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.

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.

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.

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.

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 Entamoeba histolyt/ca (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 0.2 mM 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).CuCl_2 showed the highest lethal effect on E.histolytica cysts,whereas the lowest lethal effect was observed with CoCl_2.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 24 h (P<0.01). CONCLUSION:It may be stated that the effect of ever- increasing contamination of the environment with metal waste materials on parasites should be investigated further.

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.

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.

艾滋病相关卡波西肉瘤感染溶组织内阿米巴1例

患者,男,32岁,一过性发热伴紫红色结节样皮疹6月余,HIV-Ab(+)、II期梅毒,进一步进行治疗中。卡波西肉瘤(T1I1S1)累及咽喉、皮肤、淋巴结、肺、消化道,行C1-2紫杉醇(泰素)治疗。入院后行粪便常规检查,发现溶组织内阿米巴,口服甲硝唑治疗后,4d后结果转为阴性。提示临床在患者粪便检查出现溶组织阿米巴包囊体或滋养体时,患者无论是否出现阿米巴肠炎相关症状,患者均需要积极治疗。

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。

Discharges of Wastewater Treatment Plants Needed Further Monitoring to Minimize Potential Risk of Entamoeba and Blastocystis for Public Health

The protozoan parasites Entamoeba histolytica and Blastocystis hominis are responsible for causing human amebiasis and hominis infections,respectively.These infections are highly prevalent and are often linked to waterborne diseases.Due to the absence of regulations for monitoring these protozoa at the discharge points of wastewater treatment plants(WWTPs),the effluents reaching surface waters contribute to waterborne transmission.This underscores the significance of the removal capacities of WWTPs in reducing the spread of these infectious parasites.Therefore,this study examined five different types of WWTPs in Ankara,Turkey,over a year to assess their capacities to remove E.histolytica and B.hominis.The seasonal abundances of genes specific to these protozoa in both the influents and effluents of each WWTP were measured using a quantitative polymerase chain reaction.The reduction in the number of protozoan rDNA copies between the influent and effluent samples was evaluated as the removal capacity,expressed in log10 reduction(LRV)values.The results elucidated that the removal of E.histolytica and B.hominis was highly affected by the process used.Membrane bioreactor systems displayed the highest removal capacity with LRV>3.Therefore,discharges of WWTPs with other processes could need further monitoring to minimize the potential risk for public health.

FTA-巢式PCR方法鉴定溶组织内阿米巴原虫的初步研究

目的建立一种简便、快速的FTA-巢式PCR方法用于鉴定粪便中溶组织内阿米巴原虫(Entamoeba histolytica,E.h)。方法收集门诊腹泻病人新鲜粪便,用光学显微镜进行初步检查。用FTA卡抽提镜检结果为阳性的粪便DNA,根据溶组织内阿米巴原虫的SSU-rRNA序列设计引物,进行巢式PCR扩增,对PCR产物进行琼脂糖凝胶分析,并对阳性产物进行测序和序列比对分析。结果根据光学显微镜检测结果,挑选了44例镜检结果为阿米巴原虫阳性的腹泻病人粪便。经FTA-巢式PCR扩增,其中20例样本可扩增出427bp左右的目的条带,目的条带的测序和序列分析结果表明为溶组织内阿米巴原虫。镜检方法与巢式PCR方法的阳性符合率为45.45%(20/44),将E.h与形态学相似的其他内阿米巴原虫进行了鉴定和区分。结论本研究建立的FTA-巢式PCR方法具有简单、快速、准确等优点,为临床检验和流行病学调查中E.h的鉴别诊断提供了新的技术方法。

人和动物阿米巴原虫病研究进展

阿米巴原虫病主要是由溶组织内阿米巴引起的一种人兽共患寄生虫病。自1875年Feder Losch首次在人体发现该病以来,已有9个不同种属的阿米巴被先后发现,已报道的易感动物达30多种。该类原虫多寄生于人和动物的肠道和肝脏,以滋养体形式侵袭机体,引发阿米巴痢疾或肝脓肿。文章综述了近年来人和动物阿米巴原虫病的病原学、流行病学、发病机理、诊断和防治等方面的研究进展。

中国溶组织内阿米巴感染的状况

１９８８～１９９１年全国３０个省（区市）共调查了７２６个县２８４８个点，１４７７７４２人。溶组织内阿米巴感染呈全国性分布，但分布不匀。全国平均感染率为０．９５％（±０．０４％），最低为０．０１％，最高为８．１２％，平均感染率超过２％的省、自治区有西藏（８．１２％）、云南（２．５４％）、新疆（２．３７％）、贵州（２．２５％）和甘肃（２．０４％），所调查的７２６个县中，阴性者２０１个县（占检查县的２７．６９％），阳性县５２５个，占７２．３１％。感染率在１％以内的县占阳性县的６６．０９％，感染率高于１０％者仅１４个县占阳性县的２．２９％。不同民族的感染率不一。感染率有随年龄增加而上升的趋势，至１５岁以后感染率有波动，变化不大。女性感染率（０．９４％）高于男性（０．８４％）。新疆与河北的感染有家庭聚集性现象。感染率因温度带、干湿区域及饮用水源而异。

甲硝唑酯体外抗阴道毛滴虫和阿米巴的效果

用本校药学院物理化学教研室试制的甲硝唑酯对体外培养的阴道毛滴虫和溶组织内阿米巴的作用进行观察。结果表明该药对阴道毛滴虫的致死浓度为２．６μｇ／ｍｌ，与甲硝唑的致死浓度２．０μｇ／ｍｌ相近；该药对溶组织内阿米巴的致死浓度为４０．０μｇ／ｍｌ，与甲硝唑相同。提示该药具有抗此两种原虫的作用。

溶组织内阿米巴的酶类及其解毒作用

研究发现溶组织内阿米巴含有ＳＯＤ、ＣＡＴ与ＰＯａｓｅ三种解毒酶类，前者通过歧化反应清除自由基Ｏ２，后者通过催化与分解清除Ｈ２Ｏ２。它们的解毒作用是虫体生存的重要防御手段。若虫体解毒酶类发生障碍，毒物将使虫体积蓄中毒死亡，还可能对人体造成损害。并测出虫体的碱性磷酸酶和淀粉酶含量。

溶组织内阿米巴与侵袭内阿米巴的化学元素和氨基酸的研究

本文报道以生化方法检测溶组织内阿米巴与侵袭内阿米巴的６种化学元素（Ｆｅ、Ｃｕ、Ｚｎ、Ｃａ、Ｍｇ、Ｐ）和１７种的氨基酸，及侵袭内阿米巴的ＳＯＤ、ＣＡＴ、ＡＫＰ三种酶的含量，并对它们在代谢中的作用作了讨论。