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Phylogenetic and transcriptomic study of aldo-keto reductases in Haemonchus contortus and their inducibility by flubendazole 线虫中醛酮还原酶的系统发育和转录组学研究以及氟苯咪唑对它们的诱导作用
IF 4.1 2区 医学 Q1 PARASITOLOGY Pub Date : 2024-07-08 DOI: 10.1016/j.ijpddr.2024.100555
Karolína Štěrbová, Lucie Raisová Stuchlíková, Nikola Rychlá, Kateřina Kohoutová, Markéta Babičková, Lenka Skálová, Petra Matoušková

Aldo-keto reductases (AKRs), a superfamily of NADP(H)-dependent oxidoreductases, catalyze the oxidoreduction of a wide variety of eobiotic and xenobiotic aldehydes and ketones. In mammals, AKRs play essential roles in hormone and xenobiotic metabolism, oxidative stress, and drug resistance, but little is known about these enzymes in the parasitic nematode Haemonchus contortus. In the present study, 22 AKR genes existing in the H. contortus genome were investigated and a phylogenetic analysis with comparison to AKRs in Caenorhabditis elegans, sheep and humans was conducted. The constitutive transcription levels of all AKRs were measured in eggs, larvae, and adults of H. contortus, and their expression was compared in a drug-sensitive strain (ISE) and a benzimidazole-resistant strain (IRE) previously derived from the sensitive strain by imposing benzimidazole selection pressure. In addition, the inducibility of AKRs by exposure of H. contortus adults to benzimidazole anthelmintic flubendazole in vitro was tested. Phylogenetic analysis demonstrated that the majority of AKR genes in H. contortus lack orthologues in the sheep genome, which is a favorable finding for considering AKRs as potential drug targets. Large differences in the expression levels of individual AKRs were observed, with AKR1, AKR3, AKR8, and AKR10 being the most highly expressed at most developmental stages. Significant changes in the expression of AKRs during the life cycle and pronounced sex differences were found. Comparing the IRE and ISE strains, three AKRs were upregulated, and seven AKRs were downregulated in adults. In addition, the expression of three AKRs was induced by flubendazole exposure in adults of the ISE strain. Based on these results, AKR1, AKR2, AKR3, AKR5, AKR10 and AKR19 in particular merit further investigation and functional characterization with respect to their potential involvement in drug biotransformation and anthelmintic resistance in H. contortus.

醛酮还原酶(AKRs)是一种依赖 NADP(H)的超家族氧化还原酶,可催化多种生物和异生物醛酮的氧化还原。在哺乳动物体内,AKRs 在激素和异生物代谢、氧化应激和耐药性方面发挥着重要作用,但人们对寄生线虫中的这些酶却知之甚少。在本研究中,研究人员调查了寄生线虫基因组中的 22 个 AKR 基因,并与秀丽隐杆线虫、绵羊和人类的 AKR 基因进行了系统发育分析和比较。研究人员测量了所有 AKRs 在虫卵、幼虫和成虫中的组成转录水平,并比较了它们在药物敏感株(ISE)和通过施加苯并咪唑选择压力从敏感株衍生的苯并咪唑抗性株(IRE)中的表达情况。此外,还测试了在体外将变形杆菌成虫暴露于苯并咪唑类抗虫药氟苯咪唑中诱导 AKR 的可能性。系统发育分析表明,轮虫中的大多数 AKR 基因在绵羊基因组中缺乏同源物,这一发现有利于将 AKR 作为潜在的药物靶点。观察到单个 AKR 的表达水平差异很大,其中 AKR1、AKR3、AKR8 和 AKR10 在大多数发育阶段的表达水平最高。在生命周期中,AKRs 的表达发生了显著变化,并发现了明显的性别差异。比较 IRE 和 ISE 品系,在成年期有三个 AKR 上调,七个 AKR 下调。此外,在 ISE 株系的成虫中,氟苯咪唑会诱导三个 AKRs 的表达。基于这些结果,AKR1、AKR2、AKR3、AKR5、AKR10 和 AKR19 尤其值得进一步研究,并确定其功能特征,以了解它们在 H. contortus 的药物生物转化和抗寄生虫药耐药性中的潜在参与。
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引用次数: 0
Effect of Phlebotomus papatasi on the fitness, infectivity and antimony-resistance phenotype of antimony-resistant Leishmania major Mon-25 Phlebotomus papatasi 对抗锑利什曼原虫 Mon-25 的适应性、感染性和抗锑表型的影响。
IF 4.1 2区 医学 Q1 PARASITOLOGY Pub Date : 2024-06-24 DOI: 10.1016/j.ijpddr.2024.100554
Nalia Mekarnia , Kamal-Eddine Benallal , Jovana Sádlová , Barbora Vojtková , Aurélie Mauras , Nicolas Imbert , Maryline Longhitano , Zoubir Harrat , Petr Volf , Philippe M. Loiseau , Sandrine Cojean

Leishmania major is responsible for zoonotic cutaneous leishmaniasis. Therapy is mainly based on the use of antimony-based drugs; however, treatment failures and illness relapses were reported. Although studies were developed to understand mechanisms of drug resistance, the interactions of resistant parasites with their reservoir hosts and vectors remain poorly understood. Here we compared the development of two L. major MON-25 trivalent antimony-resistant lines, selected by a stepwise in vitro Sb(III)-drug pressure, to their wild-type parent line in the natural vector Phlebotomus papatasi. The intensity of infection, parasite location and morphological forms were compared by microscopy. Parasite growth curves and IC50 values have been determined before and after the passage in Ph. papatasi. qPCR was used to assess the amplification rates of some antimony-resistance gene markers. In the digestive tract of sand flies, Sb(III)-resistant lines developed similar infection rates as the wild-type lines during the early-stage infections, but significant differences were observed during the late-stage of the infections. Thus, on day 7 p. i., resistant lines showed lower representation of heavy infections with colonization of the stomodeal valve and lower percentage of metacyclic promastigote forms in comparison to wild-type strains. Observed differences between both resistant lines suggest that the level of Sb(III)-resistance negatively correlates with the quality of the development in the vector. Nevertheless, both resistant lines developed mature infections with the presence of infective metacyclic forms in almost half of infected sandflies. The passage of parasites through the sand fly guts does not significantly influence their capacity to multiply in vitro. The IC50 values and molecular analysis of antimony-resistance genes showed that the resistant phenotype of Sb(III)-resistant parasites is maintained after passage through the sand fly. Sb(III)-resistant lines of L. major MON-25 were able to produce mature infections in Ph. papatasi suggesting a possible circulation in the field using this vector.

大利什曼原虫是人畜共患皮肤利什曼病的罪魁祸首。治疗主要以使用锑基药物为主,但也有治疗失败和疾病复发的报道。虽然研究人员已经了解了抗药性的机制,但对抗药性寄生虫与其贮存宿主和载体之间的相互作用仍然知之甚少。在这里,我们比较了通过逐步体外Sb(III)-药物压力筛选出的两个L. major MON-25三价抗锑品系与其野生型亲本品系在自然载体Phlebotomus papatasi中的发展情况。通过显微镜比较了感染强度、寄生虫位置和形态。利用 qPCR 评估了一些抗锑基因标记的扩增率。在沙蝇的消化道中,Sb(III)抗性品系在感染初期的感染率与野生型品系相似,但在感染后期则出现了显著差异。因此,与野生型品系相比,抗性品系在第 7 天时表现出较低的重度感染率,并在气孔瓣膜上形成定殖,而中生原虫的比例也较低。两个抗性品系之间的差异表明,Sb(III)抗性水平与载体的发育质量呈负相关。尽管如此,两个抗性品系都发展出了成熟的感染,几乎一半的受感染沙蝇都出现了有感染力的元簇。寄生虫通过沙蝇内脏并不会对其体外繁殖能力产生重大影响。IC50 值和抗锑基因的分子分析表明,耐 Sb(III)寄生虫的抗锑表型在通过沙蝇后仍能保持。对 Sb(III)有抗性的 L. major MON-25 株系能够在 Ph. papatasi 中产生成熟的感染,这表明该病媒可能在田间流通。
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引用次数: 0
In vitro and in vivo activity evaluation and mode of action of broxaldine on Toxoplasma gondii 溴鼠灵对弓形虫的体外和体内活性评估及作用模式。
IF 4.1 2区 医学 Q1 PARASITOLOGY Pub Date : 2024-06-20 DOI: 10.1016/j.ijpddr.2024.100552
Yanhua Qiu , Bintao Zhai , Yubin Bai , Hongling Lin , Lingyu Wu , Wei Luo , Mengyan Shi , Shulin Chen , Jiyu Zhang

Toxoplasma gondii (T. gondii) is a highly successful global parasite, infecting about one-third of the world's population and significantly affecting human life and the economy. However, current drugs for toxoplasmosis treatment have considerable side effects, and there is no specific drug to meet current needs. This study aims to evaluate the anti-T. gondii activity of broxaldine (BRO) in vitro and in vivo and explore its mechanism of action. Our results showed that compared to the control group, the invasion rate of tachyzoites in the 4 μg/mL BRO group was only 14.31%, and the proliferation rate of tachyzoites in host cells was only 1.23%. Furthermore, BRO disrupted the lytic cycle of T. gondii and reduced the size and number of cysts in vitro. A mouse model of acute toxoplasmosis reported a 41.5% survival rate after BRO treatment, with reduced parasite load in tissues and blood. The subcellular structure of T. gondii was observed, including disintegration of T. gondii, mitochondrial swelling, increased liposomes, and the presence of autophagic lysosomes. Further investigation revealed enhanced autophagy, increased neutral lipids, and decreased mitochondrial membrane potential in T. gondii treated with BRO. The results also showed a significant decrease in ATP levels. Overall, BRO demonstrates good anti-T. gondii activity in vitro and in vivo; therefore, it has the potential to be used as a lead compound for anti-T. gondii treatment.

弓形虫(T. gondii)是一种非常成功的全球性寄生虫,感染了全球约三分之一的人口,对人类生活和经济造成了重大影响。然而,目前治疗弓形虫病的药物有相当大的副作用,而且还没有一种特效药物能满足当前的需要。本研究旨在评估布洛沙定(BRO)在体外和体内的抗弓形虫活性,并探讨其作用机制。结果表明,与对照组相比,4 μg/mL BRO 组速虫的侵袭率仅为 14.31%,速虫在宿主细胞中的增殖率仅为 1.23%。此外,BRO 还能破坏淋球菌的溶解周期,减少体外包囊的大小和数量。据报道,急性弓形虫病小鼠模型经 BRO 治疗后存活率为 41.5%,组织和血液中的寄生虫量减少。观察到弓形虫的亚细胞结构,包括弓形虫解体、线粒体肿胀、脂质体增加以及自噬溶酶体的存在。进一步研究发现,用 BRO 处理的淋球菌自噬能力增强,中性脂质增加,线粒体膜电位降低。结果还显示 ATP 水平明显下降。总之,BRO 在体外和体内都表现出了良好的抗淋病活性;因此,它有可能被用作抗淋病治疗的先导化合物。
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引用次数: 0
Efficacy of the bumped kinase inhibitor BKI-1708 against the cyst-forming apicomplexan parasites Toxoplasma gondii and Neospora caninum in vitro and in experimentally infected mice 撞击激酶抑制剂 BKI-1708 在体外和实验感染小鼠体内对形成囊蚴的弓形虫和犬新孢子虫的疗效。
IF 4.1 2区 医学 Q1 PARASITOLOGY Pub Date : 2024-06-19 DOI: 10.1016/j.ijpddr.2024.100553
Maria Cristina Ferreira de Sousa , Dennis Imhof , Kai Pascal Alexander Hänggeli , Ryan Choi , Matthew A. Hulverson , Samuel L.M. Arnold , Wesley C. Van Voorhis , Erkang Fan , Sánchez-Sánchez Roberto , Luis M. Ortega-Mora , Andrew Hemphill

Toxoplasma gondii and Neospora caninum are major worldwide morbidity-causing pathogens. Bumped kinase inhibitors (BKIs) are a compound class that has been optimized to target the apicomplexan calcium-dependent protein kinase 1 (CDPK1) – and several members of this class have proven to be safe and highly active in vitro and in vivo. BKI-1708 is based on a 5-aminopyrazole-4-carboxamide scaffold, and exhibited in vitro IC50 values of 120 nM for T. gondii and 480 nM for N. caninum β-galactosidase expressing strains, and did not affect human foreskin fibroblast (HFF) viability at concentrations up to 25 μM. Electron microscopy established that exposure of tachyzoite-infected fibroblasts to 2.5 μM BKI-1708 in vitro induced the formation of multinucleated schizont-like complexes (MNCs), characterized by continued nuclear division and harboring newly formed intracellular zoites that lack the outer plasma membrane. These zoites were unable to finalize cytokinesis to form infective tachyzoites. BKI-1708 did not affect zebrafish (Danio rerio) embryo development during the first 96 h following egg hatching at concentrations up to 2 μM. Treatments of mice with BKI-1708 at 20 mg/kg/day during five consecutive days resulted in drug plasma levels ranging from 0.14 to 4.95 μM. In vivo efficacy of BKI-1708 was evaluated by oral application of 20 mg/kg/day from day 9–13 of pregnancy in mice experimentally infected with N. caninum (NcSpain-7) tachyzoites or T. gondii (TgShSp1) oocysts. This resulted in significantly decreased cerebral parasite loads and reduced vertical transmission in both models without drug-induced pregnancy interference.

弓形虫(Toxoplasma gondii)和犬新孢子虫(Neospora caninum)是全球主要的致病病原体。颠簸激酶抑制剂(BKIs)是一类针对 apicomplexan 的钙依赖性蛋白激酶 1(CDPK1)而优化的化合物--该类化合物的几个成员已被证明在体外和体内安全且活性高。BKI-1708 基于 5-aminopyrazole-4-boxamide 支架,对淋球菌的体外 IC50 值为 120 nM,对 N. caninum β-半乳糖苷酶表达株的 IC50 值为 480 nM,在浓度高达 25 μM 时不影响人包皮成纤维细胞(HFF)的活力。电子显微镜证实,在体外将受塔氏原虫感染的成纤维细胞暴露于 2.5 μM BKI-1708 会诱导形成多核裂殖子样复合物(MNC),其特征是核分裂持续进行,并包藏新形成的缺乏外质膜的胞内佐虫。这些滋生体无法完成细胞分裂,形成具有感染力的速生虫。BKI-1708 在浓度高达 2 μM 时不会影响斑马鱼(Danio rerio)卵孵化后最初 96 小时的胚胎发育。连续五天以 20 毫克/千克/天的剂量给小鼠注射 BKI-1708,其血浆药物浓度为 0.14 至 4.95 μM。在实验性感染犬细小病毒(N. caninum (NcSpain-7) tachyzoites)或淋病双球菌(T. gondii (TgShSp1))卵囊的小鼠中,从怀孕的第 9-13 天开始口服 20 毫克/千克/天的 BKI-1708 进行体内疗效评估。这两种模型中的脑寄生虫数量都明显减少,垂直传播也有所降低,而不会受到药物引起的妊娠干扰。
{"title":"Efficacy of the bumped kinase inhibitor BKI-1708 against the cyst-forming apicomplexan parasites Toxoplasma gondii and Neospora caninum in vitro and in experimentally infected mice","authors":"Maria Cristina Ferreira de Sousa ,&nbsp;Dennis Imhof ,&nbsp;Kai Pascal Alexander Hänggeli ,&nbsp;Ryan Choi ,&nbsp;Matthew A. Hulverson ,&nbsp;Samuel L.M. Arnold ,&nbsp;Wesley C. Van Voorhis ,&nbsp;Erkang Fan ,&nbsp;Sánchez-Sánchez Roberto ,&nbsp;Luis M. Ortega-Mora ,&nbsp;Andrew Hemphill","doi":"10.1016/j.ijpddr.2024.100553","DOIUrl":"10.1016/j.ijpddr.2024.100553","url":null,"abstract":"<div><p><em>Toxoplasma gondii</em> and <em>Neospora caninum</em> are major worldwide morbidity-causing pathogens. Bumped kinase inhibitors (BKIs) are a compound class that has been optimized to target the apicomplexan calcium-dependent protein kinase 1 (CDPK1) – and several members of this class have proven to be safe and highly active <em>in vitro</em> and <em>in vivo</em>. BKI-1708 is based on a 5-aminopyrazole-4-carboxamide scaffold, and exhibited <em>in vitro</em> IC<sub>50</sub> values of 120 nM for <em>T. gondii</em> and 480 nM for <em>N. caninum</em> β-galactosidase expressing strains, and did not affect human foreskin fibroblast (HFF) viability at concentrations up to 25 μM. Electron microscopy established that exposure of tachyzoite-infected fibroblasts to 2.5 μM BKI-1708 <em>in vitro</em> induced the formation of multinucleated schizont-like complexes (MNCs), characterized by continued nuclear division and harboring newly formed intracellular zoites that lack the outer plasma membrane. These zoites were unable to finalize cytokinesis to form infective tachyzoites. BKI-1708 did not affect zebrafish (<em>Danio rerio</em>) embryo development during the first 96 h following egg hatching at concentrations up to 2 μM. Treatments of mice with BKI-1708 at 20 mg/kg/day during five consecutive days resulted in drug plasma levels ranging from 0.14 to 4.95 μM. <em>In vivo</em> efficacy of BKI-1708 was evaluated by oral application of 20 mg/kg/day from day 9–13 of pregnancy in mice experimentally infected with <em>N. caninum</em> (NcSpain-7) tachyzoites or <em>T. gondii</em> (TgShSp1) oocysts. This resulted in significantly decreased cerebral parasite loads and reduced vertical transmission in both models without drug-induced pregnancy interference.</p></div>","PeriodicalId":13775,"journal":{"name":"International Journal for Parasitology: Drugs and Drug Resistance","volume":"25 ","pages":"Article 100553"},"PeriodicalIF":4.1,"publicationDate":"2024-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2211320724000344/pdfft?md5=05337ab18fdd00386b0462cc8f9ec858&pid=1-s2.0-S2211320724000344-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141450433","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Lower micromolar activity of the antifungal imidazoles on the bacterial-type bifunctional aldehyde/alcohol dehydrogenase (AdhE) in Cryptosporidium parvum and in vitro efficacy against the zoonotic parasite 抗真菌咪唑类化合物对副隐孢子虫体内细菌型双功能醛/醇脱氢酶(AdhE)的较低微摩尔活性和对人畜共患寄生虫的体外疗效
IF 4 2区 医学 Q1 PARASITOLOGY Pub Date : 2024-06-06 DOI: 10.1016/j.ijpddr.2024.100551
Haichuan Chen , Dongqiang Wang , Chenchen Wang , Peng Jiang , Mingxiao Liu , Jigang Yin , Yonglan Yu

Cryptosporidium parvum is a waterborne and foodborne zoonotic protozoan parasite, a causative agent of moderate to severe diarrheal diseases in humans and animals. However, fully effective treatments are unavailable for medical and veterinary uses. There is a need to explore new drug targets for potential development of new therapeutics. Because C. parvum relies on anaerobic metabolism to produce ATP, fermentative enzymes in this parasite are attractive targets for exploration. In this study, we investigated the ethanol-fermentation in the parasite and characterized the basic biochemical features of a bacterial-type bifunctional aldehyde/alcohol dehydrogenase, namely CpAdhE. We also screened 3892 chemical entries from three libraries and identified 14 compounds showing >50% inhibition on the enzyme activity of CpAdhE. Intriguingly, antifungal imidazoles and unsaturated fatty acids are the two major chemical groups among the top hits. We further characterized the inhibitory kinetics of selected imidazoles and unsaturated fatty acids on CpAdhE. These compounds displayed lower micromolar activities on CpAdhE (i.e., IC50 values ranging from 0.88 to 11.02 μM for imidazoles and 8.93 to 35.33 μM for unsaturated fatty acids). Finally, we evaluated the in vitro anti-cryptosporidial efficacies and cytotoxicity of three imidazoles (i.e., tioconazole, miconazole and isoconazole). The three antifungal imidazoles exhibited lower micromolar efficacies against the growth of C. parvum in vitro (EC50 values ranging from 4.85 to 10.41 μM and selectivity indices ranging from 5.19 to 10.95). The results provide a proof-of-concept data to support that imidazoles are worth being further investigated for potential development of anti-cryptosporidial therapeutics.

副隐孢子虫是一种通过水和食物传播的人畜共患原生动物寄生虫,是导致人类和动物中度至重度腹泻疾病的病原体。然而,目前还没有完全有效的医疗和兽医治疗方法。因此有必要探索新的药物靶点,以开发潜在的新疗法。由于寄生虫依靠厌氧代谢产生 ATP,因此这种寄生虫体内的发酵酶是有吸引力的探索目标。在这项研究中,我们研究了寄生虫的乙醇发酵过程,并鉴定了细菌型双功能醛/醇脱氢酶(即 CpAdhE)的基本生化特征。我们还筛选了三个文库中的 3892 个化学条目,发现了 14 个对 CpAdhE 的酶活性有 50%抑制作用的化合物。耐人寻味的是,抗真菌的咪唑类和不饱和脂肪酸是命中率最高的两大化学组。我们进一步鉴定了所选咪唑类和不饱和脂肪酸对 CpAdhE 的抑制动力学。这些化合物对 CpAdhE 的微摩尔活性较低(即咪唑类化合物的 IC50 值为 0.88 至 11.02 μM,不饱和脂肪酸的 IC50 值为 8.93 至 35.33 μM)。最后,我们评估了三种咪唑类化合物(即替康唑、咪康唑和异康唑)的体外抗隐孢子虫功效和细胞毒性。这三种咪唑类抗真菌剂对副噬菌体的体外生长具有较低的微摩尔效力(EC50 值介于 4.85 至 10.41 μM 之间,选择性指数介于 5.19 至 10.95 之间)。这些结果提供了概念验证数据,证明咪唑类化合物值得进一步研究,以开发潜在的抗隐孢子虫疗法。
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引用次数: 0
Ouabain, ATPase inhibitor, potentially enhances the effect of polyhexamethylene biguanide on Acanthamoeba castellanii ATP酶抑制剂欧贝因可增强聚六亚甲基双胍对卡斯特阿米巴的作用
IF 4 2区 医学 Q1 PARASITOLOGY Pub Date : 2024-05-22 DOI: 10.1016/j.ijpddr.2024.100550
Kuang-Yi Shih , Yao-Tsung Chang , Yu-Jen Wang , Jian-Ming Huang

Acanthamoeba, a free-living amoeba, is commonly found in various natural environments, such as rivers and soil, as well as in public baths, swimming pools, and sewers. Acanthamoeba can cause severe illness such as granulomatous amoebic encephalitis and Acanthamoeba keratitis (AK) in humans. AK, the most recognized disease, can cause permanent visual impairment or blindness by affecting the cornea. AK commonly affects contact lens wearers who neglect proper cleaning habits. The symptoms of AK include epithelial and stromal destruction, corneal infiltrate, and intense ocular pain, occasionally necessitating surgical removal of the entire eyeball. Current AK treatment involves the hourly application of eye drops containing polyhexamethylene biocide (PHMB). However, studies have revealed their ineffectiveness against drug-resistant strains. Acanthamoeba can form cysts as a survival mechanism in adverse environments, though the exact mechanism remains unknown. Our experiments revealed that sodium P-type ATPase (ACA1_065450) is closely linked to encystation. In addition, various encystation buffers, such as MgCl2 or NaCl, induced the expression of P-type ATPase. Furthermore, we used ouabain, an ATPase inhibitor, to inhibit the Na+/K+ ion pump, consequently decreasing the encystation rate of Acanthamoeba. Our primary objective is to develop an advanced treatment for AK. We anticipate that the combination of ouabain and PHMB may serve as an effective therapeutic approach against AK in the future.

棘阿米巴是一种自由生活的阿米巴,常见于各种自然环境中,如河流和土壤,以及公共浴池、游泳池和下水道。阿米巴原虫可导致严重疾病,如肉芽肿阿米巴脑炎和阿米巴角膜炎(AK)。AK是最常见的疾病,可通过影响角膜导致永久性视力损伤或失明。AK 通常会影响那些忽视正确清洁习惯的隐形眼镜佩戴者。AK 的症状包括角膜上皮和基质破坏、角膜浸润和剧烈眼痛,有时需要通过手术切除整个眼球。目前治疗 AK 的方法是每小时滴用含有聚六亚甲基生物杀灭剂(PHMB)的眼药水。但研究表明,这些药物对耐药菌株无效。棘阿米巴可以形成囊肿,作为在恶劣环境中的一种生存机制,但其确切机制尚不清楚。我们的实验发现,钠 P 型 ATP 酶(ACA1_065450)与囊肿的形成密切相关。此外,MgCl2或NaCl等各种气滞缓冲液都能诱导P型ATP酶的表达。此外,我们还使用了一种 ATPase 抑制剂--乌巴因(ouabain)来抑制 Na+/K+ 离子泵,从而降低了棘阿米巴的包囊率。我们的主要目标是开发一种先进的 AK 治疗方法。我们预计,uabain 和 PHMB 的组合将来可能会成为治疗 AK 的有效方法。
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引用次数: 0
First case of macrocyclic lactone-resistant Dirofilaria immitis in Europe - Cause for concern 欧洲首例抗大环内酯类药物的软下疳病例--值得关注
IF 4 2区 医学 Q1 PARASITOLOGY Pub Date : 2024-05-21 DOI: 10.1016/j.ijpddr.2024.100549
Donato Traversa , Anastasia Diakou , Mariasole Colombo , Sohini Kumar , Thavy Long , Serafeim C. Chaintoutis , Luigi Venco , Gianluca Betti Miller , Roger Prichard

Heartworm disease caused by the nematode Dirofilaria immitis is one of the most important parasitoses of dogs. The treatment of the infection is long, complicated, risky and expensive. Conversely, prevention is easy, safe, and effective and it is achieved by the administration of macrocyclic lactones (MLs). In recent years, D. immitis strains resistant to MLs have been described in Southern USA, raising concerns for possible emergence, or spreading in other areas of the world. The present study describes the first case of ML-resistant D. immitis in a dog in Europe. The dog arrived in Rome, Italy, from USA in 2023. Less than 6 months after its arrival in Italy, the dog tested positive for D. immitis circulating antigen and microfilariae, despite it having received monthly the ML milbemycin oxime (plus an isoxazoline) after arrival. The microfilariae suppression test suggested a resistant strain. Microfilariae DNA was examined by droplet digital PCR-based duplex assays targeting four marker positions at single nucleotide polymorphisms (SNP1, SNP2, SNP3, SNP7) which differentiate resistant from susceptible isolates. The genetic analysis showed that microfilariae had a ML-resistant genotype at SNP1 and SNP7 positions, compatible with a resistant strain. It is unlikely that the dog acquired the infection after its arrival in Europe, while it is biologically and epidemiologically plausible that the dog was already infected when imported from USA to Europe. The present report highlights the realistic risk of ML-resistant D. immitis strains being imported and possibly transmitted in Europe and other areas of the world. Monitoring dogs travelling from one area to another, especially if they originate from regions where ML-resistance is well-documented, is imperative. Scientists, practitioners, and pet owners should be aware of the risk and remain vigilant against ML-resistance, in order to monitor and reduce the spreading of resistant D. immitis.

由盘尾丝虫病线虫引起的心丝虫病是狗最重要的寄生虫病之一。该病的治疗过程漫长、复杂、风险高且费用昂贵。相反,通过服用大环内酯(ML)来预防则简单、安全、有效。近年来,美国南部出现了对大环内酯类药物有抗药性的白喉杆菌菌株,这引起了人们对白喉杆菌可能在世界其他地区出现或传播的担忧。本研究描述了欧洲首例对 ML 具有抗药性的白喉杆菌病例。这只狗于 2023 年从美国抵达意大利罗马。抵达意大利不到 6 个月后,尽管该犬在抵达后每月接受一次 ML 米尔贝霉素肟(外加一种异噁唑啉)治疗,但该犬的 D. immitis 循环抗原和微丝蚴检测结果呈阳性。微丝蚴抑制试验表明这是一种耐药菌株。微丝蚴 DNA 检测采用基于液滴数字 PCR 的双链检测法,针对单核苷酸多态性(SNP1、SNP2、SNP3 和 SNP7)的四个标记位点进行检测,这些标记位点可区分抗性和易感分离株。基因分析表明,微丝蚴在 SNP1 和 SNP7 位点上具有抗 ML 基因型,与抗性菌株相符。该犬不可能是在抵达欧洲后才感染的,而从生物学和流行病学角度来看,该犬从美国进口到欧洲时就已经感染了。本报告强调了耐甲型肝炎病毒株被输入并可能在欧洲和世界其他地区传播的现实风险。必须对从一个地区前往另一个地区的狗进行监控,尤其是当这些狗来自有充分证据表明对 ML 具有抗药性的地区时。科学家、从业人员和宠物主人都应意识到这一风险,并对耐甲流病毒保持警惕,以监测和减少耐甲流病毒的传播。
{"title":"First case of macrocyclic lactone-resistant Dirofilaria immitis in Europe - Cause for concern","authors":"Donato Traversa ,&nbsp;Anastasia Diakou ,&nbsp;Mariasole Colombo ,&nbsp;Sohini Kumar ,&nbsp;Thavy Long ,&nbsp;Serafeim C. Chaintoutis ,&nbsp;Luigi Venco ,&nbsp;Gianluca Betti Miller ,&nbsp;Roger Prichard","doi":"10.1016/j.ijpddr.2024.100549","DOIUrl":"https://doi.org/10.1016/j.ijpddr.2024.100549","url":null,"abstract":"<div><p>Heartworm disease caused by the nematode <em>Dirofilaria immitis</em> is one of the most important parasitoses of dogs. The treatment of the infection is long, complicated, risky and expensive. Conversely, prevention is easy, safe, and effective and it is achieved by the administration of macrocyclic lactones (MLs). In recent years, <em>D. immitis</em> strains resistant to MLs have been described in Southern USA, raising concerns for possible emergence, or spreading in other areas of the world. The present study describes the first case of ML-resistant <em>D. immitis</em> in a dog in Europe. The dog arrived in Rome, Italy, from USA in 2023. Less than 6 months after its arrival in Italy, the dog tested positive for <em>D. immitis</em> circulating antigen and microfilariae, despite it having received monthly the ML milbemycin oxime (plus an isoxazoline) after arrival. The microfilariae suppression test suggested a resistant strain. Microfilariae DNA was examined by droplet digital PCR-based duplex assays targeting four marker positions at single nucleotide polymorphisms (SNP1, SNP2, SNP3, SNP7) which differentiate resistant from susceptible isolates. The genetic analysis showed that microfilariae had a ML-resistant genotype at SNP1 and SNP7 positions, compatible with a resistant strain. It is unlikely that the dog acquired the infection after its arrival in Europe, while it is biologically and epidemiologically plausible that the dog was already infected when imported from USA to Europe. The present report highlights the realistic risk of ML-resistant <em>D. immitis</em> strains being imported and possibly transmitted in Europe and other areas of the world. Monitoring dogs travelling from one area to another, especially if they originate from regions where ML-resistance is well-documented, is imperative. Scientists, practitioners, and pet owners should be aware of the risk and remain vigilant against ML-resistance, in order to monitor and reduce the spreading of resistant <em>D. immitis</em>.</p></div>","PeriodicalId":13775,"journal":{"name":"International Journal for Parasitology: Drugs and Drug Resistance","volume":"25 ","pages":"Article 100549"},"PeriodicalIF":4.0,"publicationDate":"2024-05-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2211320724000307/pdfft?md5=3b7f04a849b1315ec7c73f8b0182faf0&pid=1-s2.0-S2211320724000307-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141095643","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Repurposing DrugBank compounds as potential Plasmodium falciparum class 1a aminoacyl tRNA synthetase multi-stage pan-inhibitors with a specific focus on mitomycin 将药物库化合物重新用作潜在的恶性疟原虫 1a 氨基酰 tRNA 合成酶多级泛抑制剂,特别关注丝裂霉素
IF 4 2区 医学 Q1 PARASITOLOGY Pub Date : 2024-05-20 DOI: 10.1016/j.ijpddr.2024.100548
Fisayo Olotu , Mariscal Brice Tchatat Tali , Curtis Chepsiror , Olivier Sheik Amamuddy , Fabrice Fekam Boyom , Özlem Tastan Bishop

Plasmodium falciparum aminoacyl tRNA synthetases (PfaaRSs) are potent antimalarial targets essential for proteome fidelity and overall parasite survival in every stage of the parasite's life cycle. So far, some of these proteins have been singly targeted yielding inhibitor compounds that have been limited by incidences of resistance which can be overcome via pan-inhibition strategies. Hence, herein, for the first time, we report the identification and in vitro antiplasmodial validation of Mitomycin (MMC) as a probable pan-inhibitor of class 1a (arginyl(A)-, cysteinyl(C), isoleucyl(I)-, leucyl(L), methionyl(M), and valyl(V)-) PfaaRSs which hypothetically may underlie its previously reported activity on the ribosomal RNA to inhibit protein translation and biosynthesis. We combined multiple in silico structure-based discovery strategies that first helped identify functional and druggable sites that were preferentially targeted by the compound in each of the plasmodial proteins: Ins1-Ins2 domain in Pf-ARS; anticodon binding domain in Pf-CRS; CP1-editing domain in Pf-IRS and Pf-MRS; C-terminal domain in Pf-LRS; and CP-core region in Pf-VRS. Molecular dynamics studies further revealed that MMC allosterically induced changes in the global structures of each protein. Likewise, prominent structural perturbations were caused by the compound across the functional domains of the proteins. More so, MMC induced systematic alterations in the binding of the catalytic nucleotide and amino acid substrates which culminated in the loss of key interactions with key active site residues and ultimate reduction in the nucleotide-binding affinities across all proteins, as deduced from the binding energy calculations. These altogether confirmed that MMC uniformly disrupted the structure of the target proteins and essential substrates. Further, MMC demonstrated IC50 < 5 μM against the Dd2 and 3D7 strains of parasite making it a good starting point for malarial drug development. We believe that findings from our study will be important in the current search for highly effective multi-stage antimalarial drugs.

恶性疟原虫氨基酰 tRNA 合成酶(PfaaRSs)是有效的抗疟靶标,在寄生虫生命周期的每个阶段,它们对蛋白质组的保真度和寄生虫的整体存活至关重要。迄今为止,这些蛋白中的一些已成为单一靶点,产生的抑制剂化合物受到抗药性发生率的限制,而抗药性可通过泛抑制策略加以克服。因此,在本文中,我们首次报告了丝裂霉素(MMC)作为 1a 类(精氨酰(A)-、半胱氨酸酰(C)-、异亮氨酰(I)-、亮氨酰(L)-、蛋氨酰(M)-和缬氨酰(V)-)可能的泛抑制剂的鉴定和体外抗疟验证。PfaaRSs,假设这可能是其先前报道的抑制核糖体 RNA 翻译和蛋白质生物合成的活性的基础。我们结合了多种基于硅学结构的发现策略,首先帮助确定了化合物在每个质体蛋白中优先靶向的功能和可药用位点:Pf-ARS 中的 Ins1-Ins2 结构域;Pf-CRS 中的反密码子结合结构域;Pf-IRS 和 Pf-MRS 中的 CP1 编辑结构域;Pf-LRS 中的 C 端结构域;以及 Pf-VRS 中的 CP 核心区域。分子动力学研究进一步表明,MMC 异构诱导了每种蛋白质全局结构的变化。同样,该化合物也对蛋白质的各个功能域造成了显著的结构扰动。此外,MMC 还诱导催化核苷酸和氨基酸底物的结合发生系统性变化,最终导致所有蛋白质失去与关键活性位点残基的关键相互作用,并最终降低了核苷酸结合亲和力。这些结果都证实,MMC 能一致地破坏目标蛋白质和重要底物的结构。此外,MMC 对 Dd2 和 3D7 株寄生虫的 IC50 < 5 μM,使其成为疟疾药物开发的良好起点。我们相信,我们的研究结果对于目前寻找高效的多阶段抗疟药物非常重要。
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引用次数: 0
Exploring therapeutic approaches against Naegleria fowleri infections through the COVID box 通过 COVID 盒探索奈格勒氏菌感染的治疗方法
IF 4 2区 医学 Q1 PARASITOLOGY Pub Date : 2024-05-07 DOI: 10.1016/j.ijpddr.2024.100545
Javier Chao-Pellicer , Iñigo Arberas-Jiménez , Ines Sifaoui , José E. Piñero , Jacob Lorenzo-Morales

Naegleria fowleri, known as the brain-eating amoeba, is the pathogen that causes the primary amoebic meningoencephalitis (PAM), a severe neurodegenerative disease with a fatality rate exceeding 95%. Moreover, PAM cases commonly involved previous activities in warm freshwater bodies that allow amoebae-containing water through the nasal passages. Hence, awareness among healthcare professionals and the general public are the key to contribute to a higher and faster number of diagnoses worldwide. Current treatment options for PAM, such as amphotericin B and miltefosine, are limited by potential cytotoxic effects. In this context, the repurposing of existing compounds has emerged as a promising strategy. In this study, the evaluation of the COVID Box which contains 160 compounds demonstrated significant in vitro amoebicidal activity against two type strains of N. fowleri. From these compounds, terconazole, clemastine, ABT-239 and PD-144418 showed a higher selectivity against the parasite compared to the remaining products. In addition, programmed cell death assays were conducted with these four compounds, unveiling compatible metabolic events in treated amoebae. These compounds exhibited chromatin condensation and alterations in cell membrane permeability, indicating their potential to induce programmed cell death. Assessment of mitochondrial membrane potential disruption and a significant reduction in ATP production emphasized the impact of these compounds on the mitochondria, with the identification of increased ROS production underscoring their potential as effective treatment options. This study emphasizes the potential of the mentioned COVID Box compounds against N. fowleri, providing a path for enhanced PAM therapies.

被称为食脑阿米巴的奈格勒氏菌是引起原发性阿米巴脑膜脑炎(PAM)的病原体,这是一种严重的神经退行性疾病,致死率超过 95%。此外,原发性阿米巴脑膜炎病例通常都曾在温暖的淡水水体中活动,使含有阿米巴的水通过鼻腔。因此,提高医护专业人员和公众对该病的认识是加快全球确诊率的关键。目前,两性霉素 B 和米替福新等治疗 PAM 的药物因其潜在的细胞毒性作用而受到限制。在这种情况下,现有化合物的再利用已成为一种前景广阔的策略。在这项研究中,对包含 160 种化合物的 COVID Box 进行了评估,结果表明,这些化合物对两种类型的 N. fowleri 菌株具有显著的体外阿米巴杀菌活性。在这些化合物中,特康唑、氯马斯汀、ABT-239 和 PD-144418 对寄生虫的选择性高于其他产品。此外,还对这四种化合物进行了程序性细胞死亡试验,揭示了在处理过的变形虫体内发生的兼容代谢事件。这些化合物表现出染色质凝结和细胞膜通透性的改变,表明它们具有诱导细胞程序性死亡的潜力。对线粒体膜电位破坏和 ATP 生成量显著减少的评估强调了这些化合物对线粒体的影响,而对 ROS 生成量增加的鉴定则强调了它们作为有效治疗方案的潜力。这项研究强调了上述 COVID Box 复合物对福氏奈瑟氏菌的潜在作用,为增强 PAM 疗法提供了一条途径。
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引用次数: 0
Acaricide resistance status of livestock ticks from East and West Africa and in vivo efficacy of acaricides to control them 东非和西非牲畜蜱虫对杀螨剂的抗药性状况以及杀螨剂的体内防治效果。
IF 4 2区 医学 Q1 PARASITOLOGY Pub Date : 2024-05-06 DOI: 10.1016/j.ijpddr.2024.100541
Alec Evans , Maxime Madder , Josephus Fourie , Lénaïg Halos , Bersissa Kumsa , Elikira Kimbita , Joseph Byaruhanga , Frank Norbert Mwiine , Dennis Muhanguzi , Safiou Bienvenu Adehan , Alassane Toure , Jahashi Nzalawahe , Fred Aboagye-Antwi , Ndudim Isaac Ogo , Leon Meyer , Frans Jongejan , Imad Bouzaidi Cheikhi , Maggie Fisher , Peter Holdsworth

Through a collaborative effort across six Sub-Saharan African countries, using recognized international assessment techniques, 23 stocks of three tick species (Rhipicephalus microplus, Rhipicephalus appendiculatus and Amblyomma variegatum) of economic importance for rural small holder farming communities from East and West Africa were collected from cattle, and evaluated in in vitro larval packet tests (LPT). The results demonstrated medium to high resistance to chlorfenvinphos and amitraz across species. Rhipicephalus microplus demonstrated high level alpha-cypermethrin and cypermethrin resistance. Stocks of A. variegatum (West Africa) and R. appendiculatus (Uganda) demonstrated medium level ivermectin resistance.

The four least susceptible stocks (East and West African R. microplus, A. variegatum and R. appendiculatus) were taken into in vivo controlled cattle studies where fipronil was found effective against West and East African R. microplus isolates although persistent efficacy failed to reach 90%. Cymiazole and cypermethrin, and ivermectin based acaricides were partially effective against R. microplus without persistent efficacy. Flumethrin spray-on killed A. variegatum within 72 h for up to 10 days posttreatment, however product application was directly to tick attachment sites, which may be impractical under field conditions. A flumethrin pour-on formulation on goats provided persistent efficacy against A. variegatum for up to one-month. Therapeutic control was achieved against R. appendiculatus through weekly spraying cattle with flumethrin, amitraz or combined cymiazole and cypermethrin. A fipronil pour-on product offered four-week residual control against R. appendiculatus (with slow onset of action).

Few studies have assessed and directly compared acaricidal activity in vitro and in vivo. There was some discordance between efficacy indicated by LPT and in vivo results. This observation calls for more research into accurate and affordable assessment methods for acaricide resistance.

No single active or product was effective against all three tick species, emphasising the need for the development of alternative integrated tick management solutions.

通过六个撒哈拉以南非洲国家的合作努力,采用公认的国际评估技术,从牛身上收集了对东非和西非农村小农户社区具有重要经济意义的三种蜱(Rhipicephalus microplus、Rhipicephalus appendiculatus 和 Amblyomma variegatum)的 23 个种群,并在体外幼虫包试验(LPT)中进行了评估。结果表明,不同物种对氯氟吡氧乙酸和双甲脒具有中等到较高的抗药性。Rhipicephalus microplus 对甲型氯氰菊酯和氯氰菊酯具有较高的抗药性。A. variegatum(西非)和 R. appendiculatus(乌干达)种群表现出中等水平的伊维菌素抗药性。四种抗药性最低的种群(东非和西非 R.microplus、A. variegatum 和 R.appendiculatus)被用于牛的体内对照研究,研究发现氟虫腈对西非和东非 R. microplus 分离物有效,但持续效力未达到 90%。腈菌唑、氯氰菊酯和伊维菌素类杀螨剂对小蝰蛇部分有效,但无持续效力。氟氯氰菊酯喷雾剂可在处理后 72 小时内杀死变色龙蜱长达 10 天,但该产品直接施用在蜱的附着点上,这在田间条件下可能不切实际。在山羊身上喷洒氟氯氰菊酯制剂可持续杀灭变异蜱长达一个月。通过每周给牛喷洒氟氯苯菊酯、双甲脒或氯氰菊酯和氯氰菊酯复配制剂,可实现对附翅虫的治疗控制。氟虫腈浇注产品可对阑尾蝇提供四周的残留控制(起效缓慢)。很少有研究对体外和体内的杀螨活性进行评估和直接比较。LPT 显示的药效与体内结果之间存在一些不一致。因此,需要对准确、经济的杀螨剂抗药性评估方法进行更多研究。没有一种活性物质或产品对所有三种蜱类都有效,这强调了开发替代性综合蜱类管理解决方案的必要性。
{"title":"Acaricide resistance status of livestock ticks from East and West Africa and in vivo efficacy of acaricides to control them","authors":"Alec Evans ,&nbsp;Maxime Madder ,&nbsp;Josephus Fourie ,&nbsp;Lénaïg Halos ,&nbsp;Bersissa Kumsa ,&nbsp;Elikira Kimbita ,&nbsp;Joseph Byaruhanga ,&nbsp;Frank Norbert Mwiine ,&nbsp;Dennis Muhanguzi ,&nbsp;Safiou Bienvenu Adehan ,&nbsp;Alassane Toure ,&nbsp;Jahashi Nzalawahe ,&nbsp;Fred Aboagye-Antwi ,&nbsp;Ndudim Isaac Ogo ,&nbsp;Leon Meyer ,&nbsp;Frans Jongejan ,&nbsp;Imad Bouzaidi Cheikhi ,&nbsp;Maggie Fisher ,&nbsp;Peter Holdsworth","doi":"10.1016/j.ijpddr.2024.100541","DOIUrl":"10.1016/j.ijpddr.2024.100541","url":null,"abstract":"<div><p>Through a collaborative effort across six Sub-Saharan African countries, using recognized international assessment techniques, 23 stocks of three tick species (<em>Rhipicephalus microplus, Rhipicephalus appendiculatus</em> and <em>Amblyomma variegatum)</em> of economic importance for rural small holder farming communities from East and West Africa were collected from cattle<em>,</em> and evaluated in <em>in vitro</em> larval packet tests (LPT). The results demonstrated medium to high resistance to chlorfenvinphos and amitraz across species. <em>Rhipicephalus microplus</em> demonstrated high level alpha-cypermethrin and cypermethrin resistance. Stocks of <em>A</em>. <em>variegatum</em> (West Africa) and <em>R</em>. <em>appendiculatus</em> (Uganda) demonstrated medium level ivermectin resistance.</p><p>The four least susceptible stocks (East and West African <em>R. microplus, A. variegatum</em> and <em>R. appendiculatus)</em> were taken into <em>in vivo</em> controlled cattle studies where fipronil was found effective against West and East African <em>R. microplus</em> isolates although persistent efficacy failed to reach 90%. Cymiazole and cypermethrin, and ivermectin based acaricides were partially effective against <em>R. microplus</em> without persistent efficacy. Flumethrin spray-on killed <em>A. variegatum</em> within 72 h for up to 10 days posttreatment, however product application was directly to tick attachment sites, which may be impractical under field conditions. A flumethrin pour-on formulation on goats provided persistent efficacy against <em>A. variegatum</em> for up to one-month. Therapeutic control was achieved against <em>R. appendiculatus</em> through weekly spraying cattle with flumethrin, amitraz or combined cymiazole and cypermethrin. A fipronil pour-on product offered four-week residual control against <em>R. appendiculatus</em> (with slow onset of action).</p><p>Few studies have assessed and directly compared acaricidal activity <em>in vitro</em> and <em>in vivo.</em> There was some discordance between efficacy indicated by LPT and <em>in vivo</em> results. This observation calls for more research into accurate and affordable assessment methods for acaricide resistance.</p><p>No single active or product was effective against all three tick species, emphasising the need for the development of alternative integrated tick management solutions.</p></div>","PeriodicalId":13775,"journal":{"name":"International Journal for Parasitology: Drugs and Drug Resistance","volume":"25 ","pages":"Article 100541"},"PeriodicalIF":4.0,"publicationDate":"2024-05-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2211320724000228/pdfft?md5=bda4ea540bd633855a1234e58b6eac46&pid=1-s2.0-S2211320724000228-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140957300","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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International Journal for Parasitology: Drugs and Drug Resistance
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