Ultrastructural alterations in Schistosoma mansoni juvenile and adult male worms after in vitro incubation with primaquine

BACKGROUND Praziquantel has been cited as the only drug for treating schistosomiasis. However, concerns over drug resistance have encouraged the search for novel drug leads. The antimalarial drug primaquine possesses interesting anti-schistosmal properties. OBJECTIVES This study is the first to document the potential role of primaquine as a schistosomicide and the ultrastructural changes induced by primaquine on juvenile or adult male worms of Schistosoma mansoni. METHODS Ultrastructural alterations in the tegumental surface of 21-day-old juvenile and adult male worms of S. mansoni were demonstrated following primaquine treatment at different concentrations (2, 5, 10, 15, and 20 µg/mL) and incubation periods (1, 3, 6, 24, and 48 h) in vitro, using both scanning and transmission electron microscopy. FINDINGS At low concentrations (2, 5, and 10 µg/mL) both juvenile and adult male worms were alive after 24 h of incubation, whereas contraction, paralysis, and death of all worms were observed after 24 h of drug exposure at 20 µg/mL. The tegument of juvenile and adult male worms treated with primaquine exhibited erosion, peeling, and sloughing. Furthermore, extensive damage of both tegumental and subtegumental layers included embedded spines, and shrinkage of muscles with vacuoles. The in vitro results confirmed that primaquine has dose-dependent effects with 20 µg/mL as the most effective concentration in a short incubation period. MAIN CONCLUSIONS The schistosomicidal activity of primaquine indicates that this drug possesses moderate in vitro activity against juvenile and adult male worms, since it caused high mortality and tegumental alterations. This study confirmed that the antimalarial drug primaquine possesses anti-schistosomal activity. Further investigation is needed to elucidate its mechanism of action.

THE SUSCEPTIBILITY OF RECENT ISOLATES OF Schistosoma mansoni TO PRAZIQUANTEL

Introduction: Schistosomiasis is a chronic disease caused by trematode flatworms of the genus Schistosoma and its control is dependent on a single drug, praziquantel (PZQ), but concerns over PZQ resistance have renewed interest in evaluating the in vitro susceptibility of recent isolates of Schistosoma mansoni to PZQ in comparison with well-established strains in the laboratory. Material and methods: The in vitro activity of PZQ (6.5-0.003 µg/mL) was evaluated in terms of mortality, reduced motor activity and ultrastructural alterations against S. mansoni. Results: After 3 h of incubation, PZQ, at 6.5 µg/mL, caused 100% mortality of all adult worms in the three types of recent isolates, while PZQ was inactive at concentrations of 0.08-0.003 µg/mL after 3 h of incubation. The results show that the SLM and Sotave isolates basically presented the same pattern of susceptibility, differing only in the concentration of 6.5 µg/mL, where deaths occurred from the range of 1.5 h in Sotave and just in the 3 h range of SLM. Additionally, this article presents ultrastructural evidence of rapid severe PZQ-induced surface membrane damage in S. mansoni after treatment with the drug, such as disintegration, sloughing, and erosion of the surface. Conclusion: According to these results, PZQ is very effective to induce tegument destruction of recent isolates of S. mansoni.

Antischistosomal activity of a calcium channel antagonist on schistosomula and adult Schistosoma mansoni worms

Current schistosomiasis control strategies are largely based on chemotherapeutic agents and a limited number of drugs are available today. Praziquantel (PZQ) is the only drug currently used in schistosomiasis control programs. Unfortunately, this drug shows poor efficacy in patients during the earliest infection phases. The effects of PZQ appear to operate on the voltage-operated Ca2+channels, which are located on the external Schistosoma mansoni membrane. Because some Ca2+channels have dihydropyridine drug class (a class that includes nifedipine) sensitivity, an in vitro analysis using a calcium channel antagonist (clinically used for cardiovascular hypertension) was performed to determine the antischistosomal effects of nifedipine on schistosomula and adult worm cultures. Nifedipine demonstrated antischistosomal activity against schistosomula and significantly reduced viability at all of the concentrations used alone or in combination with PZQ. In contrast, PZQ did not show significant efficacy when used alone. Adult worms were also affected by nifedipine after a 24 h incubation and exhibited impaired motility, several lesions on the tegument and intense contractility. These data support the idea of Ca2+channels subunits as drug targets and favour alternative therapeutic schemes when drug resistance has been reported. In this paper, strong arguments encouraging drug research are presented, with a focus on exploring schistosomal Ca2+channels.

Glomerulopatia esquistossomótica: comportamentos das alterações em camundongos infectados pelo schistosoma mansoni, antes e depois do tratamento com praziquantel / Schistosomal glomerulopathy: behavior changes in mice infected with schistosoma mansoni before and after treatment with praziquantel

A esquistossomose é um grave problema de saúde pública causado pelo helminto Schistosoma mansoni, sendo uma das endemias parasitárias de maior prevalência no Brasil. Apresenta diversas formas clínicas passíveis de serem reproduzidas em modelos experimentais. A lesão renal da glomerulopatia esquistossomótica está associada ao antígeno parasitário que, em consequência da presença de hipertensão portal e formação da circulação colateral, permitem que complexos imunes sejam depositados em glomérulos renais, desencadeando um processo inflamatório progressivo. O objetivo do trabalho foi avaliar o comportamento das alterações renais em camundongos infectados pelo S. mansoni, antes e após o tratamento com praziquantel, a fim de observar a resposta à presença dos complexos imunes e ao processo inflamatório formado na glomerulopatia. Foram organizados 3 grupos de camundongos BALB/c de ambos os sexos: grupo controle intacto (10 animais), grupo de reinfectados tratados (35 animais) e grupo de reinfectados não tratados (35 animais). A avaliação da estrutura renal foi analisada através da microscopia óptica, observando a histologia glomerular com as colorações de HE, PAS e PIFG, através da imunofluorescência marcando IgG e antígeno de Nash, e da microscopia eletrônica de transmissão com emprego da morfometria para avaliar áreas de depósito de complexos imunes. Foram analisadas, também, a carga parasitária, o peso do fígado, a presença dos vermes nas veias mesentéricas e a estrutura física dos órgãos. Os resultados demonstraram significativa redução da carga parasitária, do peso do fígado, das alterações estruturais nas observações histológicas e dos depósitos de complexos imunes em área e marcação no tecido renal, após tratamento com praziquantel. O tratamento específico reverteu os processos iniciais de agressão renal na glomerulopatia do modelo experimental, embora em alguns poucos camundongos, após a quimioterapia, a doença se manteve. Os achados deste trabalho reforçam a possibilidade de que o estado normal de estrutura renal pode ser restabelecido com o tratamento antiparasitário, desde que ministrado logo que se instala a glomerulopatia esquistossomótica clínica.

Schistosomiasis is an important public health problem due to infection with the helminth Schistosoma mansoni, which is also one the parasitic diseases listed as highly prevalent in Brazil. It presents several clinical forms, which are susceptible of experimental reproduction in mice. The lesion involving the kidney – Schistosomal glomerulopathy – is related to the presence of circulating parasite antigens, which can be attached to circulating antibodies to form antigen-antibody complexes with glomerular deposition. However, in the presence of portal hypertension and collateral circulation, these antigen complexes, can bypass macrophage clearance and reach the systemic circulation, thus coming to be deposited within the renal glomeruli, starting a progressive inflammatory process. The aim of this research was to evaluate renal changes in mice infected with S. mansoni, before and after treatment with praziquantel, through the presence of immune complexes in the glomeruli, and its correlation with the glomerular changes shown. BALB/c mice infected, infected and treated mice and non-infected groups were evaluated. Besides the evaluation of the renal changes by optical microscopy (HE, PAS, PIFG), immunofluorescence (IgG and Nash antigen) and transmission electron microscopy, a morphometric study of the areas containing immune complexes was also attempted. Besides parasite burden, liver weigh, number of worm pairs within mesenteric veins, and organs physical structure, were also evaluated.

Solid dispersions with hydrogenated castor oil increase solubility, dissolution rate and intestinal absorption of praziquantel / Dispersões sólidas com aumento de solubilidade de hidrogenado do óleo de mamona, taxa de dissolução e absorção intestinal de praziquantel

The solubility behavior of drugs remains one of the most challenging aspects in formulation development. Solid Dispersion (SD) has tremendous potential for improving drug solubility. Although praziquantel (PZQ) is the first drug of choice in the treatment of schistosomiasis, its poor solubility has restricted its delivery oral route. In spite of its poor solubility, PZQ is well absorbed in the gastrointestinal tract, but large doses are required to achieve adequate concentration at the target sites. The aim of this study was to improve the solubility and dissolution rate of PZQ and to evaluate its intestinal absorption. SDs were formulated with PEG-60 castor oil hydrogenated (CR-60) using a fusion and evaporation method. Pure PZQ and physical mixtures (PM) and PZQ-CR-60 (2:1; 1:1; 1:2 ratios) were compared as regards their solubility, dissolution and intestinal absorption. The experimental results demonstrated the improvement in the solubility, dissolution rate and intestinal absorption. In addition, the solubility behavior showed pH dependency and that the solubility of PZQ was slower in acidic medium than in neutral and basic mediums. The increase in PZQ solubility of the SD with the CR-60 could be attributed to several factors such as improved wettability, local solubilization, drug particle size reduction and crystalline or, interstitial solid solution reduction.

A solubilidade de fármacos ainda é um dos principais desafios no desenvolvimento de formulações farmacêuticas. As dispersões sólidas (DS) apresentam grande potencial para melhorar a solubilidade de fármacos. O praziquantel é o fármaco de primeira escolha no tratamento da esquistossomose, contudo a baixa solubilidade em água restringe seu uso à administração pela via oral. Apesar da baixa solubilidade, o PZQ é bem absorvido através do trato gastrintestinal, mas doses orais elevadas são requeridas para garantir concentrações suficientes de fármaco para o tecido alvo. O objetivo deste estudo foi melhorar a solubilidade, a dissolução e avaliar a absorção do PZQ. As DS foram formuladas com óleo de castor hidrogenado - PEG 60 (CR-60), pelo uso dos métodos de fusão e evaporação do solvente. PZQ puro, mistura física (MF) e DS de CR-60-PZQ (1:2; 1:1; 2:1) foram comparados quanto à solubilidade, dissolução e absorção intestinal. Os resultados experimentais mostraram aumento na solubilidade, na taxa de dissolução e na absorção intestinal do PZQ nas DS. A solubilidade do PZQ foi maior em meio ácido, mostrando uma dependência do pH. O aumento na solubilidade do PZQ nas DS com CR-60 foi atribuída a fatores como aumento da molhabilidade, solubilização local, redução granulométrica e redução da cristalinidade ou, ainda, a ocorrência de uma solução sólida intersticial.

Praziquantel, levamisol e diflubenzuron no controle de Dolops carvalhoi (Crustacea: Branchiura) e Anacanthorus penilabiatus (Monogenea: Dactylogyridae) em Piaractus mesopotamicus Holmberg, 1887 (Osteichthyes: Characidae) / Praziquantel, levamisole and diflubenzuron in the control of Dolops carvalhoi (Crustacea: Branchiura) and Anacanthorus penilabiatus (Monogenea: Dactylogyridae) in Piaractus mesopotamicus Holmberg, 1887 (Osteichthyes: Characidae)

Neste trabalho, avaliou-se a eficácia antiparasitária do praziquantel, levamisol e diflubenzuron administrados via oral, adicionados à ração, para pacus (Piaractus mesopotamicus) infectados por Anacanthorus penilabiatus e Dolops carvalhoi. Foram utilizadas 19 caixas d'água de 300 L de capacidade, comportando 28 peixes cada. Os tratamentos foram feitos misturando os princípios ativos nas dietas. A intensidade parasitária e eficácia foram avaliadas 1 dia antes e 3, 7 e 15 dias após o início da alimentação com ração contendo diflubenzuron, levamisol e praziquantel isolados ou associados em diferentes concentrações por 7 dias. Os resultados da eficácia terapêutica sugerem que, isoladamente ou associado com levamisol e praziquantel, o diflubenzuron é eficiente contra o crustáceo D. carvalhoi, demonstrando que a eficácia dos tratamentos nos dias 3, 7 e 15 variou de 96,2 a 100 por cento. Contra os monogenóides, as drogas não apresentaram eficácia satisfatória. Os resultados sugerem o uso do diflubenzuron para o controle de D. cavalhoi em peixes de cativeiro e em condições de quarentenário.

This assay evaluated the control efficacy of diflubenzuron, praziquantel and levamisole added to the diet of pacu (Piaractus mesoptamicus) infected with Anacanthorus penilabiatus and Dolops carvalhoi. 19 water tanks of 300 L capacity were utilized with 28 fish in each one. The treatments were made by mixing the active principles in the diet. The experiment was evaluated in four harvests done 1 day before and 3, 7 and 15 days after the treatment. The medicated feeding was applied for 7 days. The results of efficacy suggest that the diflubenzuron alone or associated with levamisole and praziquantel was efficient against the crustacean D. carvalhoi and the efficacy in the 3, 7 and 15 days evaluations ranged from 96,2 to 100 percent. Against the monogenean the drugs did not present efficacy. The results suggest the use of diflubenzuron for the control of D. carvalhoi in captive fishes in special conditions.

Association of oxamniquine praziquantel and clonazepam in experimental Schistosomiasis mansoni

The antischistosomal activity of clonazepam, when administered alone or in association with oxamniquine and praziquantel, was experimentally evaluated in mice infected with Schistosoma mansoni. The animals were treated 45 days post-infection with a single dose, by oral route, according to three treatment schedules: clonazepam 25 mg/kg and sacrificed 15 min, 1h or 4 h after treatment; clonazepam 1.0, 2.5 or 10.0 mg/kg and sacrificed 15 days post-treatment or with the dose of 10 mg/kg in association with oxamniquine 50 mg/kg or praziquantel 200 mg/kg, single dose, orally, every schedule with a control group. The efficacy of the drugs in vivo was assessed by means of worm counts and their distribution in mesentery and liver, mortality and oogram changes. In the chemotherapeutic schedules used, clonazepam did not present antischistosomal activity and the result of the association of this drug with oxamniquine or praziquantel was not significantly different from the one obtained when these two last drugs were administered alone. In the in vitro experiments, the worms exposed to 0.6 mg/mL clonazepam remained motionless throughout the 8-day-period of observation, without egg-laying, whereas the worms of the control group showed normal movements, egg-laying and hatching of miracidia on the last day of observation. The results obtained in the present study confirm the action of clonazepam on S. mansoni adult worm, in vitro, causing total paralysis of males and females. However, no additive or synergistic effects were observed when clonazepam were used in association with oxamniquine or praziquantel.

Efeito do praziquantel incorporado a lipossomas nos diferentes estágios de desenvolvimento dos ovos de Schistosoma mansoni / Effect of praziquantel in liposomes on Schistosoma mansoni eggs at different development stages

A esquistossomose mansônica é causada pelo trematódeo digenético intravascular Schistosoma mansoni. Para o tratamento dessa enfermidade o praziquantel (PZQ) e a oxamniquina (OXA) são os fármacos escolhidos. Noentanto, esses fármacos apresentam limitações quanto à ação e casos de resistência ou tolerância já foram relatados. Por esse motivo, são necessários os estudos de novas alternativas que visam melhorar os fármacos já existentes, como a incorporação desses em lipossomas. Este estudo verificou a ação do praziquantel incorporado a lipossomas (lip.PZQ) sobre os ovos de S. mansoni, linhagem BH em camundongos Mus musculus (Swiss-SPF). Para tanto, foram testadas quatro doses de PZQ elip.PZQ (47; 60; 250 e 300mg/kg) sendo que parte dos camundongos foi tratada após 30 dias de infecção e outra após 45 dias. A análise do o ograma mostrou que a dose lip.PZQ 300mg/kg administrada no 45º dia de infecção foi mais eficaz, pois reduziu a oviposição pelas fêmeas de S. mansoni.

Activity of praziquantel on in vitro transformed Schistosoma mansoni sporocysts

Praziquantel (PZQ) is effective against all the evolutive phases of Schistosoma mansoni. Infected Biomphalaria glabrata snails have their cercarial shedding interrupted when exposed to PZQ. Using primary in vitro transformed sporocysts, labeled with the probe Hoechst 33258 (indicator of membrane integrity), and lectin of Glycine max (specific for carbohydrate of N-acetylgalactosamine membrane), we evaluated the presence of lysosomes at this evolutive phase of S. mansoni, as well as the influence of PZQ on these acidic organelles and on the tegument of the sporocyst. Although the sporocyst remained alive, it was observed that there was a marked contraction of its musculature, and there occurred a change in the parasite's structure. Also, the acidic vesicles found in the sporocysts showed a larger delimited area after contact of the parasites with PZQ. Damages to the tegument was also observed, as show a well-marked labeling either with Hoechst 33258 or with lectin of Glycine max after contact of sporocysts with the drug. These results could partially explain the interruption/reduction mechanism of cercarial shedding in snails exposed to PZQ.

The effects of drugs, ions, and poly-l-lysine on the excretory system of Schistosoma mansoni

We have been able to label the excretory system of cercariae and all forms of schistosomula, immature and adult worms with the highly fluorescent dye resorufin. We have shown that the accumulation of the resorufin into the excretory tubules and collecting ducts of the male adult worm depends on the presence of extracellular calcium and phosphate ions. In the adult male worms, praziquantel (PZQ) prevents this accumulation in RPMI medium and disperses resorufin from tubules which have been prelabelled. Female worms and all other developmental stages are much less affected either by the presence of calcium and phosphate ions, or the disruption caused by PZQ. The male can inhibit the excretory system in paired female. Fluorescent PZQ localises in the posterior gut (intestine) region of the male adult worm, but not in the excretory system, except for the anionic carboxy fluorescein derivative of PZQ, which may be excreted by this route. All stages of the parasite can recover from damage by PZQ treatment in vitro. The excretory system is highly sensitive to damage to the surface membrane and may be involved in vesicle movement and damage repair processes. In vivo the adult parasite does not recover from PZQ treatment, but what is inhibiting recovery is unknown, but likely to be related to immune effector molecules.

Difilobotriasis humana por diphyllobothrium pacificum: un nuevo caso en Antofagasta, norte de Chile / Diphyllobothrium pacificum human infection: report of one case

We report a 26 years old asymptomatic female that expelled spontaneously a 39 proglottid strobila. The taxonomic study of proglottids and eggs found in the stool examination concluded that the patient was infected by Diphyllobothrium pacificum. This infection was probably acquired by the ingestion of raw fish (Sciaena deliciosa) with lemon in a traditional plate called "cebiche". She was treated with a single dose of praziquantel in a dose of 10 mg/kg body weight. After 24 h of treatment there were no scolices on feces. The patient did not have anemia. All eight family members studied, did no have the infection. A follow up stool examination three months later remained negative

Suscetibilidade aos agentes quimioterápicos de isolados de Schistosoma mansoni oriundos de pacientes tratados com oxamniquine e praziquantel e näo curados / Susceptibility to the chemotherapeutical compounds of Schistosoma mansoni from patients treated with oxamniquine and praziquantel and not cured ones

Ten inhabitants of Itaquara, Bahia, Brazil treated with oxamniquine and subsequently praziquantel were not cured. Schistosoma mansoni isolates derived from these patients were studied. Snails were infected with miracidia derived from the feces of these patients and the cercariae produced used to infect albino mice. The animals were then treated with a single oral dose of oxamniquine (25, 50 and 100mg/kg) or praziquantel (100, 200 and 400 mg/kg). The response to chemotherapy was significantly different in some of the isolates although it was not possible to characterize any of them as resistant. In addition, DNA analysis of the isolates by means of ®Random Amplified Polymorphic DNA® indicated a low degree of variability as compared with a laboratory strain, LE. Thus, it was not possible to characterize these organisms at a genetic level as a distinct strain.

Lethal effect of oxamniquine and praziquantel on mice experimentally infected with Schistosoma mansoni

Pesquisou-se a letalidade causada por administracao de drogas (oxamniquina e praziquantel) em camundongos infectados por Schistosoma mansoni e seus respectivos controles nao infectados. Os resultados indicam que os animais infectados apresentam claramente taxas de mortalidade mais altas, quando foi utilizado o praziquantel...

Enfoque terapéutico de la neurocisticercosis / Therapeutic management of neurocysticercosis

Resulta claro que todo paciente portador de neurocisticercosis debe recibir tratamiento etiológico o sintomático de acuerdo a cada caso en partícular. La indicación de terapia corticoidal está más definida para el manejo de quistes parenquimatosos y subaracnoideos gigantes, hidrocefalia por aracnoiditis activa, angeítis y encefalitis por cisticerco. La cirugía tiene su rol específico en los casos de hidrocefalia, quistes espinales y aquellos quistes subaracnoideos gigantes en el caso de que no respondan al tratamiento médico. La craneotomía descompresiva puede resultar una medida de salvataje en casos de encefalitis por cisticerco. Por último es importante mencionar que actualmente la droga de elección es el albendazol, pero el praziquantel debe ser considerado en casos especiales de resistencia y usársela como terapia única o asociada al fármaco de elección

Response of drug resistant isolates of Schistosoma mansoni to antischistosomal agents

The susceptibility of four isolates of Schistosoma mansoni (BH, MAP, MPR-1 and K) to four multiple doses of anti-schistosomal agents (hycanthone, niridazole, oxamniquire, and praziquantel) were evaluated in infected female Swiss albino mice. These schistosomal isolates had been maintained in the laboratory without further drug pressure for 20 to 30 generations. Multiple dosage regimens were used for each drug against each isolate of S. mansoni to generate ED50 (effective dose 50%) values. Results demonstrated that the K isolate is resistant to niridazole, the MPR-1 isolate to oxamniquine, and the MAP isolate to both hycanthone and oxamniquine. The BH isolate was susceptible to all drugs and was used as the reference isolate. All isolates were susceptible to praziquantel. The significance of the difference in response of the MPR-1 and MAP isolates is discussed. These results confirm the resistance of these isolates of S. mansoni of three schistosomicides and demonstrate that the resistance of these isolates are stable over long periods of time without exposure to drugs

Praziquantel inhibits Schistosoma mansoni attachment in vitro

Male adult Schistosoma mansoni worms were placed in a glass dish containing Tyrode solution and observed for 15 min after addition of praziquantel (0.01 to 1 pmicron M). Praziquantel promoted a concentrationp- and time-dependent inhibition of sucker-mediated attachment of the worm. Attachment inhibition was correlated with shortening of the parasite. We propose that the rapid and total inhibition of worm attachment observed in vitro with 1 micronM praziquantel indicates that therapeutic concentrations of this drug should promote a rapid hepatic shift, in vivo, which may facilitate host tissue reaction

Neurocisticercosis, tratamiento con prazicuantel / Neurocysticercosis, treatment with praziquantel

Se ha encontrado cisticercosis en el sistema nervioso central en el 2.3 a 6.3% y 0.7% de necropsias efectuadas en México y Colombia respectivamente; mientras que de 82.700 realizadas entre 1918 a 1965 en la Universidad de California, Estados Unidos, se presentaron 17 casos; sin embargo, el número se incrementó de manera considerable debido a la inmigración de latinoamericanos en los últimos años. También se ha informado en Europa del este, India, Sudamérica siendo la infección parasitaria más común del sistema nervioso central (SNC)

Efeito da quimioterapia sobre os ovos do Schistosoma mansoni / Effect of chemotherapy on the Schistosoma mansoni eggs

A administraçäo de praziquantel a camundongos infectados pelo Schistosoma mansoni(50 cercarias/8 semanas) causou necrose de coagulaçäo e/ou lítica, e por vezes calcificaçäo dos miracídios nos tecidos a partir do 4§ dia do início do tratamento. A administraçäo conjugada de oxamniquine/hycanthone, embora muito efetiva para eliminar os vermes adultos, näo teve açäo sobre os miracídios no interior dos granulomas, tendo os testes de eclosäo sido positivos até o 15§ dia após o tratamento curativo. A açäo do praziquantel sobre os ovos do S. mansoni pode ter repercussäo sorológica ou patogênica, facilitando uma mais rápida reabsorçäo dos granulomas pelos tecidos do hospedeiro

Failure at inducing resistance to schistosomicidal drugs in a brazilian human strain of Schistosoma mansoni

No presente estudo, realizou-se uma tentativa de induçäo de resistência a 3 drogas esquistossomicidas em uma cepa brasileira de S. mansoni, segundo o esquema de induçäo de resistência tipo II preconizado por Jansma et al. em 1977. Houve insucesso nas 3 tentativas realizadas. A geraçäo parental tratada com a droga durante o estágio imaturo do verme mostrou-se menos suscetível aos quimioterápicos do que as geraçöes F1 e F2 do verme. Uma hipótese é levantada para a explicaçäo do fato