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From laboratory to clinical practice: an update of the immunological and molecular tools for neurocysticercosis diagnosis 从实验室到临床实践:神经囊虫病诊断免疫学和分子工具的最新进展
Pub Date : 2024-07-01 DOI: 10.3389/fpara.2024.1394089
L. Toribio, J. Bustos, Hector H. Garcia
Neurocysticercosis (NCC) is caused by the invasion of Taenia solium larvae in the central nervous system (CNS) and stands as the predominant cause of epilepsy and other neurological disorders in many developing nations. NCC diagnosis is challenging because it relies on brain imaging exams (CT or MRI), which are poorly available in endemic rural or resource-limited areas. Moreover, some NCC cases cannot be easily detected by imaging, leading to inconclusive results. Multiple laboratory assays, principally immunological, have been developed to support the diagnosis and/or monitor the treatment efficacy, but its production can be costly, laborious, and non-globally accessible because they depend on parasite material. Therefore, recent advances have been focused on the implementation of recombinant or synthetic antigens as well as monoclonal antibodies for NCC immunodiagnosis purposes. Similarly, molecular diagnosis has been explored, obtaining promising results. Here we described the recent progress in the development of immunological and molecular diagnostic tools for NCC diagnosis over the past 13 years, discussing their potential application to address important challenges and how to focus future directions to improve NCC diagnosis with emphasis on enhance accessibility and the importance of test validation to provide an adequate support for clinical decisions.
神经囊尾蚴病(NCC)是由疟原虫幼虫侵入中枢神经系统(CNS)引起的,是许多发展中国家癫痫和其他神经系统疾病的主要病因。NCC 的诊断具有挑战性,因为它依赖于脑成像检查(CT 或 MRI),而这些检查在流行的农村地区或资源有限的地区很难获得。此外,有些 NCC 病例不易通过影像学检查发现,导致诊断结果不确定。目前已开发出多种实验室检测方法(主要是免疫学检测方法)来支持诊断和/或监测治疗效果,但由于这些方法依赖于寄生虫材料,因此成本高昂、费时费力,而且无法在全球范围内使用。因此,最近的进展集中在重组或合成抗原以及单克隆抗体的应用上,以达到 NCC 免疫诊断的目的。同样,分子诊断也得到了探索,并取得了可喜的成果。在此,我们介绍了过去 13 年中用于 NCC 诊断的免疫学和分子诊断工具的最新进展,讨论了这些工具在应对重要挑战方面的潜在应用,以及如何聚焦未来方向以改善 NCC 诊断,重点是提高可及性和测试验证的重要性,从而为临床决策提供充分支持。
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引用次数: 0
Transcriptome analysis reveals molecular targets of erythrocyte invasion phenotype diversity in natural Plasmodium falciparum isolates from Cameroon 转录组分析揭示喀麦隆恶性疟原虫天然分离株红细胞侵袭表型多样性的分子靶标
Pub Date : 2024-05-24 DOI: 10.3389/fpara.2024.1370615
Ines A. Ngoh, Karim Mane, Jarra Manneh, F. Bojang, A. Jawara, Theresia N. Akenji, D. Anong, Umberto D’Alessandro, A. Amambua-Ngwa
Further understanding of the molecular mediators of alternative RBC invasion phenotypes in endemic malaria parasites will support malaria blood-stage vaccine or drug development. This study investigated the prevalence of sialic acid (SA)-dependent and SA-independent RBC invasion pathways in endemic Plasmodium falciparum parasites from Cameroon and compared the schizont stage transcriptomes in these two groups to uncover the wider repertoire of transcriptional variation associated with the use of alternative RBC invasion pathway phenotypes. A two-color flow cytometry-based invasion-inhibition assay against RBCs treated with neuraminidase, trypsin, and chymotrypsin and deep RNA sequencing of schizont stages harvested in the first ex vivo replication cycle in culture were employed in this investigation. RBC invasion phenotypes were determined for 63 isolates from asymptomatic children with uncomplicated malaria. Approximately 80% of the isolates invaded neuraminidase-treated but not chymotrypsin-treated RBCs, representing SA-independent pathways of RBC invasion. The schizont transcriptome profiles of 16 isolates with invasion phenotypes revealed a total of 5,136 gene transcripts, with 85% of isolates predicted at schizont stages. Two distinct transcriptome profile clusters belonging to SA-dependent and SA-independent parasites were obtained by data reduction with principal component analysis. Differential analysis of gene expression between the two clusters implicated, in addition to the well-characterized adhesins, the upregulation of genes encoding proteins mediating merozoite organelle discharges as well as several conserved, virulent, merozoite-associated, and exported proteins. The latter majority have been shown to have structural and physiological relevance to RBC surface remodeling and immune evasion in malaria and thus have potential as anti-invasion targets.
进一步了解地方性疟疾寄生虫中替代性 RBC 侵袭表型的分子介质将有助于疟疾血液阶段疫苗或药物的开发。本研究调查了喀麦隆地方性恶性疟原虫中依赖和不依赖硅铝酸(SA)的 RBC 侵袭途径的流行情况,并比较了这两类寄生虫的裂殖期转录组,以揭示与使用替代 RBC 侵袭途径表型相关的更广泛的转录变异。这项研究采用了一种基于双色流式细胞仪的入侵抑制检测方法,针对经神经氨酸酶、胰蛋白酶和糜蛋白酶处理的红细胞进行检测,并对培养物中第一个体外复制周期收获的裂殖期进行深度 RNA 测序。对来自无症状疟疾患儿的 63 个分离株的红细胞侵袭表型进行了测定。约 80% 的分离株侵入了神经氨酸酶处理过的红细胞,但没有侵入糜蛋白酶处理过的红细胞,这表明红细胞侵入的途径与 SA 无关。16 个具有入侵表型的分离株的裂殖转录组图谱共显示了 5,136 个基因转录本,其中 85% 的分离株被预测为处于裂殖阶段。通过主成分分析对数据进行还原,得到了两个不同的转录组图谱群,分别属于依赖 SA 和不依赖 SA 的寄生虫。对这两个群组之间基因表达的差异分析表明,除了特征明显的粘附蛋白外,编码介导子实体细胞器排出的蛋白质以及几种保守的、有毒性的、与子实体相关的和输出的蛋白质的基因也出现了上调。后一类蛋白质已被证明在结构上和生理上与 RBC 表面重塑和疟疾的免疫逃避有关,因此有可能成为抗入侵的目标。
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引用次数: 0
Next step towards point-of-care molecular diagnosis of female genital schistosomiasis (FGS): evaluation of an instrument-free LAMP procedure 女性生殖器血吸虫病(FGS)护理点分子诊断的下一步:评估无仪器 LAMP 程序
Pub Date : 2024-05-13 DOI: 10.3389/fpara.2024.1297310
Kim J. M. van Bergen, E. Brienen, Bodo S. Randrianasolo, C. Ramarokoto, P. Leutscher, E. F. Kjetland, Angela van Diepen, Floris Dekker, V. Saggiomo, A. Velders, Lisette van Lieshout
Detection of Schistosoma spp. DNA in gynaecological samples by quantitative real-time polymerase chain reaction (qPCR) is considered to be the reference diagnostic test for female genital schistosomiasis (FGS). However, qPCR needs expensive laboratory procedures and highly trained technicians. Loop-mediated amplification (LAMP) is a more field-friendly isothermal procedure for the detection of parasite-specific DNA, but it still requires electrically powered equipment. Here, we validated a Schistosoma haematobium-specific Sh-LAMP procedure and tested a fully instrument-free isothermal amplification using a novel low-cost, and reusable Temperature-cup (T-cup) device. Specific primers were selected based on published assays, targeting the ribosomal intergenic spacer (IGS) region of S. haematobium. Technical validation of the IGS-Sh-LAMP was performed using 20 negative controls, including DNA extracts of soil-transmitted helminths and S. mansoni, and a 10-fold dilution series (100–10−3) of DNA extracted from a single S. haematobium egg (n=4). For clinical validation, the IGS-Sh-LAMP was tested on 125 DNA samples extracted from vaginal swabs of a previous FGS study in Madagascar. Results were compared with the quantification cycle value (Cq) of the standard ITS-2 targeting qPCR. Single S. haematobium egg DNA up to a 10–2 dilution and an ITS-2 Cq <35 tested positive in the IGS-Sh-LAMP. The specificity was found to be excellent (100%). In the clinical samples, IGS-Sh-LAMP showed comparable results with the qPCR, with 35.2% and 33.6% positives, respectively, and a concordance of 79.2% (99/125). Of the 12 false-negatives, 5 corresponded to the 7 qPCR positive samples with very low DNA levels (Cq ≥35). On the other hand, IGS-Sh-LAMP detected 14 additional cases that were not detected by qPCR. The T-cup IGS-Sh-LAMP performance was evaluated in a representative sub-selection (n=10) of IGS-Sh-LAMP positive clinical samples. The T-cup IGS-Sh-LAMP was found to be a very user-friendly method, but in different runs, it missed 1 to 4 of the 10 IGS-Sh-LAMP positive samples, specifically those with a low DNA load. Our results show that the IGS-Sh-LAMP is a suitable alternative to the ITS-2 qPCR for the diagnosis of FGS in gynaecological samples, with high potential for the T-cup as a fully instrument-free isothermal amplification device for point-of-care diagnosis in low-resource settings.
通过实时定量聚合酶链反应(qPCR)检测妇科样本中的血吸虫 DNA 被认为是女性生殖器血吸虫病(FGS)的参考诊断测试。然而,qPCR 需要昂贵的实验室程序和训练有素的技术人员。环路介导扩增(LAMP)是一种更适合现场检测寄生虫特异性 DNA 的等温扩增程序,但仍需要电力驱动设备。在这里,我们验证了血吸虫特异性 Sh-LAMP 程序,并使用新型低成本、可重复使用的温度杯(T-cup)装置测试了完全无需仪器的等温扩增。根据已发表的检测方法选择了特异引物,目标是血吸虫的核糖体基因间间隔(IGS)区域。IGS-Sh-LAMP 的技术验证使用了 20 个阴性对照,包括土壤传播蠕虫和曼森氏杆菌的 DNA 提取物,以及从单个血吸虫卵中提取的 DNA 的 10 倍稀释系列(100-10-3)(n=4)。为了进行临床验证,IGS-Sh-LAMP 在马达加斯加的一项 FGS 研究中对 125 份从阴道拭子中提取的 DNA 样品进行了测试。结果与标准 ITS-2 靶向 qPCR 的定量周期值 (Cq) 进行了比较。在 IGS-Sh-LAMP 中,稀释度达到 10-2 的单个血吸虫卵 DNA 和 ITS-2 Cq <35 的检测结果均为阳性。特异性极佳(100%)。在临床样本中,IGS-Sh-LAMP 与 qPCR 的结果相当,阳性率分别为 35.2% 和 33.6%,一致性为 79.2%(99/125)。在 12 个假阴性样本中,5 个与 7 个 DNA 含量极低(Cq ≥35)的 qPCR 阳性样本相对应。另一方面,IGS-Sh-LAMP 又检测到了 14 个 qPCR 未检测到的病例。在具有代表性的 IGS-Sh-LAMP 阳性临床样本子选择(n=10)中评估了 T-cup IGS-Sh-LAMP 的性能。我们发现 T-cup IGS-Sh-LAMP 是一种非常容易使用的方法,但在不同的运行中,它漏检了 10 个 IGS-Sh-LAMP 阳性样本中的 1 到 4 个,特别是那些 DNA 负载较低的样本。我们的研究结果表明,IGS-Sh-LAMP 是 ITS-2 qPCR 诊断妇科样本中 FGS 的合适替代方法,T-cup 作为一种完全免费的等温扩增设备,在低资源环境下用于床旁诊断具有很大的潜力。
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引用次数: 0
In vitro and in vivo antimalarial activities of the ethanol extract of Erythrina sigmoidea stem bark used for the treatment of malaria in the Western Region of Cameroon 喀麦隆西部地区用于治疗疟疾的 Erythrina sigmoidea 茎皮乙醇提取物的体内外抗疟活性
Pub Date : 2024-04-09 DOI: 10.3389/fpara.2024.1359442
Tientcheu Noutong Jemimah Sandra, Noumedem Anangmo Christelle Nadia, Yamssi Cédric, Gamago Nkadeu Guy-Armand, Mounvera Abdel Azizi, Ngouyamsa Nsapkain Aboubakar Sidiki, Tako Djimefo Alex Kevin, V. Payne, Haibo Hu
Malaria is one of the leading causes of morbidity and/or mortality in tropical Africa. The spread and development of resistance to chemical antimalarial drugs and the relatively high cost of the latter are problems associated with malaria control and are reasons to promote the use of plants to meet healthcare needs to treat malaria. The aim of this study was to evaluate antiplasmodial activities of extracts of Erythrina sigmoidea (Mah quat), which is traditionally used for the treatment of malaria in the western region of Cameroon.The ethanol extract of E. sigmoidea stem bark was obtained through the maceration process using 95% ethanol, while the aqueous extract was prepared by infusion. The in vitro antiplasmodial effect of extracts against P. falciparum chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) strains was determined using the Trager and Jensen method. On the other hand, the in vivo antimalarial activity of the extract was evaluated in mice infected with Plasmodium berghei strain NK65 using the Peters’ 4-day suppressive test and Ryley test (curative test). A total of 36 mice were used, subdivided into six groups of six mice each: one normal control, a negative control, a positive control, and three other groups for the tested product. Blood samples were collected on the 10th day of each test for hematological parameters.The aqueous extract had an in vitro antiplasmodial activity against the chloroquine-sensitive strain with an IC50 of 29.51 ± 3.63 µg/mL and against the chloroquine-resistant strain with an IC50 of 35.23 ± 3.17 µg/mL. The highest in vitro antiplasmodial activity was observed with the ethanol extract against the chloroquine-sensitive strain with an IC50 of 6.44 ± 0.08 µg/mL and against the chloroquine-resistant strain with an IC50 of 7.53 ± 0.22 µg/mL. The ethanol extract demonstrated suppressive activity in vivo with reduction rates of 87.69%, 86.79%, and 81.08% at doses of 500 mg/kg, 250 mg/kg, and 125 mg/kg, respectively; and curative activity in vivo with reduction rates of 80%, 78.5%, and 77.5% at doses of 500 mg/kg, 250 mg/kg, and 125 mg/kg, respectively. The number of white blood cells in the negative control (44.55 ± 5.02 103/µL) was higher compared to the other groups. As for the red blood cells, we observed a massive destruction of the latter in the infected and untreated group (5.82 ± 1.50 106/µL) compared to the infected and ethanol extract-treated groups (8.74 ± 1.57 106/µL for 500 mg/kg, 7.54 ± 1.77 106/µL for 250 mg/kg, and 8.9 ± 1.50 106/µL for 125 mg/kg).This study provides scientific data on the use of E. sigmoidea by the local population for the treatment of malaria. It shows that E. sigmoidea has antiplasmodial activity, and we also see that there are differences between the parameters that we have in the treated groups and those of the untreated group. However, toxicity tests are necessary to assess its safety.
疟疾是非洲热带地区发病和/或死亡的主要原因之一。化学抗疟药物抗药性的传播和发展以及后者相对较高的成本是与疟疾控制相关的问题,也是促进使用植物来满足治疗疟疾的医疗保健需求的原因。本研究旨在评估喀麦隆西部地区传统上用于治疗疟疾的 Erythrina sigmoidea(Mah quat)萃取物的抗疟活性。E. sigmoidea 茎皮的乙醇萃取物是用 95% 的乙醇浸泡获得的,而水萃取物则是通过浸泡制备的。萃取物对恶性疟原虫氯喹敏感菌株(3D7)和氯喹抗性菌株(Dd2)的体外抗疟效果采用特拉格和詹森法进行测定。另一方面,采用彼得斯 4 天抑制试验和雷利试验(治愈试验),评估了提取物在小鼠感染伯格氏疟原虫 NK65 株后的体内抗疟活性。共使用了 36 只小鼠,每只小鼠分为六组:一组为正常对照组,一组为阴性对照组,一组为阳性对照组,另外三组为受试产品组。水提取物对氯喹敏感株具有体外抗疟活性,IC50 为 29.51 ± 3.63 µg/mL,对氯喹耐药株的 IC50 为 35.23 ± 3.17 µg/mL。乙醇提取物对氯喹敏感菌株的体外抗疟活性最高,IC50 为 6.44 ± 0.08 µg/mL,对氯喹耐药菌株的 IC50 为 7.53 ± 0.22 µg/mL。乙醇提取物在体内具有抑制活性,剂量为 500 毫克/千克、250 毫克/千克和 125 毫克/千克时,抑制率分别为 87.69%、86.79% 和 81.08%;在体内具有治疗活性,剂量为 500 毫克/千克、250 毫克/千克和 125 毫克/千克时,治疗率分别为 80%、78.5% 和 77.5%。阴性对照组的白细胞数量(44.55 ± 5.02 103/µL)高于其他组。至于红细胞,我们观察到,与感染组和乙醇提取物处理组(500 毫克/千克为 8.74 ± 1.57 106/微升,250 毫克/千克为 7.54 ± 1.77 106/微升,125 毫克/千克为 8.9 ± 1.50 106/微升)相比,未处理组(5.82 ± 1.50 106/微升)的红细胞被大量破坏。这项研究提供了当地人使用 E. sigmoidea 治疗疟疾的科学数据,表明 E. sigmoidea 具有抗疟活性,而且我们还看到治疗组和未治疗组的参数之间存在差异。不过,还需要进行毒性测试,以评估其安全性。
{"title":"In vitro and in vivo antimalarial activities of the ethanol extract of Erythrina sigmoidea stem bark used for the treatment of malaria in the Western Region of Cameroon","authors":"Tientcheu Noutong Jemimah Sandra, Noumedem Anangmo Christelle Nadia, Yamssi Cédric, Gamago Nkadeu Guy-Armand, Mounvera Abdel Azizi, Ngouyamsa Nsapkain Aboubakar Sidiki, Tako Djimefo Alex Kevin, V. Payne, Haibo Hu","doi":"10.3389/fpara.2024.1359442","DOIUrl":"https://doi.org/10.3389/fpara.2024.1359442","url":null,"abstract":"Malaria is one of the leading causes of morbidity and/or mortality in tropical Africa. The spread and development of resistance to chemical antimalarial drugs and the relatively high cost of the latter are problems associated with malaria control and are reasons to promote the use of plants to meet healthcare needs to treat malaria. The aim of this study was to evaluate antiplasmodial activities of extracts of Erythrina sigmoidea (Mah quat), which is traditionally used for the treatment of malaria in the western region of Cameroon.The ethanol extract of E. sigmoidea stem bark was obtained through the maceration process using 95% ethanol, while the aqueous extract was prepared by infusion. The in vitro antiplasmodial effect of extracts against P. falciparum chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) strains was determined using the Trager and Jensen method. On the other hand, the in vivo antimalarial activity of the extract was evaluated in mice infected with Plasmodium berghei strain NK65 using the Peters’ 4-day suppressive test and Ryley test (curative test). A total of 36 mice were used, subdivided into six groups of six mice each: one normal control, a negative control, a positive control, and three other groups for the tested product. Blood samples were collected on the 10th day of each test for hematological parameters.The aqueous extract had an in vitro antiplasmodial activity against the chloroquine-sensitive strain with an IC50 of 29.51 ± 3.63 µg/mL and against the chloroquine-resistant strain with an IC50 of 35.23 ± 3.17 µg/mL. The highest in vitro antiplasmodial activity was observed with the ethanol extract against the chloroquine-sensitive strain with an IC50 of 6.44 ± 0.08 µg/mL and against the chloroquine-resistant strain with an IC50 of 7.53 ± 0.22 µg/mL. The ethanol extract demonstrated suppressive activity in vivo with reduction rates of 87.69%, 86.79%, and 81.08% at doses of 500 mg/kg, 250 mg/kg, and 125 mg/kg, respectively; and curative activity in vivo with reduction rates of 80%, 78.5%, and 77.5% at doses of 500 mg/kg, 250 mg/kg, and 125 mg/kg, respectively. The number of white blood cells in the negative control (44.55 ± 5.02 103/µL) was higher compared to the other groups. As for the red blood cells, we observed a massive destruction of the latter in the infected and untreated group (5.82 ± 1.50 106/µL) compared to the infected and ethanol extract-treated groups (8.74 ± 1.57 106/µL for 500 mg/kg, 7.54 ± 1.77 106/µL for 250 mg/kg, and 8.9 ± 1.50 106/µL for 125 mg/kg).This study provides scientific data on the use of E. sigmoidea by the local population for the treatment of malaria. It shows that E. sigmoidea has antiplasmodial activity, and we also see that there are differences between the parameters that we have in the treated groups and those of the untreated group. However, toxicity tests are necessary to assess its safety.","PeriodicalId":73098,"journal":{"name":"Frontiers in parasitology","volume":"29 4","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140722517","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Genetic variation of the Plasmodium falciparum circumsporozoite protein in parasite isolates from Homabay County in Kenya 肯尼亚霍马贝县寄生虫分离株中恶性疟原虫周孢子虫蛋白的遗传变异
Pub Date : 2024-04-08 DOI: 10.3389/fpara.2024.1346017
M. Maina, S. Musundi, Josiah O. Kuja, Harrison Waweru, Daniel Kiboi, B. Kanoi, Jesse Gitaka
The Plasmodium falciparum Circumsporozoite Protein (PfCSP) has been used in developing the RTS,S, and R21 malaria vaccines. However, genetic polymorphisms within Pfcsp compromise the effectiveness of the vaccine. Thus, it is essential to continuously assess the genetic diversity of Pfcsp, especially when deploying it across different geographical regions. In this study, we assessed the genetic diversity of the Pfcsp on isolates from Homabay County, a malaria-endemic region in western Kenya, and compared it against other isolates from Kenya. We extracted DNA from 27 microscopically confirmed P. falciparum positive samples and conducted Illumina sequencing to generate paired-end short reads. The sequences were then mapped to the Pf3D7 reference genome, and genetic variation was analyzed using bcftools. Additionally, we retrieved isolates from two other malaria-endemic regions in Kenya, Kisumu (n=58) and Kilifi (n=596), from MalariaGEN version 7 and compared their genetic diversity and natural selection. We also evaluated the predicted binding affinities for HLA class I and II supertype alleles for the identified haplotypes using NetMHCpan and NetMHCIIpan. Our results show that the N-terminal of PfCSP was relatively conserved with a notable mutation at A98G across all isolates. The number of NANP repeats varied across the three Kenyan sites within the central repeat region. Furthermore, the C-terminal region showed polymorphism within the Th2R and Th3R regions. Haplotype network analysis of the Kenyan isolates revealed 69 haplotypes, with the 3D7 reference being found in the most prevalent haplotype. When assessing the predicted binding affinities between supertypes in HLA class I and II with the identified haplotypes, we observed stronger predicted binding affinities to multiple haplotypes except for those containing the 3D7 reference. The results suggest the need to take into account the existing changes occurring in Pfcsp while developing malaria vaccines.
恶性疟原虫圆孢子虫蛋白(PfCSP)已被用于开发 RTS、S 和 R21 疟疾疫苗。然而,Pfcsp 的基因多态性会影响疫苗的效果。因此,持续评估 Pfcsp 的遗传多样性至关重要,尤其是在不同地理区域使用时。在这项研究中,我们评估了来自肯尼亚西部疟疾流行地区霍马拜县的Pfcsp分离株的遗传多样性,并与肯尼亚的其他分离株进行了比较。我们从 27 个经显微镜确认的恶性疟原虫阳性样本中提取了 DNA,并进行了 Illumina 测序,以生成成对的短读数。然后将序列映射到 Pf3D7 参考基因组,并使用 bcftools 分析遗传变异。此外,我们还从 MalariaGEN 第 7 版中检索了肯尼亚另外两个疟疾流行地区基苏木(n=58)和基利菲(n=596)的分离株,并比较了它们的遗传多样性和自然选择。我们还使用 NetMHCpan 和 NetMHCIIpan 评估了已确定的单倍型与 HLA I 类和 II 类超级等位基因的预测结合亲和力。我们的结果表明,PfCSP 的 N 端相对保守,所有分离株的 A98G 有明显突变。在中央重复区域内,三个肯尼亚位点的 NANP 重复序列数量各不相同。此外,C末端区域在Th2R和Th3R区域内显示出多态性。对肯尼亚分离物进行的单倍型网络分析发现了 69 种单倍型,其中以 3D7 参考单倍型最为普遍。在评估 HLA I 类和 II 类超型与已确定的单倍型之间的预测结合亲和力时,我们观察到除了含有 3D7 参考型的单倍型外,其他多个单倍型都有更强的预测结合亲和力。这些结果表明,在开发疟疾疫苗时需要考虑到 Pfcsp 中发生的现有变化。
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引用次数: 0
Immunomodulatory properties of Leishmania tarentolae extracellular vesicles containing the Spike protein of SARS-CoV-2 含有 SARS-CoV-2 Spike 蛋白的透明利什曼原虫胞外囊泡的免疫调节特性
Pub Date : 2024-04-08 DOI: 10.3389/fpara.2024.1306478
Ana Catalina Medina, Hamlet Adolfo Acevedo Ospina, Albert Descoteaux
Extracellular vesicles released by the protozoan parasite Leishmania display immunomodulatory properties towards mammalian immune cells. In this study, we have evaluated the potential of extracellular vesicles derived from the non-pathogenic protozoan Leishmania tarentolae towards the development of a vaccine adjuvant. As a proof of concept, we expressed in L. tarentolae a codon-optimized SARS-CoV-2 Spike protein fused to the L. mexicana secreted acid phosphatase signal peptide in the N-terminal and to a 6×-His stretch in the C-terminal. Extracellular vesicles released by the engineered L. tarentolae were isolated by ultracentrifugation and fast protein liquid chromatography and were characterized via nanoparticle tracking analysis and transmission electron microscopy. The recombinant S protein was present in extracellular vesicles released by L. tarentolae, as determined by Western blot analyses and immunoelectron microscopy. Next, we evaluated the immunomodulatory potential of extracellular vesicles containing the S protein towards bone-marrow-derived macrophages and bone-marrow-derived dendritic cells. Our data show that in bone-marrow-derived dendritic cells, extracellular vesicles containing the S protein induced an increased expression of proinflammatory genes compared to plain extracellular vesicles whereas the opposite was observed in bone-marrow-derived macrophages. These findings reveal the immunomodulatory potential of L. tarentolae extracellular vesicles and provide a proof of concept that they can be used as adjuvant in the context of dendritic cell stimulation.
原生动物利什曼原虫释放的胞外囊泡对哺乳动物免疫细胞具有免疫调节特性。在这项研究中,我们评估了从非致病性原生动物透明利什曼原虫(Leishmania tarentolae)中提取的胞外囊泡开发疫苗佐剂的潜力。作为概念验证,我们在透明利什曼原虫中表达了一种经过密码子优化的 SARS-CoV-2 Spike 蛋白,其 N 端融合了墨西哥利什曼原虫分泌型酸性磷酸酶信号肽,C 端融合了 6×His 片段。通过超速离心和快速蛋白质液相色谱法分离了工程化的透明带状病毒释放的胞外囊泡,并通过纳米粒子跟踪分析和透射电子显微镜对其进行了表征。通过 Western 印迹分析和免疫电子显微镜测定,重组 S 蛋白存在于透明带绦虫释放的细胞外囊泡中。接下来,我们评估了含有 S 蛋白的细胞外囊泡对骨髓源性巨噬细胞和骨髓源性树突状细胞的免疫调节潜力。我们的数据显示,在骨髓树突状细胞中,与普通细胞外囊泡相比,含有 S 蛋白的细胞外囊泡会诱导促炎基因的表达增加,而在骨髓巨噬细胞中则相反。这些发现揭示了L. tarentolae胞外囊泡的免疫调节潜力,并证明了它们可用作树突状细胞刺激的佐剂。
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引用次数: 0
EhVps35, a retromer component, is involved in the recycling of the EhADH and Gal/GalNac virulent proteins of Entamoeba histolytica EhVps35是一种反转录蛋白成分,参与了EhADH和Gal/GalNac毒蛋白的回收利用
Pub Date : 2024-03-26 DOI: 10.3389/fpara.2024.1356601
Joselin Díaz-Valdez, R. Javier‐Reyna, Sarita Montaño, Daniel Talamás-Lara, Esther Orozco
The retromer is a highly conserved eukaryotic complex formed by the cargo selective complex (CSC) and the sorting nexin (SNX) dimer subcomplexes. Its function is protein recycling and recovery from the endosomes to conduct the target molecules to the trans-Golgi network or the plasma membrane. The protozoan responsible for human amoebiasis, Entamoeba histolytica, exhibits an active membrane movement and voracious phagocytosis, events in which the retromer may be fully involved. In this work, we studied the structure of EhVps35 the central member of the CSC retromeric subcomplex as it binds EhVps26 and EhVps29, the other two CSC members, allowing the position of the retromer in the membranes. We also studied the EhVps35 role in the recycling of virulence proteins, particularly those involved in phagocytosis. Confocal microscopy assays revealed that EhVps35 is located in the plasmatic and endosomal membranes and in the phagocytic cups and channels. In addition, it follows the target cell from the moment it is in contact with the trophozoites. Molecular docking analyses, immunoprecipitation assays, and microscopy studies revealed that EhVps35 interacts with the EhADH, Gal/GalNac lectin, and actin proteins. In addition, experimental evidence indicated that it recycles surface proteins, particularly EhADH and Gal/GalNac proteins, two molecules highly involved in virulence. Knockdown of the Ehvps35 gene induced a decrease in protein recycling, as well as impairments in the efficiency of adhesion and the rate of phagocytosis. The actin cytoskeleton was deeply affected by the Ehvps35 gene knockdown. In summary, our results revealed the participation of EhVps35 in protein recycling and phagocytosis. Furthermore, altogether, our results demonstrated the concert of finely regulated molecules, including EhVps35, EhADH, Gal/GalNac lectin, and actin, in the phagocytosis of E. histolytica.
retromer 是一种高度保守的真核生物复合物,由货物选择复合物(CSC)和分拣毒素(SNX)二聚体亚复合物组成。它的功能是从内体回收蛋白质,并将目标分子引导至跨高尔基网络或质膜。导致人类阿米巴病的原生动物--组织溶解恩塔米巴虫(Entamoeba histolytica)--表现出活跃的膜运动和贪婪的吞噬作用,而 retromer 可能完全参与了这些活动。在这项工作中,我们研究了 EhVps35 的结构,它是 CSC retromeric 亚复合物的核心成员,它与另外两个 CSC 成员 EhVps26 和 EhVps29 结合,从而确定了 retromer 在膜中的位置。我们还研究了 EhVps35 在毒力蛋白循环中的作用,特别是那些参与吞噬作用的蛋白。共聚焦显微镜检测显示,EhVps35 位于质膜和内体膜以及吞噬杯和吞噬通道中。此外,它从目标细胞与滋养体接触的那一刻起就跟随目标细胞。分子对接分析、免疫沉淀试验和显微镜研究显示,EhVps35 与 EhADH、Gal/GalNac 凝集素和肌动蛋白相互作用。此外,实验证据表明,它能回收表面蛋白,尤其是 EhADH 和 Gal/GalNac 蛋白,这两种分子与毒力密切相关。敲除 Ehvps35 基因会导致蛋白质循环减少,并影响粘附效率和吞噬率。肌动蛋白细胞骨架深受 Ehvps35 基因敲除的影响。总之,我们的研究结果表明,EhVps35 参与了蛋白质回收和吞噬作用。此外,我们的研究结果表明,在组织溶解虫的吞噬过程中,EhVps35、EhADH、Gal/GalNac凝集素和肌动蛋白等调控精细的分子共同发挥作用。
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引用次数: 0
Toward the elimination of NTDs: application of cost-effective and sensitive molecular environmental surveillance tools—a pilot study 消除非传染性疾病:应用具有成本效益和敏感性的分子环境监测工具--试点研究
Pub Date : 2024-03-26 DOI: 10.3389/fpara.2024.1340161
Juliet Hodgson, Gideon Twieku, Gerard Quarcoo, Emmanuel Armah, M. Osei-Atweneboana, S. Armoo
Neglected tropical diseases (NTDs) affect over a billion people worldwide. The 2021–2030 NTD road map calls for innovative and highly efficient interventions to eliminate or significantly reduce the burden of NTDs. These include sensitive and cost-effective diagnostic techniques for disease surveillance. Environmental surveillance has been employed effectively in this regard to measure and track infectious diseases such as polio on a population-wide scale. In this study, environmental surveillance was used as a cost-effective tool for the detection of soil-transmitted helminths (STHs) in Accra, Ghana, in an area that is populated by urban vegetable farmers. The activities of urban farmers expose them to the risk of STH infection, as well as impact the transmission in urban areas since leafy vegetables could carry infective stages of STHs. A total of 32 wastewater samples were collected from eight points on the Nima Creek (the main source of irrigation for the farmers) over a 7-week period. Real-time PCR and melt peak analysis were used to screen four STHs (i.e., Ascaris lumbricoides, Necator americanus, Ancylostoma duodenale, and Trichuris trichiura). This study revealed that A. lumbricoides (17 out of 32 wastewater samples, 53.3%) was the most prevalent STH, followed by A. duodenale (31.2%), T. trichiura (21.9%), and N. americanus (12.5%). Environmental surveillance helps in the detection of the types of STH pathogens circulating within the community and in the design of mass drug administration (MDA) strategies. This surveillance technique can also provide preliminary information for environmental modifications to help reduce STH transmission in line with the One Health approach recommended in the 2021–2030 NTD road map.
被忽视的热带疾病(NTDs)影响着全球十多亿人。2021-2030 年非传染性疾病路线图呼吁采取创新和高效的干预措施,以消除或大幅减轻非传染性疾病的负担。这些措施包括用于疾病监测的灵敏且具有成本效益的诊断技术。在这方面,环境监测已被有效地用于在全人口范围内测量和跟踪脊髓灰质炎等传染病。在这项研究中,环境监测被用作一种具有成本效益的工具,用于检测加纳阿克拉一个由城市菜农居住的地区的土壤传播蠕虫(STHs)。城市菜农的活动使他们面临感染 STH 的风险,同时也影响了城市地区的传播,因为叶菜可能携带 STH 的感染阶段。在为期 7 周的时间里,我们从尼玛溪(农民的主要灌溉水源)上的 8 个点共采集了 32 份废水样本。利用实时 PCR 和熔峰分析筛选出四种 STHs(即蛔虫、美洲蛲虫、十二指肠疽和毛滴虫)。这项研究显示,蛔虫(32 个废水样本中的 17 个,占 53.3%)是最普遍的性传播疾病,其次是十二指蛔虫(31.2%)、毛滴虫(21.9%)和美洲奈卡虫(12.5%)。环境监测有助于检测社区内流行的性传播疾病病原体类型,并有助于设计大规模用药(MDA)策略。这种监测技术还能为环境改造提供初步信息,以帮助按照 2021-2030 年非传染性疾病路线图中建议的 "一体健康 "方法减少性传播疾病的传播。
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引用次数: 0
Field testing of recombinant subunit vaccines against Teladorsagia circumcincta in lambing ewes demonstrates a lack of efficacy in the face of a multi-species parasite challenge 在产羔母羊身上对预防圆环端螺旋体(Teladorsagia circumcincta)的重组亚单位疫苗进行的现场测试表明,面对多物种寄生虫的挑战,这种疫苗缺乏效力
Pub Date : 2024-03-25 DOI: 10.3389/fpara.2024.1360029
A. Nisbet, T. N. McNeilly, D. Price, Y. Bartley, Margaret Oliver, D. McBean, Leigh Andrews, Gillian Mitchell, Rachael Duncan, Sarah Brocklehurst, Fiona Kenyon
We previously demonstrated efficacy of an 8-antigen recombinant subunit vaccine against a single species homologous Teladorsagia circumcincta challenge in lambs and in lambing ewes in pen trials. We subsequently demonstrated efficacy of a simplified, 2-antigen, version of this vaccine in lambs in pen trials. Here, we test both vaccines in lambing ewes in a field setting.In the work presented here, 12 adjacent plots were seeded with a mixed infection of several common species of parasitic nematodes of sheep in temperate regions, including T. circumcincta. Ewes (n = 144), in groups of 12, grazed for 2 years on these plots and, in the first year, six of these groups of ewes were vaccinated with a 2-antigen prototype vaccine against T. circumcincta prior to mating and then again prior to lambing. In the following year these ewes were immunised again, this time with the 8-antigen prototype vaccine against T. circumcincta prior to mating and then prior to lambing. Throughout both seasons antigen-specific serum antibody levels in ewes and faecal worm egg counts (FEC) in ewes and their lambs were monitored, along with nematode species diversity at lambing.Immunised ewes produced elevated serum antibody levels to each of the vaccine antigens following immunisation but their FEC levels were not statistically significantly impacted by vaccination with either vaccine. FEC levels were also not impacted in lambs co-grazing the pastures with these immunised ewes. Nematode species diversity was not significantly impacted by vaccination in either year.The immunosuppressive effects of co-infecting gastrointestinal nematodes, the absence of vaccine cross-protection against co-infecting species and the influence of the periparturient relaxation in immunity probably all contributed to the inability of either vaccine to protect against T. circumcincta infection in field trials in the work presented here.
此前,我们曾在羔羊和产羔母羊的疫苗接种试验中证明了一种 8 抗原重组亚单位疫苗对单一物种同源的圆环带绦虫(Teladorsagia circumcincta)挑战的有效性。随后,我们又在羔羊圈舍试验中证明了这种疫苗的简化版(2 个抗原)的有效性。在本文介绍的工作中,我们在 12 块相邻的地块上播种了温带地区绵羊常见的几种寄生线虫,包括圆环线虫。母羊(n = 144)每 12 只为一组,在这些地块上放牧 2 年,第一年,其中 6 组母羊在交配前接种了针对圆线虫的 2 抗原原型疫苗,然后在产羔前再次接种。第二年,再次对这些母羊进行免疫接种,这次是在交配前和产羔前接种 8 抗原的圆环病毒原型疫苗。在这两个季节中,都对母羊的抗原特异性血清抗体水平、母羊及其羔羊的粪便虫卵计数(FEC)以及产羔时线虫种类的多样性进行了监测。免疫后的母羊对每种疫苗抗原的血清抗体水平都有所提高,但两种疫苗对其粪便虫卵计数水平都没有显著影响。与这些免疫母羊共同放牧的羔羊的FEC水平也未受到影响。合并感染的胃肠道线虫的免疫抑制作用、疫苗对合并感染物种缺乏交叉保护以及围产期免疫松弛的影响,可能都是本文介绍的工作中两种疫苗都无法在田间试验中保护母羊免受圆线虫感染的原因。
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引用次数: 0
Development of a rapid and highly sensitive nucleic acid-based diagnostic test for schistosomes, leveraging on identical multi-repeat sequences 利用相同的多重复序列,开发基于核酸的血吸虫快速、高灵敏诊断检测方法
Pub Date : 2024-03-14 DOI: 10.3389/fpara.2024.1361493
Ombeni Ally, B. Kanoi, S. Kamath, Clement Shiluli, E. M. Ndombi, Maurice Odiere, Gerald Misinzo, S. Nyanjom, Chunduri Kiran Kumar, Lucy Ochola, Srinivasa Raju Lolabattu, Jesse Gitaka
Schistosomiasis (Bilharzia), a neglected tropical disease caused by Schistosoma parasites, afflicts over 240 million people globally, disproportionately impacting Sub-Saharan Africa. Current diagnostic tests, despite their utility, suffer from limitations like low sensitivity. Polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR) remain the most common and sensitive nucleic acid amplification tests. Still, the sensitivity of nucleic acid amplification tests is significantly affected by the copy number of amplification targets, resulting in underestimation of true Schistosoma infections, especially in low transmission settings. Additionally, lengthy qPCR run times pose challenges when dealing with large sample volumes and limited resources. In this study, the identical multi-repeat sequences (IMRS) were used as a novel approach to enhance the sensitivity of nucleic acid-based Bilharzia diagnosis.To identify novel genomic repeat regions, we utilized the IMRS algorithm, with modifications to enable larger target region (100-200bp) identification instead of smaller sequences (18-30bp). These regions enabled customised primer-probe design to suit requirements for qPCR assay. To lower the qPCR amplification times, the assay was conducted using fast cycling condition. Regression analysis, and qPCR data visualization was conducted using Python programming.Using Schistosoma mansoni and S. haematobium, we found that IMRS-based qPCR, employing genus-specific primers and TaqMan probes, offers exceptional analytical sensitivity, detecting as little as a single genome copy per microliter within 36 minutes.The lowest concentration of DNA detected using IMRS-based PCR and qPCR represented tenfold improvement over conventional PCR. As part of further development, there is a need to compare IMRS-based qPCR against other qPCR methods for Schistosoma spp. Nonetheless, IMRS-based diagnostics promise a significant advancement in bilharzia diagnosis, particularly in low-transmission settings, potentially facilitating more effective control and treatment strategies.
血吸虫病(Bilharzia)是由血吸虫寄生引起的一种被忽视的热带疾病,全球有超过 2.4 亿人深受其害,撒哈拉以南非洲受到的影响尤为严重。目前的诊断测试尽管有用,但存在灵敏度低等局限性。聚合酶链式反应(PCR)和定量实时 PCR(qPCR)仍然是最常见、最灵敏的核酸扩增检测方法。不过,核酸扩增检测的灵敏度受扩增目标拷贝数的影响很大,导致对真正的血吸虫感染估计不足,尤其是在低传播环境中。此外,在处理大量样本和有限资源时,冗长的 qPCR 运行时间也构成了挑战。为了识别新的基因组重复区域,我们使用了 IMRS 算法,并对其进行了修改,以识别更大的目标区域(100-200bp),而不是更小的序列(18-30bp)。通过这些区域,可以定制引物-探针设计,以满足 qPCR 检测的要求。为了缩短 qPCR 扩增时间,检测在快速循环条件下进行。通过使用曼氏血吸虫和血吸虫,我们发现基于 IMRS 的 qPCR 采用了属特异性引物和 TaqMan 探针,具有极高的分析灵敏度,在 36 分钟内每微升可检测到一个基因组拷贝。尽管如此,基于 IMRS 的诊断方法有望在血吸虫病诊断方面取得重大进展,尤其是在低传播环境中,这有可能促进更有效的控制和治疗策略。
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引用次数: 0
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Frontiers in parasitology
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