Different Detection and Treatment Methods for <i>Entamoeba histolytica</i>and <i>Entamoeba dispar</i>in Water/Wastewater: A Review

Entamoeba histolytica is an anaerobic parasitic protozoan and well known as a human pathogen, while its close relative, Entamoeba dispar, also possesses similar characteristics as an infectious agent. These microorganisms are generally transmitted in fecal-contaminated water. However, E. dispar present in industrial wastewater is also capable of creating biofilms that can cause adverse impacts in piping networks. Therefore, it is important to detect both of these protozoan species in water and to find a cost-effective technique for inactivation or management control. This review article summarizes the available detection methods in water and wastewater matrices along with feasible disinfection techniques.

Development of loop-mediated isothermal amplification for rapid detection of Entamoeba histolytica

Objective:To develop a loop-mediated isothermal amplification(LAMP) assay for the detection of Entamoeba histolytica(E.histolytica),the causative agent of amebiasis.Methods:The LAMP primer set was designed from E.histolytica hemolysin gene HLY6.Genomic DNA of E.histolytica trophozoites strain HK9 was used to optimize the LAMP mixture and conditions.Amplification of DNA in the LAMP mixture was monitored through visual inspection for turbidity of the LAMP mix as well as addition of fluorescent dye.Results:Positive LAMP reactions turned turbid while negative ones remained clear.Upon addition of a fluorescent dye,all positive reactions turned green while the negative control remained orange under ambient light After elecrophoresis in 1.5% agarose gels,a ladder of multiple bands of different sizes can be oliserved in positive samples while no bands were detected in the negative control.The sensitivity of the assay was found to be S parasites per reaction which corresponds to approximately 1S.8 ng/μL DNA.The specificity of the assay was verified by the absence of amplified products when DNA from other gastrointestinal parasites such as the morphologically similar but non-pathogenic species,Entamoeba dispar. and other diarrhea-causing organisms such as Blastocystis hominis and Escherichia coli were used.Conclusions:The I.AMP assay we have developed enables the detection of E.histolytica with rapidity and ease,therefore rendering it is suitable for laboratory and field diagnosis of amebiasis.

Detection of Entamoeba histolytica in experimentally induced amoebic liver abscess:comparison of three staining methods

Objective:To compare the efficacy of three different tissue stains,namely haematoxylin and eosin(H&E),periodic-acid Schiff(PAS)and immunohistochemical(1HC)stains for detection of Entamoeba histolytica(E.histolytica)trophozoites in abscessed liver tissues of hamster.Methods:Amoebic liver abscess was experimentally induced in a hamster by injecting 1×10~6of axenically cultured virulent E.histolytica trophozoites(HM1-IMSS strain)into the portal vein.After a week post-inoculation,the hamster was sacrificed and the liver tissue sections were stained with H&E,PAS and IHC stains to delect the amoebic trophozoite.Results:The three stains revealed tissue necrosis and amoebic trophozoites,but with varying clarity.H&E and PAS stained the trophozoites pink and magenta,respectively,however it was difficult to differentiate the stained trophozoites from the macrophages because of their similarity in size and morphology.On the other hand,IHC stain revealed distinct brown appearance of the trophozoites in the infected liver tissues.Conclusions:It can be concluded that out of the three stains.IHC is the best for identification of E.histolytica trophozoites in tissue sections.

Amebic liver abscess by Entamoeba histolytica

Amebic liver abscesses(ALAs)are the most commonly encountered extraintestinal manifestation of human invasive amebiasis,which results from Entamoeba histolytica(E.histolytica)spreading extraintestinally.Amebiasis can be complicated by liver abscess in 9%of cases,and ALAs led to almost 50000 fatalities worldwide in 2010.Although there have been fewer and fewer cases in the past several years,ALAs remain an important public health problem in endemic areas.E.histolytica causes both amebic colitis and liver abscess by breaching the host’s innate defenses and invading the intestinal mucosa.Trophozoites often enter the circulatory system,where they are filtered in the liver and produce abscesses,and develop into severe invasive diseases such as ALAs.The clinical presentation can appear to be colitis,including upper-right abdominal pain accompanied by a fever in ALA cases.Proper diagnosis requires nonspecific liver imaging as well as detecting anti-E.histolytica antibodies;however,these antibodies cannot be used to distinguish between a previous infection and an acute infection.Therefore,diagnostics primarily aim to use PCR or enzyme-linked immunosorbent assay to detect E.histolytica.ALAs can be treated medically,and percutaneous catheter drainage is only necessary in approximately 15%of cases.The indicated treatment is to administer an amebicidal drug(such as tinidazole or metronidazole)and paromomycin or other luminal cysticidal agent for clinical disease.Prognosis is good with almost universal recovery.Establishing which diagnostic methods are most efficacious will necessitate further analysis of similar clinical cases.

Entamoeba histolytica acetyl-CoA synthetase:biomarker of acute amoebic liver abscess

Objective:To characterize the Entamoeba histolytica(E.histolytica)antigen(s)recognized by moribound amoebic liver abscess hamsters.Methods:Crude soluble antigen of E.histolytica was probed with sera of moribund hamsters in 1D-and 2D-Westem blot analyses.The antigenic protein was then sent for tandem mass spectrometry analysis.The corresponding gene was cloned and expressed in Escherichia coli BL21-AI to produce the recombinant E.histolytica ADP-forming acetyl-CoA synthetase(EhACS)protein.A customised ELISA was developed to evaluate the sensitivity and specificity of the recombinant protein.Results:A^75 kDa protein band with a pl value of 5.91-6.5 was found to be antigenic;and not detected by sera of hamsters in the control group.Tandem mass spectrometry analysis revealed the protein to be the 77 kDa E.histolytica ADP-forming acetyl-CoA synthetase(EhACS).The customised ELISA results revealed 100%sensitivity and 100%specificity when tested against infected(n=31)and control group hamsters(n=5)serum samples,respectively.Conclusions:This rinding suggested the significant role of EhACS as a biomarker for moribund hamsters with acute amoebic liver abscess(ALA)infection.It is deemed pertinent that future studies explore the potential roles of EhACS in better understanding the pathogenesis of ALA;and in the development of vaccine and diagnostic tests to control ALA in human populations.

Profiles of Entamoeba histolytica—specific imunoglobulins in human sera

Objective:To determine the profdes of anti-Entamoeba histolytica(E.histolytica) IgA.IgC. and IgM in sera of diarrheic and non-diarrheic individuals and partially characterize target antigens.Methods:Serum samples from thirty diarrheic and thirty non-diarrheic individuals were subjected to IgA,IgG,and IgM profiling through enzyme-linked immunosorbent assay (EI.ISA),flow cytometry,and immunoblot.Results:ELBA titer results showed that both diarrheic and non-diarrheic individuals possess high levels of E.histolytica-specific IgG compared to IgA and IgM.How cytometry data showed that diarrheic serum samples had higher mean reaction percentages against E.histolytica cells compared to non-diarrheic samples.Immunoreactive E.histolytica proteins with molecular weights ranging belween 7 kDa and 292 kDa were recognized by diarrheic serum IgG,and 170 kDa and 250 kDa by non-diarrheic serum IgG. Conclusions:Our findings suggest that serum anti-E.histolytica IgG,compared with serum anti-E.histolytica IgA and IgM responses,was generally high in both diarrheic and nondiarrheic sera,indicating a past exposure to the organism both in symptomatic patients as well as in asymptomatic carriers,respectively.In addition,serum IgG from diarrheic and non-diarrheic patients were able to detect immunogenic E.histolytica proteins.

Identification and Characterization of Heparan Sulphate Binding Proteins of <i>Entamoeba histolytica</i>

A large number of microbial pathogens bind to heparan sulphate on eukaryotic cell surfaces. Heparan Sulphate Binding Proteins (HSBPs) from Entamoeba histolytica culture lysates were obtained by sequential ammonium sulphate precipitation and Protein purify. SDS-PAGE and immunoblotting experiments indicated the presence of two major extracellular proteins in E. histolytica (51.2 kDa and 61.0 kDa). Characterization of HSBPs by 2D Gel electrophoresis of 40% (NH4)2SO4 precipitated lysate of E. histolytica revealed that the isoelectric point of 51.2 kDa HSBP was at pH3.0. The protein of 61.0 kDa HSBP showed three spots in 40% (NH4)2SO4 lysate which had isoelectric point between pH 4.0 - 7.0. While in 80% (NH4)2SO4 precipitated lysate, 51.2 kDa HSBP showed only one spot which had isoelectric point at pH 3. However, 61.0 kDa HSBP revealed 2 spots which had isoelectric point between pH 4 and 5. The result showed that this parasite has proteins which interact with heparan sulphate whose molecular formula is C14H23NO21S-23. These proteins may have a role in binding of parasite to heparan sulphate on host cells. Further characterization by MALDI-TOF analysis of HSBPs from E. histolytica demonstrated HSBPs to be novel protein in this parasite which has been isolated, purified and characterized first time by our group in the present study.

Antioxidant enzyme profile of two clinical isolates of Entamoeba histolytica varying in sensitivity to antiamoebic drugs

AIM To study the sensitivity and antioxidant enzyme response in two clinical isolates of Entamoeba histolytica(E. histolytica) during treatment with antiamoebic drugs,auranofin and metronidazole. METHODS E. histolytica were isolated from stool samples and maintained in Robinson's biphasic culture medium. Clinial isolates were maintained in xenic culture medium,and harvested for determination of minimum inhibitory concentrations to the two antiamoebic drugs,Metronidazole and Auranofin using microtiter plate tests. The percent survival of the two isolates were determined using the trypan blue cell count. Isolate 980 was treated with 70 μmol/L and 2 μmol/L while isolate 989 was treated with 20 μmol/L and 0.5 μmol/L of metronidazole and auranofin respectively for 24 h. Fifty thousand cells of each isolate were harvested after 24 h of treatment for analysis of the mRNA expressions of the antioxidant enzymes,thioredoxin reductase,peroxiredoxin and FeSOD using the specific primers. Cell lysate was used for determination of enzyme activity of thioredoxin reductase by measuring DTNB reduction spectrophotometrically at412 nm.RESULTS Minimum inhibitory concentration of the clinical isolates 980 and 989 for auranofin was 3 μmol/L and 1 μmol/L respectively while that for metronidazole was 80 μmol/L and 30 μmol/L respectively. Thioredoxin reductase,peroxiredoxin and FeSOD expression levels were significantly reduced in the isolate 980 when treated with Auranofin. Metronidazole treatment showed a down regulation of thioredoxin reductase. Though not significant both at the mRNA and the enzyme activity levels. Peroxiredoxin and FeSOD however remained unchanged. Auranofin treatment of isolate 989,showed an upregulation in expression of thioredoxin reductase while Peroxiredoxin and FeSOD did not show any change in expression. Upon treatment with metronidazole,isolate 989 showed an increase in thioredoxin reductase expression. Peroxiredoxin and FeSOD expressions however remain unchanged both at mRNA and enzyme activity level.CONCLUSION Clinical isolates from New Delhi NCR region show different sensitivities to antiamoebic drugs. Auranofin is effective against isolate showing higher tolerance to metronidazole as shown by its inhibition in thioredoxin reductase activity.

Prevalence of Two Gastrointestinal Parasites <i>Entamoeba histolytica</i>and <i>Giardia lamblia</i>within Samarra City, Iraq

The prevalence of two gastrointestinal parasites the Entamoeba histolytica and Giardia lamblia parasites and their impact on some blood parameters, i.e. packed cell volume (PCV), hemoglobin (Hb%) and total protein (TP) of a total 780 patients (children and adults) admitted to Samarra General Hospital were assessed. Samples of fresh feces were collected in normal physiological saline and examined using Olympic microscopes. The frequency of the parasite E. histolytica was 12.8% (46.3% male and 53.6% female). The highest frequency of infection of E. histolytica (13.8%) was found at age group (1 - 5 years old) followed by <1 year old children while the lowest (7.4%) was at ages (>41 years old). The highest rate of infection (33.9%) was found in September and the lowest (2.2%) in January. Similarly, the general infection frequency of the parasite G. lamblia was 3.9% with the highest rate at ages 1 - 20 years old and the lowest rate was 7.3% for >50 years old. The monthly, highest rate of infection (5.2%) was in August and least (2.2%) in January (2.2%). The frequency of total protein (TPD) in the blood relevant to the presence of parasite E. histolytica and G. lamblia was 4.6% and 1%, respectively. It is concluded that the above two parasites are the most common gastrointestinal parasite in Iraq whose pathogenesis to be which is likely to escalate during the summer seasons and at low hygienic services environment. There has been an irrelevance neither to anemia nor total protein deficiency. It is recommended that Ministry of Health in Iraq should not share the global idea of defining the giardiasis as a neglected disease.

Effect of human milk and colostrum on Entamoeba histolytica

AIM:Many defense factors of the mother's colostrum or milk protect infants from intestinal, respiratory and systemic infections. In the present study, we investigated the effect of colostrum and mature human milk on E. histolytica parasites in vitro.METHODS:Samples of human milk were collected from 5 healthy lactating mothers.The medium with human milk at concentrations of 2%, 5% and 10% was obtained.RESULTS:The lethal effect of E. histolytica on the medium supplemented with different concentrations of both colostrum and mature human milk was significant during the first 30min. We also detected that the results of colostrum and mature human milk were similar. No statistically significant differences were found between same concentrations of colostrum and mature human milk at the same times.CONCLUSION:Colostrum and mature human milk have significant lethal effect on E. histolytica and protect against its infection in breast fed children.

Effects of Cd^(+2),Cu^(+2),Ba^(+2) and Co^(+2) ions on Entamoeba histolytica cysts

AIM:The effects of cobalt, copper, cadmium and barium ions on the cysts of Entarnoeba histolytica (E.histolytica),an amebic dysentery agent,cultured in Robinson medium were investigated.METHODS: E.histolytica cysts and trophozoites isolated from a patient with amebiasis were cultivated in the medium,incubated at 37℃ for a period of 4 days and 40×10^4/ml amebic cysts were then transferred to a fresh medium. At the second stage, 0.05, 0.1 and 0.2mM of selected metal ions were added to the medium,and the effects of these ions on parasitic reproduction compared with the control group were observed.RESULTS:It was determined that the number of living parasites in all the groups containing metal ions decreased significantly starting from 30 minutes (P<0.01). CuCl2 showed the highest lethal effect on E. histolytica cysts, whereas the lowest lethal effect was observed with CoCl2. It was also seen that the number of living cells was decreased as the ion concentration and exposure time were increased, and that there were no living parasites in the medium at the end of 24h (P<0.01).CONCLUSION:It may be stated that the effect of everincreasing contamination of the environment with metal waste materials on parasites should be investigated further.

Evolutionary status of Entamoeba

In addition to its medical importance as para- sitic pathogen, Entamoeba has aroused people’s interest in its evolutionary status for a long time. Lacking mitochondrion and other intracellular organelles common to typical eu- karyotes, Entamoeba and several other amitochondrial pro- tozoans have been recognized as ancient pre-mitochondriate eukaryotes and named “archezoa”, the most primitive extant eukaryotes. It was suggested that they might be living fossils that remained in a primitive stage of evolution before acqui- sition of organelles, lying close to the transition between prokaryotes and eukaryotes. However, recent studies re- vealed that Entamoeba contained an organelle, “crypton” or “mitosome”, which was regarded as specialized or reductive mitochondrion. Relative molecular phylogenetic analyses also indicated the existence or the probable existence of mi- tochondrion in Entamoeba. Our phylogenetic analysis based on DNA topoisomerase II strongly suggested its divergence after some mitchondriate eukaryotes. Here, all these recent researches are reviewed and the evolutionary status of En- tamoeba is discussed.

腹泻患者溶组织内阿米巴感染现状调查

目的探究阳春市阿米巴病的临床特征及诊治结果。方法回顾性分析阳春市中医院2018年1月—2021年1月收治的434例腹泻患者临床资料,采用碘染涂片镜检法检测溶组织内阿米巴感染情况,对患者临床特征、检测过程、诊治结果进行分析。结果434例腹泻患者中检出溶组织内阿米巴原虫病原学阳性15例(3.46%),全部有寒战、高热、咳嗽症状,10例有慢性结肠炎伴黏液便,3例有腹泻、腹痛、肝区疼痛等症状,2例有急性腹泻伴下消化道出血症状;溶组织内阿米巴包囊10例,阿米巴滋养体5例;肠内阿米巴病13例,肠外2例;以慢性结肠炎反复发作而进行检查、治疗导致原发病被掩盖8例;以肠外阿米巴感染为代表的肝脓肿,因病情复杂,诊断为急性肝炎2例;诊断为急性胃肠炎1例;以急性感染发作而被误诊为急性爆发性痢疾4例。15例患者确诊阿米巴病后均采用抗阿米巴药物与临床对症治疗的综合疗法,甲硝唑治疗有效3例,甲硝唑联合黄连素治疗有效10例,甲硝唑联合白头翁灌肠治疗有效2例。结论阳春市中医院近3年腹泻患者溶组织内阿米巴感染情况较为严重,患者全部伴寒战、高热、咳嗽症状,甲硝唑联合黄连素治疗效果较好。

云南省怒江州独龙牛内阿米巴原虫的感染情况调查

为了解云南省怒江州独龙牛内阿米巴原虫的感染情况,按照春、夏、秋、冬4个季节采集到的625份独龙牛粪便样本,用常规PCR方法,基于内阿米巴原虫的18S rRNA基因对粪便样本中虫体基因进行扩增。结果显示,检出内阿米巴原虫阳性样本共61份,总感染率为9.76%(61/625)。其中,鸠门当村、亚左洛村、古泉村、茨开镇、独龙江乡的感染率分别为4.12%(4/97)、7.59%(6/79)、11.18%(17/152)、13.54%(31/229)和4.41%(3/68),5个采样点之间,其感染率差异显著(P<0.05)。怒江流域与独龙江流域的内阿米巴原虫感染率分别为10.41%(58/557)和4.41%(3/68),差异不显著(P>0.05)。春、夏、秋、冬4个季节,独龙牛内阿米巴原虫的感染率分别为11.93%(26/218)、23.66%(22/93)、3.33%(4/120)和4.64%(9/194),差异极显著(P<0.01)。在阳性样本中,有51份鉴定为Entamoeba bovis,10份鉴定为Entamoeba sp.MG107/BEL。结果表明,云南省怒江州独龙牛存在内阿米巴原虫感染,应给予重视并建议采取有效措施进行防控。

齿龈内阿米巴致病作用的动物实验

目的 研究齿龈内阿米巴 (Entamoeba gingivalis,E.g.)的致病作用及其与牙周病的关系。 方法 大鼠经免疫抑制剂处理 1wk,并于切牙颈部结扎钢丝后 ,随机分为 3组 ,分别于龈缘涂抹 E.g.、共生菌 (symbioticbacteria,s.b.)及生理盐水 ,观察各组大鼠牙周组织发病情况 ,并查脓液中有无活的病原体。 结果 E.g.组大鼠牙周组织发病率高于 s.b.组和对照组 (P<0 .0 5 ) ,s.b.组大鼠发病率亦高于对照组 (P<0 .0 5 ) ,脓液镜检或经培养均查见相应的病原体。 结论 齿龈内阿米巴是一种条件致病病原体 ,当宿主免疫力下降时 。

齿龈内阿米巴的超微结构与溶酶体酶细胞化学研究

目的 研究齿龈内阿米巴的细胞器与功能 ,探讨虫体与宿主细胞的关系。方法 用透射电镜结合电镜酶细胞化学技术观察虫体细胞超微结构 ;将虫体与口腔上皮细胞孵育 ,观察虫体对上皮细胞的粘附。结果 虫体及其伪足呈多形态 ;胞质内细胞器主要为泡状和管状的溶酶体、一些滑面内质网 ,以及丰富的糖原颗粒 ;溶酶体标志酶CMPase显示溶酶体酶丰富 ;可见虫体粘附于上皮细胞表面和小伪足伸入上皮细胞微绒毛间。结论 虫体的溶酶体水解酶消化作用及虫体伪足的机械作用对宿主牙周上皮组织的损伤 ,是虫体的致病机制之一。

齿龈内阿米巴的连续培养与致病性研究

[目的 ]建立齿龈内阿米巴 (Entamoeba gingivalis,E.g.)的连续培养方法和研究虫体的致病性。 [方法 ]比较不同培养条件下虫体生长繁殖情况 ,调查人群感染 E.g.情况及观察感染 E.g.与唾液中酸性磷酸酶含量的关系 ,大鼠实验观察 E.g.感染与牙周脓肿的关系 ,E.g.体外实验观察甲硝唑的杀虫效果。 [结果 ]虫体最适培养条件为 :改良的 L ES培养基中加含 2 0 %小牛血清的 L ocke液、青霉素、链霉素和米粉 ,p H6 .7于 35℃培养 ,第4天虫体繁殖达高峰 ,可存活 12 0～ 16 8h;E.g.检测阳性的牙周病患者其酸性磷酸酶和脂质过氧化的终产物(MDA)含量高于阴性者 ,牙周脓肿患者 E.g.感染率高于健康人群。E.g.可致大鼠牙周脓肿 ,甲硝唑与绣球花叶(SBF)杀 E.g.有良效。 [结论 ]E.g.在改良的 L ES培养基中生长良好 ,当宿主免疫力低下时 E.g.

唐山市高校学生齿龈内阿米巴感染状况

目的调查唐山市高校在校学生齿龈内阿米巴的感染状况,并分析齿龈内阿米巴感染与生活饮食习惯及口腔健康状况的关系。方法2008年随机选择唐山市6所高校1～3年级学生进行问卷调查,内容包括口腔健康状况、刷牙习惯、是否经常嚼口香糖、饮食偏好、是否吸烟等。用消毒牙签取调查者口腔齿垢物或病灶表面附着物进行涂片,镜检齿龈内阿米巴感染情况。分析口腔健康状况、生活饮食习惯等与齿龈内阿米巴感染的关系。结果共调查551名大学生,齿龈内阿米巴感染率为28.3%(156/551)。其中男、女生感染率分别为30.4%(55/181)和24.6%(91/370),两者差异无统计学意义(χ2=2.09,P>0.05);口腔健康者的齿龈内阿米巴感染率为20.8%(72/347),有口腔疾患者的感染率为41.2%(84/204),两者差异有统计学意义(χ2=26.41,P<0.01);经常刷牙者齿龈内阿米巴感染率为22.5%(53/236)明显低于不经常刷牙者的32.7%(103/315),差异有统计学意义(χ2=6.97,P<0.01);经常嚼口香糖者齿龈内阿米巴感染率为18.3%(17/93),低于不经常嚼口香糖者的30.4%(139/458),差异有统计学意义(χ2=5.55,P<0.05)。结论齿龈内阿米巴在唐山市高校学生中感染普遍,并与口腔卫生习惯和口腔疾病存在一定关系。

齿龈内阿米巴的连续培养及其生长情况

目的： 建立齿龈内阿米巴（ Ｅｎｔａｍｏｅｂａ ｇｉｎｇｉｖａｌｉｓ， Ｅｇ） Ｆ Ｊ４ 的连续培养方法， 并观察其生长情况。方法： 在不同培养条件下观察 Ｅｇ虫体大小及其存活时间。结果： Ｅｇ在改良的 Ｌｏｃｋｅｅｇｇｓｅｒｕｍ 与改良的ｙｏｌｋｅｇｇｓｅｒｕｍ 培养基中生长良好， 建立了福建３ 株 Ｅｇ（ Ｆ Ｊ２ 、 Ｆ Ｊ３ 及 Ｆ Ｊ４） ， 虫体大小均值为１３１９ μｍ ～４３９３ μｍ ×９８８ μｍ ～３０７４ μｍ 。 Ｅｇ最适宜培养条件为ｐ Ｈ 在６４ ～６７ 、改良的 Ｌｏｃｋｅｅｇｇｓｅｒｕｍ 或蛋黄液含２０ ％ 小牛血清加青霉素、链霉素及米粉。虫体繁殖高峰为ｄ４ ， 其在上述培养液体中可存活１２０ ｈ ～１６８ ｈ 。结论： Ｅｇ在 Ｌｏｃｋｅｅｇｇｓｅｒｕｍ 与 Ｙｏｌｋｅｇｇｓｅｒｕｍ 培养基中生长良好， 每４ ｄ 转种１ 次， 可达到长期培养的效果。