In vitro effect of curcumin on Schistosoma species viability, tegument ultrastructure and egg hatchability.

Schistosomiasis remains a severe problem of public health in developing countries. The development of resistance to praziquantel (PZQ) has justified the search for new alternative chemotherapies with new formulations, more effective, and without adverse effects. Curcumin (CUR), the major phenolic compound present in rhizome of turmeric (Curcuma longa L.), has been traditionally used against various diseases including parasitic infections. Here, the antischistosomal activity of CUR (50-500 µM), evaluated in parallel against S. mansoni and S. haematobium adult worms, appeared significant (P < 0.05 to < 0.0001) in a time- and dose-dependent manner. Two h incubation with CUR (500 µM) caused 100% irreversible killing of both schistosomal species. CUR (250 µM) caused the death of S. haematobium and S. mansoni worms after 2 h and 4 h, respectively. As CUR concentration decreases (50 µM), all coupled adult worms were separated into individual male and female but the worms remained viable up to 4 h. Scanning and transmission electron microscopy revealed that S. haematobium are more sensitive than S. mansoni to CUR schistosomicidal effects. In support, CUR was found to affect the antigenicity of surface membrane molecules of S. haematobium, but not S. mansoni. Of importance, CUR significantly (P < 0.05 to < 0.0001) affected S. mansoni eggs hatchability and viability, a ground for its use in chemotherapy of schistosomiasis mansoni and japonicum because of its increased bioavailability in the gastrointestinal tract. The data together emphasize that CUR is a promising potential schistosomicidal drug.

Efficacy of artemisinin-naphthoquine phosphate against Schistosoma haematobium adult flukes: dose-effect relationship and tegumental alterations.

Schistosoma haematobium and Schistosoma mansoni infections have broadly overlapping geographical distributions. Praziquantel is the only treatment for human schistosomiasis, so drug tolerance and/or resistance are major concerns. Artemisinin-naphthoquine phosphate (CO-ArNp), an artemisinin-based combination therapy endorsed by the World Health Organization as a gold standard therapy for malaria, has also been identified as a promising treatment for S. mansoni. In this in vitro study, we tested the effect of 1-40 µg/ml CO-ArNp on S. haematobium worms, and inspected tegumental changes by using scanning electron microscopy (SEM), aiming to determine if this combination therapy has a broad-spectrum antischistosomal activity. Incubation of S. haematobium adults with 20 or 30 µg/ml CO-ArNp caused 100% mortality of worms within 72 or 48 h, respectively. SEM examination showed extensive tegumental alterations such as oedema, constriction, shortening and loss of spines, fissuring, sloughing and perforation, resulting in exposure of the underlying basal lamina, mainly in treated male schistosomes. Besides the well-established potent efficacy, bioavailability, tolerability and safety of the antimalarial artemisinin-naphthoquine phosphate combined therapy, these results may also suggest its possible utilization as a new broad-spectrum antischistosomal agent.

Urogenital schistosomiasis in Cabo Delgado, northern Mozambique: baseline findings from the SCORE study.

BACKGROUND: The results presented here are part of a five-year cluster-randomised intervention trial that was implemented to understand how best to gain and sustain control of schistosomiasis through different preventive chemotherapy strategies. This paper presents baseline data that were collected in ten districts of Cabo Delgado province, northern Mozambique, before treatment. METHODS: A cross-sectional study of 19,039 individuals was sampled from 144 villages from May to September 2011. In each village prevalence and intensity of S. haematobium were investigated in 100 children first-year students (aged 5-8 years), 100 school children aged 9-12 years (from classes 2 to 7) and 50 adults (20-55 years). Prevalence and intensity of S. haematobium infection were evaluated microscopically by two filtrations, each of 10 ml, from a single urine specimen. Given that individual and community perceptions of schistosomiasis influence control efforts, community knowledge and environmental risk factors were collected using a face-to-face interview. Data were entered onto mobile phones using EpiCollect. Data summary was made using descriptive statistics. Chi-square and logistic regression were used to determine the association between dependent and independent variables. RESULTS: The overall prevalence of urogenital schistosomiasis was 60.4% with an arithmetic mean intensity of infection of 55.8 eggs/10 ml of urine. Heavy infections were detected in 17.7%, of which 235 individuals (6.97%) had an egg count of 1000 eggs/10 ml or more. There was a significantly higher likelihood of males being infected than females across all ages (62% vs 58%; P < 0.0005). Adolescents aged 9-12 years had a higher prevalence (66.6%) and mean infection intensity (71.9 eggs/10 ml) than first-year students (63.1%; 58.2 eggs/10 ml). This is the first study in Mozambique looking at infection rates among adults. Although children had higher levels of infection, it was found here that adults had a high average prevalence and intensity of infection (44.5%; 23.9 eggs/10 ml). Awareness of schistosomiasis was relatively high (68.6%); however, correct knowledge of how schistosomiasis is acquired was low (23.2%) among those who had heard of the disease. Schistosomiasis risk behaviour such as washing (91.3%) and bathing (86.7%) in open water sources likely to be infested with host snails was high. CONCLUSIONS: Urogenital schistosomiasis is widespread in Cabo Delgado. In addition, poor community knowledge about the causes of schistosomiasis and how to prevent it increases the significant public health challenge for the national control program. This was the first study in Mozambique that examined infection levels among adults, where results showed that S. haematobium infection was also extremely high. Given that this controlled trial aims to understand the impact of different combinations of schistosomiasis control through treatment of communities, schools, and treatment holidays over a five-year period, these findings highlight the importance of examining the impact of different treatment approaches also in adults. TRIAL REGISTRATION: The trials have been registered with the International Standard Randomised Controlled Trial registry under ISRCT 14117624 Mozambique (14 December 2015).

In Vitro Schistosomicidal Activity of Phytol and Tegumental Alterations Induced in Juvenile and Adult Stages of Schistosoma haematobium.

There is renewed interest in natural products as a starting point for discovery of drugs for schistosomiasis. Recent studies have shown that phytol reveals interesting in vivo and in vitro antischistosomal properties against Schistosoma mansoni adult worms. Here, we report the in vitro antischistosomal activity of phytol against Schistosoma haematobium juvenile and adult worms and alterations on the tegumental surface of the worms by means of scanning electron microscopy. The assay, which was carried out with 6 concentrations (25, 50, 75, 100, 125, and 150 µg/ml) of phytol, has shown a promising activity in a dose and time-dependent manner. There was a significant decline in the motility of the worms and a mortality rate of 100% was found at 48 hr after they had been exposed to phytol in the concentration of 150 µg/ml. Male worms were more susceptible. On the ultrastructural level, phytol also induced tegumental peeling, disintegration of tubercles and spines in addition to morphological disfiguring of the oral and ventral suckers. This report provides the first evidence that phytol is able to kill S. haematobium of different ages, and emphasizes that it is a promising natural product that could be used for development of a new schistosomicidal agent.

Bioactivity of miltefosine against aquatic stages of Schistosoma mansoni, Schistosoma haematobium and their snail hosts, supported by scanning electron microscopy.

BACKGROUND: Miltefosine, which is the first oral drug licensed for the treatment of leishmaniasis, was recently reported to be a promising lead compound for the synthesis of novel antischistosomal derivatives with potent activity in vivo against different developmental stages of Schistosoma mansoni. In this paper an in vitro study was carried out to investigate whether it has a biocidal activity against the aquatic stages of Schistosoma mansoni and its snail intermediate host, Biomphalaria alexandrina , thus being also a molluscicide. Additionally, to see whether miltefosine can have a broad spectrum antischistosomal activity, a similar in vitro study was carried out on the adult stage of Schistosoma haematobium, the second major human species, its larval stages and snail intermediate host, Bulinus truncutes. This was checked by scanning electron microscopy. RESULTS: Miltefosine proved to have in vitro ovicidal, schistolarvicidal and lethal activity on adult worms of both Schistosoma species and has considerable molluscicidal activity on their snail hosts. Scanning electron microscopy revealed several morphological changes on the different stages of the parasite and on the soft body of the snail, which further strengthens the current evidence of miltefosine's activity. This is the first report of mollusicidal activity of miltefosine and its in vitro schistosomicidal activity against S.haematobium. CONCLUSIONS: This study highlights miltefosine not only as a potential promising lead compound for the synthesis of novel broad spectrum schistosomicidal derivatives, but also for molluscicidals.

In vitro and in vivo activities of arachidonic acid against Schistosoma mansoni and Schistosoma haematobium.

The development of arachidonic acid (ARA) for treatment of schistosomiasis is an entirely novel approach based on a breakthrough discovery in schistosome biology revealing that activation of parasite tegument-bound neutral sphingomyelinase (nSMase) by unsaturated fatty acids, such as ARA, induces exposure of parasite surface membrane antigens to antibody binding and eventual attrition of developing schistosomula and adult worms. Here, we demonstrate that 5 mM ARA leads to irreversible killing of ex vivo 1-, 3-, 4-, 5-, and 6-week-old Schistosoma mansoni and 9-, 10-, and 12-week-old Schistosoma haematobium worms within 3 to 4 h, depending on the parasite age, even when the worms were maintained in up to 50% fetal calf serum. ARA-mediated worm attrition was prevented by nSMase inhibitors, such as CaCl(2) and GW4869. Scanning and transmission electron microscopy revealed that ARA-mediated worm killing was associated with spine destruction, membrane blebbing, and disorganization of the apical membrane structure. ARA-mediated S. mansoni and S. haematobium worm attrition was reproduced in vivo in a series of 6 independent experiments using BALB/c or C57BL/6 mice, indicating that ARA in a pure form (Sigma) or included in infant formula (Nestle) consistently led to 40 to 80% decrease in the total worm burden. Arachidonic acid is already marketed for human use in the United States and Canada for proper development of newborns and muscle growth of athletes; thus, ARA has potential as a safe and cost-effective addition to antischistosomal therapy.

Ultrastructural alterations of adult schistosoma haematobium harbored in mice following artemether administration.

OBJECTIVE: To perform a temporal examination of ultrastructural alterations in adult Schistosoma haematobium due to artemether. METHODS: Eight mice infected with 100-120 S. haematobium cercariae for 81 days were treated intragastrically with 400 mg/kg artemether. At 24 hours, 3, 7 and 14 days post-treatment, groups of 2 mice were sacrificed and schistosomes collected by the perfusion technique. Worm samples were fixed and examined by transmission electron microscopy. Schistosomes were also obtained from 2 untreated mice that served as control. RESULTS: Typical ultrastructural alterations included swelling, lysis and vacuolization of the tegumental matrix, and disappearance of basal membrane. In sensory organelles and tubercles, there was extensive or local lysis of internal structure. In the musculature, parenchymal tissues, syncytium and gut epithelial cells, focal or extensive lysis, decrease in granular endoplasmic reticulum, vacuolization and degeneration of mitochondria were observed. These alterations became apparent both in male and female worms 24 hours post-treatment. In female worms, severe damage to the vitelline cells was also observed, resulting in the emergence of vacuoles, a decrease in granular endoplasmic reticulum, fusion of vitelline balls or even collapse of damaged vitelline cells. The most extensive tegumental alterations were observed 3-7 days post-treatment. Whilst 14 days post-treatment ultrastructural damage was still apparent, the tegument of some worms showed similar features to those recovered from untreated control mice. CONCLUSION: Administration of artemether to mice infected with adult S. haematobium results in extensive damage to the ultrastructure in the tegument and subtegument tissues of the worms, confirming previous results with other schistosome species.

Effect of artemether on the tegument of adult Schistosoma haematobium recovered from mice.

OBJECTIVE: To assess the effect of artemether on the tegument of adult Schistosoma haematobium harbored in mice. METHODS: Ten mice were infected subcutaneously with 100-120 S. haematobium cercariae each. At day 81 post-infection, 8 mice were treated orally with 400 mg/kg artemether. Mice were sacrificed at 1, 3, 7 and 14 days post-treatment, and schistosomes were collected by the perfusion technique, fixed and examined under a scanning electron microscope. Schistosomes obtained from the 2 untreated mice served as a control. RESULTS: Twenty-four hours post-treatment, tubercles on the tegument of male worms showed lesions, characterized by enlargement, collapse and partial peeling off from the border with the tegument. In both male and female worms, the tegument showed focal or extensive swelling, fusion, vacuolization, erosion, peeling, and destruction of sensory structures. Three days post-treatment, tegumental alterations further aggravated; particularly severe damage was the swelling or collapse of the oral sucker observed in both sexes. In addition, extensive swelling, erosion and peeling of tegumental ridges and destruction of discoid-like sensory structures were seen in female worms. Seven to 14 days post-treatment, moderate-to-severe damage was still evident in some worms, whereas other worms surviving the treatment showed apparent recovery in most parts of their tegument. CONCLUSION: Artemether causes extensive and severe tegumental damage in adult S. haematobium.

Antischistosomal action of atorvastatin alone and concurrently with medroxyprogesterone acetate on Schistosoma haematobium harboured in hamster: surface ultrastructure and parasitological study.

Aiming to study the influence of long-term administration of lipid lowering agents (atorvastatin; AV), and to study the action of combined treatment with injectable contraceptive (medroxyprogesterone acetate; MPA) on tegumental ultrastrucutre and survival of Schistosoma worms, this study was established. AV (0.9 mg kg-1) was administered orally for 49 successive days to Schistosoma heamatobium-infected hamster starting from day 35 post-infection (pi). Another group of infected hamster was administrated MPA intramuscularly (0.1 ml kg-1) at days 7 and 35 pi followed by AV treatment regimen. Both treatment regimens significantly affected the surface ultrastructure of the male worms more pronouncedly than the female ones. Combined treatment was more severe in action compared to single one. The combined treatment was characterized by losing of spines and damaging of tubercles throughout the tegument, severe erosion and peeling and appearance of deep crakes in different parts of the tegument. Moreover, mild to sever destruction to the oral suckers of both female and male worms was noticed. On the other hand, both treatment regimens significantly reduced numbers of recovered S. haematobium worms and tissue egg load. Oogram pattern was affected only in case of combined treatment with high percentage of dead eggs. In conclusion, AV, if given continuously for long time, has a pronounced antischistosomal action especially when accompanied with contraceptive intake. These promising results may encourage further investigation with the intention of their possible application on treatment of schistosomiasis as a complement strategy to praziquantel chemotherapy.

Schistosomal appendicitis in pregnancy.

Acute appendicitis is the most common acute surgical infection during pregnancy. Although usually pyogenic in origin, parasitic infections account for a small percentage of cases. Despite the relatively high prevalence of acute appendicitis in our environment, it is not commonly associated with schistosomiasis. We report here the association of pregnancy and appendicitis caused by Schistosoma haematobium. Schistosomiasis is very common complication of pregnancy in hyperendemic areas. Schistosome egg masses can lodge throughout the body and cause acute inflammation of the appendix, liver and spleen. Congestion of pelvic vessels during pregnancy facilitates passage of eggs into the villi and intervillous spaces, causing an inflammatory reaction. Tourism and immigration make this disease a potential challenge for practitioners everywhere.

Tegumental alterations in juvenile Schistosoma haematobium harboured in hamsters following artemether treatment.

We report the findings of a detailed temporal study on tegumental alterations in juvenile Schistosoma haematobium, induced by artemether, using scanning electron microscopy. Hamsters infected with S. haematobium cercariae for 28 days were treated intragastrically with a single dose of 300 mg/kg artemether. Groups of two hamsters were killed 24 h, 72 h and 7 days after treatment, and schistosomula were recovered from livers by perfusion and subsequent systematic examination of the tissue, before routinely processing for scanning electron microscopic examination. Most schistosomula collected 24 h after artemether administration showed severe tegumental damage, usually including swelling, fusion, vesiculation, peeling and collapse of enlarged sensory structures. After 72 h, tegumental damage had increased and schistosomula generally showed contraction with extensive swelling, erosion and peeling of the tegument. Seven days post-treatment, severe tegumental damage was only seen in a single male specimen with swelling of the worm body and destruction of the oral sucker. The other schistosomula showed only light to moderate damage, suggesting that schistosomula surviving the treatment began to recover. Our findings of tegumental damage following artemether treatment correlate with the efficacy of this novel antischistosomal drug in killing the juvenile stages of S. haematobium and complement recent findings with S. japonicum and S. mansoni.

Schistosomiasis of the urinary bladder in an African immigrant to North Carolina.

A 30-year-old black man came to the emergency department with gross hematuria, prostatitis, and urethritis 4 months after immigrating to this country from the Sudan in Africa. Urinalysis revealed hematuria and no ova or parasites. An intravenous urogram showed normal kidneys and ureters. The patient had cystoscopy with biopsies of an inflamed and ulcerated bladder mucosa. The nematode Schistosoma haematobium and schistosome eggs were identified without evidence of urothelial malignancy. The patient was treated with praziquantel and is currently asymptomatic. Over 200 million people are infected with schistosomiasis worldwide, yet this disease is rarely encountered in this country. The differential diagnosis and assessment of patients must include exposure to uncommon diseases that are endemic to other geographic regions.

Morphometric variability of Schistosoma intercalatum eggs: a diagnostic dilemma.

Variability of Schistosoma intercalatum eggs in shape and size, and their similarity to those of S. haematobium presented a problem of species identification when egg morphology was the diagnostic criterion used in a study of human schistosomiasis conducted on São Tomé and Principe. More than 2500 egg measurements were obtained by light micoscopy to gather data relating to size variability of S. intercalatum eggs, to evaluate whether factors such as age of host, sex of host and intensity of infection are correlated with variability, and the data were compared with previously published measurements on different isolates and strains of S. intercalatum: the range in length (104-203 microns) embraces most of the measurements reported in other studies of S. intercalatum eggs. There was no correlation either between age and sex of the host, or intensity of infection with variability of egg size. Comparison between measurements of the eggs of S. haematobium, S. intercalatum and S. bovis eggs are presented.

Human bilharzial ureters: I. Fine structure of eggs deposited in the submucosa and muscularis.

The internal fine structure of the partially calcified Schistosoma haematobium eggs in the lower ureteral segments of Saudi patients with chronic urinary schistosomiasis is described. The egg shell is penetrated by cribriform pores and consists of the three previously described layers: outer microspinous, middle intermediately dense, and inner dense layers. The space between the egg shell and the developing embryo is partitioned by three layers. An outer acellular Reynolds' layer of unknown origin and function consists of a fibrillar material mixed with a finely granular matrix extending to the egg-shell pores via racimose channels. The middle von Lichtenberg's envelope consists of a single layer of flattened epithelial cells containing several mitochondria in other previously described Schistosoma eggs suggesting an active, and perhaps selective, transport in or out of the egg shell. The inner fluid filled cavity, or Lehman's lacuna, between the von Lichtenberg's envelope and the embryo contains numerous lipoid bodies suggesting a relation to vitelline cells. - Four systems (out of eight previously described for the free, mature Schistosoma miracidium) have been recognized for the first time in the developing miracidium within S. haematobium eggs and include: 1) ciliated epidermal plates representing the epithelial system, 2) an outer circular and an inner longitudinal muscle layers forming the musculatures, 3) lateral penetration glands, and 4) ciliated flame cells representing the excretory system.

Interaction of schistosome eggs with vascular endothelium.

The interaction of schistosome eggs with venular endothelium was studied using primary cultures of human umbilical vein endothelial cells. Freshly oviposited and embryonated eggs of Schistosoma mansoni, Schistosoma japonicum, and Schistosoma haematobium were used. The cultures were evaluated by light, scanning, and transmission electron microscopy. Endothelial cell monolayers were found to retain eggs by actively migrating over those that came to lie on top of them. The monolayers then reestablished confluency and their lumenal polarity. Eggs deposited directly by adult worms elicited a more rapid and complete response than embryonated eggs isolated from the liver tissues of infected rodents or latex beads. Cell migration was shown to be more complete in the presence of sera than in serum-free media. It is concluded from these observations that eggs can be passively transferred to the perivenular space by the nonspecific response of endothelial cells.

[Sterility as a sequela of tubal schistosomiasis]. / Sterilität als Folge einer tubaren Schistosomiasis.

A 21-year old african women presented with primary sterility in the outpatient department of our university hospital. Screening examinations, including ultrasound, demonstrated a tumour of 5.8 x 4.1 cm diameter on the left ovary. While performing laparotomy to extirpate this tumour, bilateral occlusion of the tubes was discovered by chromopertubation.This diagnosis led us to perform bilateral salpingostomy and reimplantation of the Fallopian tubes. In addition to the ovarian tumour (dermoid), fragments of the Fallopian tubes were sent for histological examination, which revealed eggs of schistosomes probably of the species Schistosoma haematobium. Since tourism to and immigration from endemic areas is increasing, schistosomiasis should be considered when it is not possible to elucidate the cause of sterility by classical means.

Human bilharzial ureters: IV. Ultrastructural interaction between multinucleate giant cells and the parasite eggs.

The ultrastructural interaction between multinucleate giant cells (MGCs) and the Schistosoma haematobium eggs is described at the transmission electron microscopy (TEM) level, in granulomas of the lower ureteral segments of Saudi patients. The MGCs were found only within the parasite empty egg-shells which were embedded in the ureter submucosa and muscularis. For the first time, two morphologically different types of MGCs were recognized: the moderate to electron-dense type I contained two types of 8-12 nuclei and cytoplasm poor in cell organelles which appeared non-functional. Type 1 MGCs were considered to be "old" and non-active cells. The electron-lucent to moderately dense type II MGCs contained 3-4 nuclei of one type, and cytoplasm rich in cell organelles. According to the performance of their organelles, type II MGCs were considered to be "young" and highly active cells. The human type II MGCs did not form the cytoplasmic membranous labyrinths previously described for the free moving MGCs obtained from experimental mice, and were suggested to be involved in the cell motility. As in murine MGCs, type II cells were incorporated in exocytic and endocytic activities aimed the degradation of the inner soft layer of the egg-shell. This study shows that individual macrophages were responsible for eliminating the outer and possibly the middle hard layers of the egg-shell.

Schistosoma haematobium (Egyptian strain) transmission microscopy of normal and praziquantel treated worms.

The ultrastructure of the tegument and subtegumental structures of S. haematobium worms was examined by TEM. The effect of praziquantel on residual worms which remained after a curative dose was also studied. The male S. haematobium worms were characterized by deep tortuous pits and sharp apically directed spines. Spherical and elliptical bodies were also observed. The esophageal tegument was described. The effect of praziquantel on the male worms revealed flattening of spines and their disappearance in some regions as well as disruption and vacuolization of the tegument in others.

Scanning electron microscopy of normal and praziquantel treated S. haematobium worms (Egyptian strain).

The ultrastructure of the tegument of S. haematobium was examined before and after treatment with Praziquantel using scanning electron microscopy. The surface of the adult male worms prior to treatment showed numerous tubercles with apically directed spines and the lateral border showed highly pitted folds. The oral and ventral suckers showed well developed spines. Praziquantel administration caused various structural changes in the various groups studied. Blebs and spine deformities appeared as early as half an hr. after administration. Changes were also observed when the drug was administered prior to worm maturation resulting in generalized deformities in the worms which survived treatment, loss of spines and tegumental swellings.

Variation in the morphology of the dorsal and dorso-lateral tegument of male Schistosoma haematobium from southern Africa.

The teguments of Schistosoma haematobium males from three localities in the Eastern Transvaal and one in the eastern Caprivi were studied by means of scanning electron microscopy. Eastern Transvaal S. haematobium, which occurs sympatrically with S. mattheei, a bovine schistosome also infecting man and which hybridizes with S. haematobium, exhibited certain S. mattheei characteristics. The occurrence of these characteristics were neither related to the prevalence of human S. mattheei infections nor could they be attributed exclusively to phenotypic plasticity. The variation therefore may be geographical and possibly related to the phylogeny of the two species.