chronic diarrhea ^[11] . Patients treated with corticosteroids, cancer patients, and persons infected with the human T-cell-lymphotropic virus 1 may develop Strongyloides hyperinfection syndrome (SHS) ^[12],[13] , which is estimated to happen in 1.5-2.5% of the patients with strongyloidiasis ^[14] . The reported mortality of SHS reaches up to 87% ^[6],[15] .

The clinical features of SHS are nonspecific; therefore, a high index of suspicion is required for early diagnosis and appropriate therapy ^[16] . Any factors that suppress Th ₂ response may potentially trigger hyperinfection or disseminated infection, which could be fatal ^[10] . However, SHS is known by exacerbation of gastrointestinal and pulmonary symptoms, and detection of increased numbers of larvae in stool and/or sputum is a hallmark of the disease. The condition was well recognized since 1966 and has increased in frequency during the past decades as a result of increased use of immunosuppressive therapy ^[17],[18] . A patient with SHS presents with severe pulmonary disease that necessitates respiratory support, followed by acute abdomen and intestinal obstruction ^[19] . Pulmonary manifestations include asthma-like symptoms, pneumonia, pulmonary hemorrhage, pleural effusion, and acute respiratory failure ^[5],[20] . The intestinal manifestations include severe cramping, abdominal pain, watery diarrhea, weight loss, nausea, vomiting, occasionally gastrointestinal bleeding, and small bowel obstruction ^[5] .

Definitive diagnosis of strongyloidiasis relies basically on detection of larvae in stool or sputum ^[21],[22] . However, a single stool sample examination is inadequate and is said to be ˜25-50% sensitive ^{[22],[23],[24]} . The agar plate culture (APC) method, in which serpiginous tracks of motile larvae or free-living adults become apparent after 3 or 5 days of incubation at room temperature, is a preferred method with high sensitivity and easy implementation ^[25],[26] . Recent advances in diagnosis of S. stercoralis include a luciferase immunoprecipitation system that shows increased sensitivity and specificity to detect S. stercoralis-specific antibodies, and a real-time quantitative PCR method to detect S. stercoralis in fecal samples ^[27] .

Albendazole, mebendazole, and thiabendazole have been shown to be effective for treatment of S. stercoralis infection ^[28],[29] . Recently, ivermectin (IVM) was shown to be the first line of treatment ^[8] for strongyloidiasis and was confirmed as its drug of choice ^[24] . IVM was also shown to be an effective treatment of SHS ^[30],[31] . It is a semisynthetic macrocyclic lactone that was developed for safe and efficacious veterinary antihelminthic treatment ^[32] .

The current study aimed to investigate the effect of IVM on S. stercoralis larvae in culture and to assess the parasitological, histopathological, and ultrastructural changes in the lungs of mice with strongyloidiasis and SHS before and after treatment with IVM.

Materials and methods

Type of study

The study was designed as a case - control experimental study.

Parasite collection

Stool samples were collected from patients attending the Clinical Pathology Laboratory of Menoufiya University hospital from different areas in Menoufiya Governorate during the period from 2010 to 2011. S. stercoralis larvae were obtained from samples found positive during routine examination by zinc sulfate concentration ^[33] . Few grams of homogenized fresh positive stool samples were cultured in the center of nutrient agar plates ^[34] .

Agar plate culture

The cultured plates were incubated with stool samples at room temperature for 48 h then the plates were examined by inverted microscope for S. stercoralis-infective filariform larvae (L3) ^[35] . Larvae were collected ^[25],[26] for further in-vitro and in-vivo studies by washing the surface of positive APCs with PBS solution. This was then centrifuged, and larvae in sediment were counted under microscope at magnification ×100 ^[25],[26] . Counted larvae were recultured in six agar plates with an approximate number of 50 larvae per plate. Larvae in two plates served as control, whereas larvae in the other four plates were exposed to IVM (15 μg/ml) ^[36] . After exposure to IVM for 2 h, larvae from two plates were fixed in 2.5% glutaraldehyde for 24 h for SEM examination in the Electron Microscopy Unit, Faculty of Medicine, Tanta University ^[37] . Larval motility was assessed after 24 h of exposure to IVM by microscopic examination.

Experimental animals

Eight-week-old female BALB/c mice weighing 18-20 g were obtained from the Department of Natural Biological Products, Theodor Bilharz Institute (Cairo, Egypt), and kept at the animal house in Forensic and Toxicology Department in Faculty of Medicine, Menoufyia University. Mice were fed with commercial diet and tap water and were kept at the animal house for 2 weeks before initiation of experiment for stool examination to exclude other intestinal parasitic infection and for adaptation.

Experimental infection

Number of S. stercoralis L3 obtained from APCs was adjusted to 5000 larvae/ml PBS ^[25],[26] . Each mouse was inoculated subcutaneously with 1500 L3 in 300 ml PBS in the abdominal region ^[38] . Ninety-six laboratory bred mice were divided into four groups, 24 mice each: group I (GI) were immunosuppressed and infected with S. stercoralis larvae; group II (GII) mice were immunosuppressed, infected with S. stercoralis larvae, and treated by IVM; Mice in group III (GIII) were infected with S. stercoralis larvae; and those in group IV (GIV) were infected with S. stercoralis larvae and treated by IVM.

For detection of intestinal hyperinfection, stools collected from each group on 1 ^st , 6 ^th , 11 ^th , and 13 ^th days postinfection (dpi) were examined using the Baermann technique, and the mean number of larvae/g was calculated ^[39] . Migratory phase and pulmonary passage of S. stercoralis larvae were evaluated on 2 ^nd , 7 ^th , 12 ^th , and 14 ^th dpi. Six mice from each group were killed at each time point. Lungs were removed from killed mice for histopathological study and transmission electron microscopy (TEM) examination in Electron Microscopy Unit in Faculty of Medicine, Tanta University.

Drugs

IVM was commercially purchased in the form of 6 mg tablets (BioPharm, Cairo, Egypt). For in-vitro study, a stock solution of the drug was prepared at 10 mg/ml with dimethylsulfoxide and used at a concentration of 15 μg/ml ^[36] . For in-vivo study, a single dose of 0.5 mg/kg was given by oral route after 2 days of infection ^[39] . Dexamethasone in the form of ampules (Sigma-Aldrich, St. Louis, Missouri, USA) was given by intraperitoneal injection at a dosage of 5 mg/kg body weight 1 day before infection, and given for 6 days/week until the end of the study ^[40] .

Scanning electron microscopic study

Control larvae as well as those exposed to IVM for 2 h were collected ^[25],[26] , fixed in 2.5% glutaraldehyde, washed in PBS, and fixed in 1% osmium tetraoxide. The specimens were rewashed, dehydrated in graded acetone, and adhered to SEM stubs. Samples were coated with a 20 nm thick gold layer ^[37] , examined, and digital images were stored in a computer.

Histopathological examination

Samples of the lungs were taken from all the studied groups for histopathological examination at each time point. Tissue specimens were fixed in 10% formalin, washed, dehydrated in ascending grades of alcohol, cleared in xylene, embedded in paraffin wax, sectioned, and stained with hematoxylin and eosin ^[41] .

Transmission electron microscopic study ^[37]

For TEM lung samples were taken from mice killed on 7 ^th and 14 ^th dpi. The specimens were fixed in 2.5% glutaraldehyde, rinsed twice in PBS, postfixed with 1% osmium tetraoxide, washed twice with PBS, and dehydrated in ascending grades of ethanol. Specimens were placed in absolute alcohol then in acetone for 2 h before transfer to a ratio of 1:3 resin:acetone for 24 h, 1:1 for 24 h, and subsequently 3:1 for 24 h. Finally, the specimens were submerged in resin and embedded in resin blocks. Ultrathin sections of 1 μm thickness were prepared from these embedded blocks. Sections were poststained with uranyl acetate 0.5% and examined by TEM (Jeol Ltd., Tokyo, Japan) at Electron Microscopy Unit in Faculty of Medicine, Tanta University.

Statistical analysis

Results were collected, tabulated, and statistically analyzed using the SPSS version 16 computer software statistical package (SPSS Inc., Chicago, USA). Two types of statistics were performed. Descriptive statistics included the mean (χ) and SD. Analytic statistics, the F-test or ANOVA, was used to compare means and their SD from three or more deviations. P value of less than 0.05 was considered statistically significant ^[42] .

Ethical consideration

Stool samples were collected from the patients according to instructions of the committee of Research, Publication and Ethics of the Faculty of Medicine, Menoufyia University. All procedures were explained to individuals in local language, and written or thumb-pointed informed consent was obtained. The experimental animal studies were conducted in accordance with the international valid guidelines, and the animals were maintained under convenient conditions.

Results

Agar plate culture

Culture of S. stercoralis larvae on agar plates succeeded in yielding satisfactory profuse numbers. After 48 h of culture, tracks left by the movement of larvae were observed. These tracks could be seen by the naked eye and L3 appeared bright and actively motile by inverted microscope [Figure 1]a and b. After exposure to IVM, the larvae immediately contracted and showed very sluggish movement. After 24 h, no movement was detected with darkness of larval appearance [Figure 1]c.

Figure 1: Agar plate cultures of Strongyloides stercoralis. (a) Larvae in 2-dayold culture (×400). (b) Eggs (green arrows), larvae (red arrows), and adults (yellow arrow) in 4-day-old culture (×400). (c) Dark nonmotile larvae treated with ivermectin for 24 h (× 400).

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Scanning electron microscope

Results showed that larvae exposed to IVM for 2 h were affected; their cuticles appeared edematous with loss of normal features [Figure 2]b. Moreover, severe damage and sloughing of the cuticle were also seen [Figure 2]c.

Figure 2: SEM of Strongyloides stercoralis larvae. (a) The control group larva with normal cuticle. (b) Larva treated with ivermectin for 2 h showing edematous cuticle with loss of normal features. (c) Larva showing severe damage and sloughing of the cuticle after 2 h.

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In-vivo findings

S. stercoralis larvae were detected in stools from all groups of mice from the 1 ^st dpi. [Table 1] shows nonsignificant difference between both groups of immunosuppressed mice (GI and GII) (P > 0.05) and between both groups of immune mice (GIII and GIV) (P > 0.05). However, there was significant difference between immunosuppressed mice in GI and GII in comparison with immune mice in each of GIII and GIV (P < 0.0001). Starting from 6 ^th dpi, the treated groups (GII and GIV) showed significant decrease in larva count in comparison with the untreated groups (GI and GIII) (P < 0.0001). No larva was detected in stool of mice in GIV on 11 ^th and 13 ^th dpi. Moreover, GI showed progressive increase in larva output until the end of study, whereas GIII showed obvious decrease in larva output recording significant difference compared with GI (P < 0.0001) on 1 ^st , 6 ^th , 11 ^th , and 13 ^th dpi.

Table 1: Mean number (± SD) of S. stercoralis larvae/g in stool samples of mice at different dpi in all groups

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Histopathological findings

Lung sections obtained from all the studied groups on 2 dpi showed mild degree of hemorrhage, congestion, edema, and destruction of alveolar walls. Larvae were detected in alveolar spaces [Figure 3]. In the following days, the immunosuppressed infected untreated group (GI) revealed progressive hemorrhage and destruction of lung parenchyma associated with larvae in all studied sections as seen on 7 ^th , 12 ^th , and 14 ^th dpi [Figure 4]a and b, respectively. Lung sections of the immunosuppressed infected treated group (GII) showed prominent intra-alveolar edema, moderate hemorrhage, and few cellular infiltrates with detection of larvae only on 7 ^th dpi [Figure 5]a, in addition to the presence of collagen fibers on 12 ^th dpi [Figure 5]b. Mild improvement in lung pathology was seen in the sections obtained on 14 ^th dpi [Figure 5]c.

Figure 3: Lung tissue section of GI larva: (L) mild degree of congestion and hemorrhage; (H) edema and thickened alveolar walls together with emphysematous changes (E) (H&E, ×100). Similar findings were seen in the other studied groups on 2n d dpi.

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Figure 4: Lung tissue sections of GI. (a) Hemorrhage (H) and destruction of alveolar walls with Strongyloides stercoralis larvae (arrow) within alveoli on seventh dpi (H&E, ×200). (b) Massive hemorrhage (H) and congestion with S. stercoralis larvae (arrows) within alveoli on 12th and 14th dpi (H&E, ×200).

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Figure 5: Lung tissue sections of GII: (a) Larva (arrow); prominent intraalveolar edema (E); mild destruction of alveolar walls; and mild cellular infiltration on seventh dpi (H&E, ×200). (b) Diffuse infiltration by foamy macrophages (black arrows); fragmented collagen fibers (red arrow); area of hemorrhage (asterisk) on 12th dpi (H&E, ×400). (c) Mild enlargement of alveolar spaces with pale pink homogenous transudate fl uid seen within some of the alveolar spaces and lung septum. The lung septum also revealed mild inflammatory infiltrate (arrow) on 14th dpi (H&E , ×100).

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The infected untreated group (GIII) showed severe inflammatory infiltrates in lung parenchyma on 7 ^th dpi [Figure 6]a with areas of fibrosis on 12 ^th dpi [Figure 6]b. On the 14 ^th dpi, there was mild destruction of alveolar architecture with progressive decrease in cellular infiltrates and RBCs [Figure 6]c. The infected treated group (GIV) showed obvious amelioration of lung tissue with mild hemorrhage and cellular infiltration at all times following the treatment [Figure 7]a and b, mainly on 14 ^th dpi with greater preservation of alveolar architecture [Figure 7]c.

Figure 6: Lung sections of GIII: (a) Nodular collection of heavy peribronchiolar infiltration of chronic inflammatory cells including lymphocytes, plasma cells, and eosinophils (yellow arrow). Bronchiolar wall
displayed destruction, focal ulceration, thickened hyalinized basement membrane, and newly formed fibrous tissue. Area of fibrosis (red arrow), few foamy macrophages (black arrow), and wide dilated congested capillary were also detected on seventh dpi (H&E, ×400). (b) Peribronchiolar chronic inflammatory infiltrates including foamy macrophages, lymphocytes, plasma cells, and few eosinophils (arrow). The bronchiole showed destructed wall (asterisk) surrounded by dense newly formed fi brous tissue on the 12th dpi (H&E, ×400). (c) Mild destruction of alveolar architecture with emphysematous (yellow arrow), hemorrhagic changes and moderate cellular infiltrates (red arrow) on the 14th dpi (H &E, ×200).

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Figure 7: Lung tissue section of GIV: (a) Mild distortion of the alveolar architecture in the form of distended alveolar space filled with RBCs together with mild inflammatory infiltrate in the lung septum on 7th dpi (H&E, ×100). (b) Infiltration by inflammatory cells formed of lymphocytes, plasma cells (arrows), and eosinophils (circle) on 12th dpi (H&E, ×400). (c) Improved preservation of alveolar architecture with mild inflammatory infiltrate in the lung septum on 14th dpi ( H&E, ×100).

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Transmission electron microscopy

Results of lung sections obtained from GI on 7 ^th and 14 ^th dpi are shown in [Figure 8]. There was persistence of infection on 14 ^th dpi denoted by the numerous electron-dense variable size and shape structures that seemed to be part of the larvae of S. stercoralis [Figure 8]c. In GII, parts of larvae were detected only on 7 ^th dpi and not seen in sections studied on 14 ^th dpi. Pneumocytes type II showed characteristic structure with presence of microvilli [Figure 9]a. Mitochondria in cells were swollen with disintegration of cristae [Figure 9]b and c. Dilatation of endoplasmic reticulum was also seen on 7 ^th dpi [Figure 9]b. In addition, collagen fibers were seen in the lung sections studied on 14 ^th dpi [Figure 9]c. Meanwhile, TEM of lung sections obtained from GIII on 7 ^th dpi showed different inflammatory cells. Macrophage cells contained numerous variable-sized vacuoles and hydropsy of the Clara cell. Pneumocytes type II showed swelling with destruction of mitochondrial cristae [Figure 10]a and b. More obvious findings were seen in the lung sections studied on 14 ^th dpi, where bundles of collagen fibers and areas of fibrosis were detected [Figure 10]c. Meanwhile, the epithelial lining of the terminal bronchiole including ciliated and nonciliated epithelial cells presented normal characteristic features [Figure 10]d. In GIV, active cellular reaction around terminal bronchioles was detected involving different types of inflammatory cells with presence of phagocytic cells containing variable vacuoles. Collagen fibers were also seen on 7 ^th dpi [Figure 11]a and b, whereas on 14 ^th dpi, the epithelial lining of the terminal bronchioles were more or less normal [Figure 11]c.

Figure 8: TEM micrographs of GI: (a) Lung edema with degeneration of the alveolar cells (1); endothelial cells lining the blood vessels (2); amorphous light electron-dense material (3) in the lumen of the alveoli and air bubbles (4) (7th dpi). (b) Hypertrophied pneumocytes type II with numerous vacuoles and swelling of mitochondria (1); septal capillary (2) (7th dpi). (c) Septal capillary (1); RBCs (2); electron-dense variable size and shape structures that seem to be parts of larvae in the septal wall and perivascular under the flattened alveolar cells type I (3); alveolar lumen (4); pneumocytes type II (5) (14th dpi).

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Figure 9: TEM micrograph of GII: (a) Small blood vessel (1); RBCs (2); part from the Strongyloides stercoralis larvae (3); an outer undulating light electron-dense membrane surrounding membranous structures having variable shape and size of electron-dense structures (4); pneumocytes type II (5); microvilli (7th dpi). (b) Plasma cells (1); swollen mitochondria with disintegration of their cristae (2); dilated variable rough endoplasmic reticulum in longitudinal or cross-sections (3); part from another cell with numerous swollen mitochondria (4); mitochondria with loss of cristae (7th dpi). (c) Higher magnification of a part from a cell containing numerous mitochondria showing disintegration of cristae (7th dpi). (d) Plasma cell (1); abundant rough endoplasmic reticulum (2); a bundle of collagen fi ber (3); and amorphous light electron-dense material (4) (14th dpi).

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Figure 10: TEM micrograph of GIII lung tissue section: (a) Fibroblast cells (1); collagen fibers (2); lymphoid cells (3); plasma cells (4); swollen pneumocytes type II with destruction of mitochondrial cristae and presence of lamellar bodies (5); part from macrophage cells containing electron-dense bodies and numerous vacuoles (6) (7th dpi). (b) Lining epithelium of the terminal bronchiole showing Clara cells (1) (containing numerous secretory granules as well as swelling and hydropsy of the cell); Goblet cells (2) (7th dpi). (c) Bundles of collagenous fibers running in different directions (1); part of fibroblast cell (2) (14th dpi). (d) Epithelial lining of terminal bronchiole showing ciliated epithelial cells (1); nonciliated epithelial cells containing electron-dense secretory granules with microvilli on their apical surface (2) (14th dpi).

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Figure 11: TEM micrograph of GIV: (a) Cellular reaction surrounding the terminal bronchiole containing lymphocytes (1); plasma cells (2); Clara cells (3) (seventh dpi). (b) Septal wall with abundant collagen fibers (1); phagocytic cells containing variable sized and shaped vacuoles (2); alveolar lumen (3); septal capillary containing RBC (4) (seventh dpi). (c) Epithelial lining of the terminal bronchiole showing ciliated epithelial cells (1); nonciliated epithelial cells containing electron-dense secretory granules and microvilli on their apical surface (2) (14th dpi).

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Discussion

In vitro results revealed that APC was very effective in detection and culture of S. stercoralis worms [Figure 1]a and b. It was reported that APC is the method of choice especially in immunocompromised patients with increased sensitivity for detection of the parasite ^[25],[43] . Exposure of S. stercoralis larvae to IVM in APC-induced loss of larval motility after 24 h [Figure 1]c. Similar results were reported for Strongyloides ratti and Strongyloides venezuelensis after 24 h of exposure to IVM ^[44] . Another study carried out on IVM-treated S. ratti larvae also showed that, as the drug concentration increased, marked and rapid decreased motility was observed ^[36] . It was suggested that IVM binds to glutamate-activated chloride channels existing in the nerve or muscle cells of nematodes. With specific and high affinity, the drug causes hyperpolarization of the nerve or muscle cells by increasing permeability of chloride ion through the cell membrane, and as a result the parasites are paralyzed to death ^[45] .

Our SEM results showed severe destruction of cuticle in larvae exposed to the drug in vitro [Figure 2]b and c. Similar changes were caused by IVM in Capillaria. hepatica in the form of disorganized cuticle and absence of surface uniformity ^[46] . In addition, SEM of Fasciola gigantica worms exposed to 50 μg/ml of IVM showed severe deformity with tegument swelling, furrows, shrinkage, and loss of spines after 48 h ^[47] .

In our study, mice were used as an experimental model for S. stercoralis hyperinfection. Abraham et al. ^[48] also used mice in their study to induce immunization with the recombinant antigen Ss-IR against infection with S. stercoralis. Notably, as fecal samples from humans with S. stercoralis infection were not available, most of other studies were undertaken using samples from related rodent species ^[36] . For this reason, the primary in vitro culture of positive human fecal samples on APC was carried out in our study aiming to induce high numbers of larvae needed for further in vivo study.

Fecal larval output was determined to assess the intestinal stage of hyperinfective strongyloidiasis. The study was conducted until 14 ^th dpi where the larval output was detected in all the studied groups on first dpi. In GIII, the maximum larval output was detected on the 6 ^th dpi [Table 1]. Incidentally, similar results were reported ^[38] where larvae were recovered from the intestine on the 2 ^nd dpi reaching the maximum number around 7 ^th dpi, then started to decline on 12 ^th dpi. In contrast and as expected, there was a prolonged and high larval output observed throughout the days of the study in immunosuppressed GI. Similar results were recorded in dexamethasone-treated mice infected by S. venezuelensis ^[49] . The significant reduction in the treated groups (II and IV), especially GIV in which there was complete elimination of larva from stool on 11 ^th and 13 ^th dpi, supported the efficacy of IVM as compared with GI in which there was progressive increase in larval output indicating persistence of intestinal hyperinfection. This was in agreement with the study by Keiser et al. ^[39] who reported 90% larval reduction in rats infected with S. ratti after treatment with 0.5 mg/kg single oral dose of IVM. It was also reported that two doses of IVM resulted in worm burden reduction of 91-96% ^[50] and complete eradication of adult S. ratti in mice ^[51] . A study conducted on Thai patients with chronic strongyloidiasis, in which a 7-day course of oral albendazole (800 mg daily) was compared with a single or double oral dose of IVM (200 μg/kg body weight, given 2 weeks apart), showed that both a single and a double dose of IVM were more effective than a 7-day course of high dose albendazole ^[52] .

Hyperinfection syndrome is a result of the numerous multiplication and migration of infective larvae especially in immunosuppressed state ^[8],[10] . Immunosuppression in this study was achieved by administration of dexamethasone. Corticosteroids have been shown to affect immunity by increasing the apoptosis of Th ₂ cells, reducing eosinophilic count, and inhibiting mast cells response ^[49] . Corticosteroids also increase ecdysteroid-like substances that act as molting signals leading to increased production of autoinfective filariform larvae with dissemination of infection ^[10] . Siddiqui et al. ^[53] demonstrated the presence of a steroid receptor on S. stercoralis, which could also play a role in the pathogenesis of hyperinfection syndrome and more systemic disseminated infection.

As the lung is frequently involved in SHS, we studied the effect of IVM on the migratory and lung stages of this parasite at different durations postinfection. On the 2 ^nd dpi, mild lung pathology including congestion, edema, focal areas of hemorrhage, and cellular infiltration was detected in all the studied groups [Figure 3]. The only evidence of damage due to the presence of larvae 36 h following subcutaneous inoculation was the presence of occasional areas of intra-alveolar hemorrhage, probably related to the vascular injury caused by exit of larvae from the vessels ^[54] . Moreover, in studying mechanical damage and inflammation caused by S. venezuelensis larvae migrating through the pulmonary environment, it was explained that the average width of larvae (15-20 μm) is larger than the size of lung capillaries (7-10 μm); hence, larvae in the circulation would embolize and break lung capillaries ^[55] . However, the intense cellular infiltration with a large number of eosinophils and lymphoid reaction was seen mainly in infected nontreated GIII mice killed on the 7 ^th dpi [Figure 6]a, which may be attributed to the immune-mediated pulmonary pathology that occurred in response to the parasite antigen ^[56] . These results were similar to the histopathological findings reported in sheep lungs infected with Protostrongylida ^[57] . In immunosuppressed GI mice, the lung showed extensive areas of hemorrhage and edema when killed on the 7 ^th , 12 ^th , and 14 ^th dpi [Figure 4]b and c, mostly resulting from migration of larvae in the lungs. This coincided with the increase in intestinal worm burden indicated by increasing fecal larval output observed in our study. The mechanical effect of large numbers of larvae breaking out of the vessels could result in such severe hemorrhage in this group. It was reported that numbers of intestinal worms, which become enormous, find their way to the lungs ^[10] . Similarly, the hemorrhagic aspect of the lung in dexamethasone-treated animals appeared more prominent and confluent ^[55] . In addition, the findings of our experimental study are similar to those seen in human lungs infected with S. stercoralis ^[58] .

IVM in the infected treated GIV proved very effective, as no migratory stages were detected on the 7 ^th dpi [Figure 7]a. In lung tissues of immune-infected nontreated GIII mice, only a few larvae were detected [Figure 6]a. On 12 ^th and 14 ^th dpi, there was mild to moderate lung pathology in GIII, whereas in GIV there was marked improvement with great preservation of lung tissues [Figure 6] and [Figure 7]a and b. It was reported that, in treatment of hyperinfection, the optimal antihelminthic activity of IVM requires an intact immune system, whereas in patients with various immunodeficiencies and recurrent autoinfective cycles, treatment should continue until the clinical syndrome resolves and larvae are no longer detectable ^[5] . The intact immune response is able to damage parasites reducing their fecundity and increasing their premature mortality that negatively affects worm growth ^[59],[60] .

The previous findings in the lung were confirmed by TEM. The infection was shown to produce variable pathological changes in the lung parenchyma that was more extensive in immunosuppressed GI mice [Figure 8], associated with edema and degeneration of alveolar cells and endothelial cells lining the blood vessels and with RBCs in septal capillaries. However, in infected nontreated GIII mice [Figure 10]a, pneumocytes type II showed swelling and destruction of mitochondrial cristae with presence of lamellar bodies. Trichinella spiralis larvae migrating through the lungs evoked destruction of type I epithelial cells and damage of lamellar bodies of epithelial cells or extracellular alveolar lining layer; severity of these changes was dependent on the number of infective larvae and possibly resulted from mechanical damage in the lung parenchyma ^[61] . Significant degenerative changes were also observed in cat and dog lungs infected by Dirofilaria immitis showing vacuolization, moderate type I cell damage, and type II cell hypertrophy ^[62] .

Lymphoid cells, plasma cells, Clara cells, and macrophages were revealed mainly in immune-infected mice whether nontreated (GIII) or treated (GIV) [Figure 1]0 and [Figure 11], reflecting the pronounced immune response to S. stercoralis infection in pulmonary tissue. Macrophages showed vacuoles and contained variable electron-dense microscopic bodies similar to those recorded for foamy macrophages isolated from day 2 after Nocardia brasiliensis infection in which the cytoplasm was dominated by large vacuoles that contained material of varying densities ^[63] . The treated groups, whether immunosuppressed (GII) or immune (GIV), showed increased collagen fibers, but this reaction was less extensive than in other groups [Figure 9] and [Figure 11]. Moreover, the presence of fibroblast and bundles of collagen fibers mostly in the nontreated infected GIII [Figure 10]c indicated the intense fibroblast reaction in lung parenchyma with intact immune system. These results are in agreement with the observations noted in a TEM study of sheep lung infected by Protostrongylus, which recorded the presence of destructive fibroblasts in lung parenchyma ^[58] . Infected rats with S. venezuelensis also showed a conspicuous increase in reticular fibers that related to granuloma composition, which serve to entrap the parasite promoting anchorage of inflammatory cells and facilitating elimination of the parasite ^[56] .

Conclusion

From the current study, it was concluded that dexamethasone-immunosuppressed mice infected by S. stercoralis showed pulmonary hyperinfection with pronounced histopathological and ultrastructural changes in the lung tissues. IVM was very effective on S. stercoralis motility in vitro with damaged topographic changes and showed improvement in all studied parameters mainly in the intact immune group. However, the immunosuppressed host may need other doses of the drug to achieve complete eradication of the worms.

Authors contribution

S.A. Shams Eldin shared in designing the research methodology, shared in laboratory work, analyzed and interpreted the results and the ultrastructural work, and shared in writing and revision of manuscript. N.M. Harba shared in designing the research methodology, collected the samples, wrote the manuscript, and shared in the practical work. R.M. Samka performed the histopathological and ultrastructural work and interpreted their results.

Acknowledgement

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