Domestic and Peri-Domestic Study Reveals the Presence of Probably <i>Acanthamoeba castellanii</i>

Acanthamoeba is free living amoeba consisting of many species that are naturally pathogenic and have been isolated from different environmental sources. The purpose of this study was to determine the prevalence and relative abundance of Acanthamoeba species in soil and water samples within the University of Jos environment and to create public health awareness on the dangers of this parasitic protozoan. The study was conducted in the eleven (11) Faculties of the University of Jos. Soil and water samples were collected from each of the faculties, cultured and morphologically identified for positive samples. In addition, quantitative data on occurrence were examined to help better understand the potential risk to the university community. The prevalence of Acanthamoeba based on the chi-square analysis indicates that there is a significant difference between the number of Acanthamoeba species in the soil and water samples collected (P < 0.05). Relative abundance of Acanthamoeba based on the Man-Whitney test indicates that there was no significant difference in the distribution of Acanthamoeba species in the water and soil samples between different faculties (P > 0.05). Morphological identification indicates the presence of probably Acanthamoeba castellani. The demonstration of the presence of Acanthamoeba species in soil and water sources calls for awareness among the clinical community, as cases of keratitis and granulomatous amoebic encephalitis might have never been recorded due to lack of expertise or unawareness amongst the clinical community. An improved treatment of water supply and strict adherence to water act needs to be strictly encouraged.

Non-related contact lens coinfection with Acanthamoeba and Fusarium

Rationale: Microbial keratitis caused by coinfection with more than one species of pathogens is a severe condition with an unfavorable prognosis. Patient concerns: An immunocompetent Nepali woman complained of pain in the left eye, redness, watering and decreased vision for 5 months. Interventions: The patient was discarded and accurately diagnosed with coinfection with Fusarium sp. and Acanthamoeba sp. The habit of washing the eyes with tap water from a domestic storage tank was the most likely source of infection since it was found to be contaminated with cysts of Acanthamoeba sp. The woman received eye drops of fluconazole and natamycin(5%), cefazoline(50 mg/m L), atropine, and tablets of itraconazole(100 mg), which were later switched to eye drops of clotrimazole(1%), natamycin(5%) and voriconazole(1%), and tablets of itraconazole. A full thickness penetrating keratoplasty was performed followed by treatment with eye drops of voriconazole(1%), natamet(5%), ofloxacin, atropine and carboxymethylcellulose for one week.Outcomes: After treatment, the condition of the patient significantly improved and was discharged one week after keratoplasty. Lessons: This is the first report of Acanthamoeba keratitis in Nepal and the first report of coinfection with Fusarium in this country and highlights the importance of early diagnosis of microbial keratitis both in single microorganism infections and coinfections, even in no contact lens wearers.

Presence of Acanthamoeba spp.in water purification plants in southern England

Objective:To identify the prevalence of Acanthamoeba in drinking water treatment plants during the course of the purification processes.Methods:Samples were taken from two drinking water purification plants and monitored for the presence of Acanthamoeba in order to estimate the removal capacity of treatment methods employed. Water samples were collected at each step in the purification,during the one year survey,and analysed for the presence of Acanthamoeba spp.by plating on bacterial-seeded plates.Results:The results showed that amoebae were present in surface raw waters in 100%of the samples tested.Acanthamoeba spp.were isolated from 71%and 57%of the water samples collected from post flat-bottom clarifier 1 and post-sedimentation plant respectively.Considering the outflow drinking waters,the removal capacity was 100%in both purification plants monitored.The occurrence of Acanthamoeba was not associated with seasonality.Conclusion: These findings confirm that water purification plants employing methods of flocculation,sedimentation,and filtration in combination with activated charcoal filtration,ozonisation and chlorination exhibited sufficient Acanthamoeba removal capacity and the presence of amoebae in the tap water may be due to older plumbing,water storage tanks,tap water hygiene,and/or environmental settings.

Phylogeography,genetic variability and structure of Acanthamoeba metapopulations in Iran inferred by 18S ribosomal RNA sequences:A systematic review and meta-analysis

Objective: To verify phylogeography and genetic structure of Acanthamoeba populations among the Iranian clinical isolates and natural/artificial environments distributed in various regions of the country.Methods: We searched electronic databases including Medline, Pub Med, Science Direct, Scopus and Google Scholar from 2005 to 2016. To explore the genetic variability of Acanthamoeba sp, 205 sequences were retrieved from keratitis patients, immunosuppressed cases and environmental sources as of various geographies of Iran.Results: T4 genotype was the predominant strain in Iran, and the rare genotypes belonged to T2, T3, T5(Acanthamoeba lenticulata), T6, T9, T11, T13 and T15(Acanthamoeba jacobsi).A total of 47 unique haplotypes of T4 were identified. A parsimonious network of the sequence haplotypes demonstrated star-like feature containing haplogroups IR6(34.1%) and IR7(31.2%) as the most common haplotypes. In accordance with the analysis of molecular variance, the high value of haplotype diversity(0.612–0.848) of Acanthamoeba T4 represented genetic variability within populations. Neutrality indices of the 18 S ribosomal RNA demonstrated negative values in all populations which represented a considerable divergence from neutrality. The majority of genetic diversity belonged to the infected contact lens and dust samples in immunodeficiency and ophthalmology wards, which indicated potential routes for exposure to a pathogenic Acanthamoeba sp. in at-risk individuals. A pairwise fixation index(FST) was from low to high values(0.024 33–0.418 92). The statistically FST points out that T4 is genetically differentiated between north-west, north-south and centralsouth metapopulations, but not differentiated between west-central, west-south, centralsouth, and north-central isolates.Conclusions: An occurrence of IR6 and IR7 displays that possibly a gene flow of Acanthamoeba T4 occurred after the founder effect or bottleneck experience through ecological changes or host mobility. This is the first systematic review and meta-analysis providing new approaches into gene migration and transmission patterns of Acanthamoeba sp, and targeting at the high-risk individuals/sources among the various regions of Iran.

Reactive uveitis, retinal vasculitis and scleritis as ocular end-stage of Acanthamoeba keratitis: a histological study

We analysed histologically two Acanthamoeba keratitis(AK) eyes with anterior and posterior segment inflammation and blindness. Two enucleated eyes of 2 patients(age 45 and 51y) with AK(PCR of epithelial abrasion positive) were analysed. Histological analysis was performed using hematoxylin-eosin, periodic acid-Schiff and G?m?rimethenamine silver staining. We could not observe Acanthamoeba trophozoites or cysts neither in the cornea nor in other ocular tissues. Meanwhile, we found uveitis, retinal vasculitis and scleritis in these eyes, due to the long-standing, recalcitrant AK. So in this stage of AK, systemic immune suppression may be necessary for a longer time period.

Comparison of anterior segment optical coherence tomography findings in acanthamoeba keratitis and herpetic epithelial keratitis

This study is to investigate the characteristic features of Acanthamoeba keratitis(AK) that differentiating it from herpetic epithelial keratitis(HEK) using anterior segment optical coherence tomography(AS-OCT). Medical records of three eyes of each AK and herpetic keratitis who had AS-OCT examination were reviewed in this study. Slitlamp biomicroscopy and AS-OCT was performed on the initial visit and on every follow-up visits in all patients. In all three AK cases, reflective bands in the corneal stroma that correspond to the area of radial keratoneuritis were observed. The depth of the reflective bands varied in each case. After AK treatment, slit-lamp biomicroscopy confirmed that radial keratoneuritis had resolved and AS-OCT confirmed that reflective bands in the corneal stroma had also disappeared in all patients. Unlike the AS-OCT results found in AK, highly reflective HEK lesions were observed only in the subepithelial area, not in the stroma. AS-OCT seems to be helpful analyzing the specific depth of the lesion which enables to distinguish AK from HEK.

A review of Acanthamoeba keratitis in the Middle East and Iran

Acanthamoeba keratitis, a vision-threatening disease, is caused by a free-living amoeba of genus Acanthamoeba. Amoeba is widely distributed all around the world. Several cases of Acanthamoeba keratitis have been reported from the Middle East and Iran as well. The infection is mainly caused by inappropriate use of contact lens;therefore, it is more common among lens wearers. The number of reported cases worldwide is increasing annually due to the increasing number of contact lens wearers for medical or cosmetic reasons. It is known that early diagnosis and treatment can reduce consequent damages, while the delay in these processes will deteriorate the vision. Nowadays contact lens wearing, surgery and use of corticosteroids are known to predispose to Acanthamoeba keratitis, and it was also believed that trauma was the main cause. Increasing public knowledge about Acanthamoeba infection, inquiring the history of patients, clinical signs and laboratory findings can be helpful to early diagnosis and better treatment. Regardless of the increasing knowledge of diagnosis and treatment, this disease is still a challenge. Considering the long curative time, and insufficient efficacy of available treatments, it seems that the prevention is more important than the treatment. This review aimed to explain Acanthamoeba keratitis in the Middle East and Iran from the aspects of epidemiology, diagnosis, and therapeutic treatment.

Atypical Presentation of Acanthamoeba Keratitis in a Contact Lens Wearer

Acanthamoeba keratitis is a serious infection that can lead to loss of vision. It is highly challenging and often poses a diagnostic dilemma, causing delay in diagnosis and treatment. We report herewith the clinical and histopathology findings of a patient with an atypical presentation of acanthamoeba keratitis in Bahrain. The patient is a 16-year-old Bahraini teenager who was a cosmetic contact lens wearer. She presented with clinical signs and symptoms of microbial keratitis, which was initially misdiagnosed elsewhere as a case of herpetic corneal infection. Her corneal biopsy confirmed the clinical diagnosis as acanthamoeba keratitis. The patient was started on anti amoebic treatment. The infection got eradicated. The cornea healed with a central scar. Eventually, she underwent penetrating keratoplasty. This case report serves to raise awareness of this rare condition. Clinicians should have a high index of suspicion when diagnosing such cases among contact lens wearers. Early diagnosis and treatment are crucial to prevent serious complications.

Riboflavin-UVA collagen cross-linking for the treatment of acanthamoeba keratitis

In this review,recent studies regarding riboflavin-ultraviolet A(UVA)collagen cross-linking for the treatment of acanthamoeba keratitis(AK)were reviewed.English written studies about acanthamoeba,keratitis,riboflavin and collagen cross-linking were retrieved from PubMed search engine(www.ncbi.nlm.nih.gov/pubmed).Although there were significant numbers of cases reporting the effectiveness of riboflavin-UVA collagen cross-linking in AK,experimental studies(in vivo and in vitro)failed to verify amoebicidal or cysticidal effect of riboflavin-UVA collagen cross-linking.In conclusion,the efficacy of riboflavin-UVA collagen cross-linking for the treatment of AK is still debatable.It is necessary to conduct a prospective case-control study for clear guidance for clinicians.

In Vitro Activity of Squaramides and Acyclic Polyamine Derivatives against Trophozoites and Cysts of Acanthamoeba castellanii

Pathogenic strains of Acanthamoeba cause keratitis (AK), granulomatous amoebic encephalitis (GAE), amoebic pneumonitis (AP), and skin infection in human and animals. The treatment of an Acanthamoeba infection is invariably very difficult and not always effective, and compounds that are amebicidic or amebistatic are frequently toxic and/or irritating for humans. Squaramides and polyamine derivatives have been demonstrated to have antitumor and antiprotozoal activity. The aim of this study was to investigate the activity of 5 squaramides and 5 acyclic polyamines against trophozoites and cysts of A. castellanii Neff. Amoebicidal activity against the trophozoites and cytotoxicity against Vero cells were evaluated with a colorimetric assay, using Alamar Blue?, and chlorhexidine digluconate was assayed as the reference drug. The squaramides 3 and 5 and the acyclic polyamine 6 appeared to be the most active against the trophozoites and their cytotoxicity was low, showing selectivity indexes of 28.3, 26, and 25.7, respectively, similar to the control drug, chlorhexidine digluconate (27.6). But only the squaramide 3 showed complete cysticidal activity at the concentrations of 100 and 200 μM, as the chlorhexidine digluconate. Further studies of the mechanism of action and in vivo assays are needed, but squaramide 3 could be used for developing novel therapeutic approaches against Acanthamoeba infections.

卡氏棘阿米巴肌动蛋白1的免疫学特性和细胞黏附功能

目的:探讨卡氏棘阿米巴(Ac)肌动蛋白1 (Actin 1)(Ac-Actin 1)的免疫学特性,初步阐明Ac-Actin 1介导Ac虫体黏附宿主细胞并参与Ac虫体入侵的作用。方法:以Ac滋养体cDNA为模板,构建原核表达载体pET 22b (+)-Ac-Actin 1,转化大肠埃希菌感受态细胞BL21 (DE3);1 mmol·L^(-1)异丙基硫代半乳糖苷(IPTG)体外诱导表达重组Ac-Actin 1蛋白(rAc-Actin 1),十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)对rAc-Actin 1进行可溶性分析,通过亲和层析方法纯化带有His标签的rAc-Actin 1。3只新西兰白兔随机分为对照组(1只)和实验组(2只);实验组兔以rAc-Actin 1为免疫原皮下注射400μg,对照组兔注入等量生理盐水,制备抗rAc-Actin 1多克隆兔血清;采用酶联免疫吸附试验(ELISA)法检测2组兔抗rAc-Actin 1多克隆抗体效价并测定IgG亚型,Western blotting法检测抗rAc-Actin 1多克隆兔血清与rAc-Actin 1的免疫反应性,间接免疫荧光实验(IFA)检测Ac-Actin 1在Ac滋养体中的定位。以正常滋养体为对照,抗rAc-Actin 1多克隆兔血清阻断Ac滋养体后与Vero细胞共孵育,显微镜观察Ac-Actin 1对Vero细胞的黏附作用。结果:SDSPAGE和BCA蛋白浓度检测,获得高浓度rAc-Actin 1 (1.7 g·L^(-1))蛋白。ELISA法检测,制备的兔抗rAc-Actin1特异性多克隆抗体效价为1∶6 400, IgG1和IgG2a浓度分别为116.76 g·L^(-1)和1 136.15 mg·L^(-1)。Western blotting法检测,兔抗rAc-Actin 1多克隆抗体可与rAc-Actin 1发生特异性结合,具有良好的免疫反应性。IFA检测,rAc-Actin 1主要定位于Ac滋养体的细胞膜上。显微镜观察,与对照组比较,随着抗体与虫体孵育时间的延长,实验组Ac滋养体对Vero细胞的黏附率明显降低(P<0.01),抗rAc-Actin 1特异性多克隆抗体可有效阻断Ac滋养体与Vero细胞的黏附。结论:rAc-Actin 1具有良好的免疫原性和免疫反应性,参与Ac虫体与宿主细胞的黏附作用。

棘阿米巴角膜炎诊断与治疗专家共识(2023)

棘阿米巴角膜炎(AK)是一种严重的致盲角膜病,起病隐匿,临床表现主要为眼表刺激症状、角膜浸润和溃疡,如未得到及时合理诊疗可引起患者视力损害。AK早期体征不典型,临床医生和患者目前对该病的认知尚不足,易误诊或漏诊,造成患眼视力不可逆损害,影响患者视觉质量和生活质量。目前国内外对AK的诊断和治疗尚缺少统一、规范的方案,导致治疗效果不明确,治疗方法不统一。中国医师协会眼科医师分会眼感染学组组织国内眼感染和角膜病专家对我国AK诊疗中存在的问题进行广泛调查和归纳,并在大量检索和复习国内外相关文献的基础上,采用德尔菲法联合面对面讨论的方法,结合我国相关专家的临床实践和经验,就AK的流行病学、发病危险因素、病原学及其发病机制、诊断和鉴别诊断、治疗方案及其流程等问题进行认真讨论,形成本专家共识意见,以指导和规范AK的诊治工作。

棘阿米巴感染治疗药物的研究进展

棘阿米巴(Acanthamoeba)是一类分布广泛、侵入人体后可引起严重、甚至致命感染的机会致病性寄生原虫。药物治疗仍是目前棘阿米巴感染首选的治疗方法。但滋养体抗药性的产生及药物治疗过程中滋养体可转变为休眠形式的抗药性包囊,导致感染复发,使治疗十分困难。近年来,传统药物不断改进,新型药物持续研发,为棘阿米巴感染的精准、高效治疗提供了广阔前景。该文概述抗棘阿米巴药物如抗细菌类药物、抗真菌药物、双胍类和新型制剂(如纳米粒子和树枝状大分子)等的研究现状,以期为棘阿米巴感染的有效治疗提供参考,为新型药物的研发提供思路。

棘阿米巴滋养体对HeLa细胞的细胞毒性作用

目的 探讨处于不同分类地位的 5株棘阿米巴滋养体对人HeLa细胞的细胞毒性作用。方法 将 5株不同虫株的棘阿米巴滋养体分别与体外培养的人HeLa细胞混合 ,观察其对肿瘤细胞的影响。并采用光镜确定肿瘤细胞的形态学变化特征 ,采用MTT(四甲基偶氮唑盐 )法检测并比较这 5种棘阿米巴对肿瘤细胞作用的差异。结果 MTT法显示 ,当棘阿米巴滋养体与HeLa细胞以 1∶1比例混合时 ,对肿瘤细胞表现出较强的细胞毒性作用。该作用不仅具时间依赖性 ,且因棘阿米巴的虫株不同而异。光镜下染色标本显示 ,经 5株棘阿米巴滋养体作用后的肿瘤细胞均出现了核染色质边集浓缩和胞膜出空泡等细胞凋亡的形态学特征。结论 这 5株棘阿米巴滋养体对HeLa细胞均有不同程度的细胞毒性作用 ,并诱导肿瘤细胞发生细胞凋亡。

棘阿米巴角膜炎的诊断和治疗探讨

目的 探讨棘阿米巴角膜炎的临床与实验室诊断方法 ,寻找有效滴眼液用以治疗。方法 观察分析 2 5例棘阿米巴角膜炎感染各阶段的临床表现 ,通过角膜细胞学检查、阿米巴分离培养、角膜活检及组织病理学检查确诊 ,检测药物对棘阿米巴的抗原虫作用及临床疗效。结果 感染自角膜上皮层开始 ,进行性侵入基质致盲。细胞学检查见包囊和 /或滋养体 (88 9% )。棘阿米巴培养阳性率 5 7 9%。洗必泰、甲硝唑滴眼液治疗棘阿米巴角膜炎有良效。抗原虫治疗2 4例 (2 5眼 ) ,治愈 2 1眼 ,无复发。结论 棘阿米巴角膜炎并非罕见 ,常因被误诊、误治导致视力丧失。重要的是早期诊断与抗原虫治疗。

大蒜素体外对卡氏棘阿米巴超微结构的影响

用不同浓度大蒜素处理处于对数生长期的卡氏棘阿米巴(Acanthamoeba castellanii,T4型),24h后在透射电镜下观察其超微结构损伤情况。结果表明,大蒜素浓度为50μg/ml时,卡氏棘阿米巴的超微结构轻度破坏,浓度为500μg/ml时,其结构严重破坏,虫体坏死。大蒜素对体外培养的卡氏棘阿米巴(T4型)超微结构有破坏作用。

棘阿米巴对黑色素瘤细胞毒性作用初探

目的 :探讨棘阿米巴滋养体分泌物中对小鼠黑色素瘤细胞B16起细胞毒作用的物质。方法 :采用MTT (四甲基偶氮唑盐 )法检测棘阿米巴滋养体分泌物对B16的细胞毒作用。结果 :MTT法表明棘阿米巴分泌物对靶细胞有一定的细胞毒作用 ,丝氨酸酶抑制剂可抑制大部分的细胞毒作用。 6 0 %硫酸铵可沉淀大部分起细胞毒作用的成分 ,主要为丝氨酸酶类。结论 :棘阿米巴滋养体分泌物在棘阿米巴致靶细胞损伤中起重要的作用 。

家兔棘阿米巴角膜炎动物模型的建立

目的:建立棘阿米巴角膜炎的动物模型。方法:在6 只新西兰家兔角膜基质层内注射地塞米松3 d 后,再注入棘阿米巴原虫悬液。结果:6 只家兔均发生角膜炎。经家兔角膜刮片用10% 氢氧化钾封片镜检、角膜组织原虫培养和病理切片染色检查证实,成功地建立了家兔棘阿米巴角膜炎动物模型。结论:通过角膜基质内注射建立的家兔棘阿米巴角膜炎动物模型。

角膜表面镜片术建立兔棘阿米巴角膜炎模型

目的建立一种理想的模拟临床人角膜棘阿米巴感染的动物模型,并对其角膜组织损伤的临床表现和组织病理学改变进行观察。方法20只新西兰大白兔利用角膜表面镜片术建立棘阿米巴角膜炎的动物模型,在角膜植片内皮面作"#"形划痕,用10-0尼龙缝线将其固定于受体角膜上,将棘阿米巴滋养体混悬液注入植片与植床间,建立棘阿米巴角膜炎模型,右眼为实验眼,左眼为对照眼;并在接种后第1天、第3天、第5天、第7天、第9天、第14天、第28天行裂隙灯显微镜检查及临床评分、角膜刮片湿封片镜检、角膜组织病理检查及培养。结果应用角膜表面镜片术成功建立兔眼棘阿米巴性角膜炎的动物模型,并经角膜刮片及培养、组织病理切片染色检查证实。实验眼角膜出现水肿,基质环形浸润,角膜新生血管等表现,对照眼均未见感染;实验眼病理组织切片上可见基质层内的棘阿米巴滋养体或包囊,伴有中性粒细胞、单核巨噬细胞、嗜酸性粒细胞等浸润。结论应用角膜表面镜片术建立的兔棘阿米巴角膜炎动物模型模拟了临床人角膜棘阿米巴感染过程,为研究棘阿米巴角膜炎的发病机制及临床治疗奠定了可靠的实验基础。