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PubMedDate not supplied

An improved PCR-based method for detection and differentiation of Entamoeba histolytica and Entamoeba dispar in formalin-fixed stools.

Detection of infectious agents in faecal samples by polymerase chain reaction (PCR) can be limited by the presence of substances which inhibit DNA amplification. Here, an improved protocol is reported for directly isolating DNA from fresh and aged formalin-fixed stools, after concentration by formalin-ethyl acetate (FEA). The protocol was successfully applied to detect DNA of Entamoeba histolytica/Entamoeba dispar complex in stools by nested PCR, showing high specificity and low detection limit. Extended time of specimen storage in formalin had no influence on PCR yields. This PCR-based method offers technical advantages for routine detection and discrimination of invasive E. histolytica and non-invasive E. dispar.

Animals↗
PubMed2005

Entamoeba histolytica and/or Entamoeba dispar: infection frequency in HIV+/AIDS patients in Mexico city.

The objective of this work was to evaluate the frequency of Entamoeba histolytica/Entamoeba dispar intestinal infection in HIV+/AIDS subjects and their HIV- close relatives or sexual partners. Enteric parasites were investigated in stool samples by microscopic examination and E. histolytica and E. dispar were identified by PCR. We found by microscopic analysis in HIV+/AIDS group that the E. histolytica/E. dispar complex was present in 5.9% of the members, while in the HIV- group was 2.9%. With PCR we found that the E. histolytica prevalence was 25.3% in the HIV+/AIDS group and 18.5% in the HIV-group. The difference in the results obtained with the microscopic and PCR is due to the different sensibility of the procedures. Besides, we found patients who were infected with E. histolytica in both groups were asymptomatic cyst passers. Our results suggest that E. histolytica strains prevalent in the studied community appear to be of low pathogenic potential.

AIDS-Related Opportunistic Infections↗
PubMed2000

Recent developments in amoebiasis:the Gal/GalNAc lectins of Entamoeba histolytica and Entamoeba dispar.

Amoebiasis is responsible for 50000-100000 deaths annually. Invasive amoebic disease begins with the attachment of Entamoeba histolytica trophozoites to colonic mucin, a process mediated by the amoebic Gal/GalNAc lectin. The non-pathogenic counterpart, E. dispar, is morphologically identical but genetically distinct. Investigations comparing the Gal/GalNac lectin from these two organisms are under way.

Amebiasis↗
PubMedDate not supplied

A serum-free, partly defined medium, PDM-805, for axenic cultivation of Entamoeba histolytica Schaudinn, 1903 and other Entamoeba.

We describe the first serum-free, partly defined medium (PDM-805) for cultivating the human enteric pathogen, Entamoeba histolytica, and the reptilian amebae E. barreti, E. invadens, and E. terrapinae. PDM-805 was developed by the stepwise replacement of yeast extract, bovine serum, and a casein peptone digest in TYI-S-33, a medium widely used for the axenic cultivation of these parasites. The defined components include amino acids, carbohydrates, B vitamins, ascorbic acid, tocopherol, thioctic acid, nucleic acid precursors, trace metals, and phosphate buffers. The undefined components include a highly purified bovine serum albumin, a lipoprotein-cholesterol solution from bovine serum, and a dialyzable, autoclavable, water-soluble growth factor(s) having a molecular weight of less than 3,500 prepared from casein peptone. To date, studies on the growth requirements of E. histolytica, strain 200:NIH, show the following are essential for sustained multiplication of this ameba: iron, glucose, biotin, folic acid, niacinamide, pantothenate, pyridoxal, riboflavin, thiamine, cysteine, an ammonium moiety (in addition to that present in cysteine), bovine serum albumin, lipoprotein-cholesterol, and casein peptone dialysate.

Animals↗
PubMed1999

Chitinase secretion by encysting Entamoeba invadens and transfected Entamoeba histolytica trophozoites: localization of secretory vesicles, endoplasmic reticulum, and Golgi apparatus.

Entamoeba histolytica, the protozoan parasite that phagocytoses bacteria and host cells, has a vesicle/vacuole-filled cytosol like that of macrophages. In contrast, the infectious cyst form has four nuclei and a chitin wall. Here, anti-chitinase antibodies identified hundreds of small secretory vesicles in encysting E. invadens parasites and in E. histolytica trophozoites overexpressing chitinase under an actin gene promoter. Abundant small secretory vesicles were also identified with antibodies to the surface antigen Ariel and with a fluorescent substrate of cysteine proteinases. Removal of an N-terminal signal sequence directed chitinase to the cytosol. Addition of a C-terminal KDEL peptide, identified on amebic BiP, retained chitinase in a putative endoplasmic reticulum, which was composed of a few vesicles of mixed sizes. A putative Golgi apparatus, which was Brefeldin A sensitive and composed of a few large, perinuclear vesicles, was identified with antibodies to ADP-ribosylating factor and to epsilon-COP. We conclude that the amebic secretory pathway is similar to those of other eukaryotic cells, even if its appearance is somewhat different.

ADP-Ribosylation Factors↗
PubMed1997

An improved colorimetric PCR-based method for detection and differentiation of Entamoeba histolytica and Entamoeba dispar in feces.

The epidemiological implications of the recent separation of "Entamoeba histolytica" into two separate species, pathogenic E. histolytica sensu stricto and commensal E. dispar, will not become apparent without methods of distinguishing between them which are applicable to large numbers of specimens. We have modified a PCR-based method to produce such a technique which may be completed in 1 day while still identifying 10(-1) E. histolytica and 1 to 10 E. dispar trophozoites per g of feces when present separately and 10 E. histolytica and 100 E. dispar trophozoites per g in the presence of 10(6) trophozoites per g of the other species. Applied to fecal specimens from 18 patients from which E. histolytica or E. dispar had been grown and identified to the species level by hexokinase isoenzyme analysis, the method in every case yielded the correct result. Positive and negative results are easily distinguished by eye, and we are now applying this technique to a large-scale epidemiological study of amebiasis in the eastern Mediterranean region.

Animals↗
PubMed1996

Differentiation of Korean isolates of Entamoeba histolytica from Entamoeba dispar.

Cysts of Entamoeba histolytica are still found from humans in Korea, but not all of the cysts are known as pathogenic. The non-pathogenic strain is regarded as a different species, E. dispar. In this study, Korean isolates of conventional E. histolytica were subjected for the differentiation by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. Human stools were screened by routine microscopic examination, and cyst or trophozoite positive stools were inoculated into Robinson media. The cultivated trophozoite positive stools were inoculated into Robinson media. The cultivated trophozoites were prepared for DNA extraction, and the DNAs were used for PCR with common primers of P1 gene. The PCR products were digested with 3 restriction enzymes and RFLP was observed. Also anti-sense primers containing the cleavage site of each restriction enzyme were designed for differentiation only by PCR. The PCR products of Korean isolates S9, S12, YS-6, and YS-27 were spliced by Taq I and Xmn I but not by Acc I, and the isolates S1, S3, S11, S15, S16, S17, S20, YS-17, and YS-44 were spliced by Acc I but not by Taq I and Xmn I. These RFLP pattern correlated well with PCR products by the species specific primers. The findings confirm that the Korean isolates S9, S12, YS-6, and YS-27 are E. histolytica and others are E. dispar. In Korea, most of the asymptomatic cyst carriers are infected by E. dispar, not by E. histolytica.

Animals↗
PubMed1996

Differentiation of Entamoeba histolytica and Entamoeba dispar in cyst-passers by immunoblot.

Differentiation of invasive strains of Entamoeba histolytica according to their pathogenicity has been a topic of long debate, but now the pathogenic species only is regarded as E. histolytica while the non pathogenic species is E. dispar. The present study applied immunoblot to differentiate infections of the two species among microscopically-detected cyst-passers in Korea. The crude extract of E. histolytica separated in 5.20% gradient gels, revealed many fractions of 94, 81, 71, 50, 44, 38.5, 37.5, 29, 19, and 18 kDa when the cysteine proteinase inhibitor, E64, was supplemented. The scrum IgG antibody of 3 proven E, histolytica cases reacted with the antigenic fractions of 117, 110, 99, 68, 66, 60, 54, 52, 46, and 45 kDa. Sera of PCR confirmed 3 cases of E. dispar reacted only to the 117 kDa fraction of the E. histolytica crude extract which was regarded as non-specific. To the antigen of monoxenic E. dispar, sera of E. dispar and E. histolytica cases showed the same immunoblot reactions. The serum IgA antibody reacted with several antigenic fractions of both E. histolytica and E. dispar, but IgM and IgE antibodies showed no reaction to either antigen. Sera of 24 symptomless amebic cyst passers were screened with the E. histolytica antigen; two were found to be infected by E. histolytica and 22 were by E. dispar. The present findings suggest that in Korea most of asymptomatic cyst passers of E. histolytica are carriers of E. dispar. Immunoblot using E. histolytica antigen is a good technique for the differentiation of E. histolytica and E. dispar infections.

Animals↗
PubMed1976

Double-blind test of metronidazole and tinidazole in the treatment of asymptomatic Entamoeba histolytica and Entamoeba hartmanni carriers.

One hundred and fifteen persons with asymptomatic Entamoeba histolytica or E. hartmanni infection, or both, were given metronidazole (750 mg three times daily for 5 days), tinidazole (1 g twice daily on 2 consecutive days), or a starch placebo. Three post-treatment stools were examined in the 2 weeks following initiation of treatment. Cysts of E. histolytica reappeared in the stools of 37% of 30 given metronidazole, 62% of 34 given tinidazole, and 70% of 31 given placebo. Cysts of E. hartmanni reappeared in the stools of 46% of 24 given metronidazole, 69% of 16 given tinidazole, and 90% of 10 given placebo. Rapid absorption and short duration of treatment make both drugs ineffective for the treatment of ameba carriers.

Adolescent↗
PubMed1997

Differentiation of Entamoeba histolytica and Entamoeba dispar from German travelers and residents of endemic areas.

Entamoeba histolytica and E. dispar detected in fecal samples of German travelers and residents of tropical and subtropical countries were cultured and differentiated by the polymerase chain reaction (PCR) and hexokinase isoenzyme typing. Twenty-one of 60 isolates were identified as E. histolytica. It was shown that 13 of 18 German short-term travelers were infected with E. histolytica, whereas only three of 22 of the examined inhabitants of tropical countries and five of 20 German long-term travelers harbored E. histolytica. It appears that short-term travelers represent a high risk group for infection with the pathogen E. histolytica.

Animals↗
PubMed1998

Field study on the distribution of Entamoeba histolytica and Entamoeba dispar in the northern Philippines as detected by the polymerase chain reaction.

We used the polymerase chain reaction (PCR) to study the distribution of Entamoeba histolytica and E. dispar in 1,872 individuals in 14 communities in the northern Philippines. Here we report a field study using a DNA extraction protocol from formalin-fixed stool specimens as previously reported. This assay detected 137 stools (7.318%) containing E. dispar and 18 stools (0.961%) containing E. histolytica. The most affected age group for E. histolytica/E. dispar infections were those 5-14 years of age. There was no significant difference in the sex distribution of E. histolytica, while in the case of E. dispar, a higher prevalence was observed in females (9.186%) than in males (5.731%) (P < 0.01). An apparent clustering of stool-positive cases of E. histolytica and E. dispar was also observed in the northern part of the study area. The results of this survey demonstrate that E. dispar is highly prevalent in the communities studied. Moreover, it offers promise for the PCR using DNA extracted from formalin-fixed stools as a sensitive epidemiologic tool for detecting E. histolytica and E. dispar infections.

Adolescent↗
PubMed2001

Evaluation of laboratory techniques for differentiation between Entamoeba histolytica and Entamoeba dispar.

Incorporation of clinical, epidemiological and serological parameters to the studies of Entamoeba histolytica resulted in the conclusion that species complex comprising two morphologically identical amoeba were implicated with the disease. The two organisms are E. histolytica and E. dispar. The former is a pathogen and responsible for invasive amoebiasis while the later is a gut commensal. Clearly accurate diagnostic tools are required to distinguish the two species. The use of a stool ELISA has been shown to be useful. A monoclonal enzyme linked immunosorbent assay based kit to detect antigen of E.histolytica only in stool was evaluated in comparison to the results of the microscopical examination of the stools and that of enzyme linked immunosorbent assay used to detect the anti- E. histolytica IgG in serum. This study demonstrated that E. dispar is prevalent in the community and offers promise for E. histolytica monoclonal enzyme immunoassay for the qualitative and semiquantitative determination of sensu lato antigen in stool as a sensitive tool for detection and distinction of E. histolytica from E. dispar infections.

Animals↗
PubMed2005

Entamoeba histolytica or Entamoeba dispar among children in Gaza, Gaza Strip?

Most physicians in Gaza prescribe medicaments for patient's suffering from Entamoeba histolytica/dispar without parasitologic diagnosis. Additionally, stool analysis performed by the routine methods usually reports the species as E. histolytica without con-firmation. In this study, 92 stool specimens were collected and analyzed by wet mount, iron haematoxylin staining, antigen detection of E. histolytica and polymerase chain reaction (PCR). The total number of E. histolytica identified by PCR was 64 (69.6%) that of E. dispar was 21 (22.8%). Mixed infection with both E. histolytica and E. dispar was evident in 7 specimens (7.6%). In the light of these results approximately 30% of suspected clinical amoebiasis cases were negative for E. histolytica. It is recommended to use PCR for diagnosis of stool specimens from patients with E. histolytica/dispar and that treatment should be prescribed for only patients positive for E.

Animals↗
PubMed2005

Entamoeba moshkovskii and Entamoeba dispar-associated infections in pondicherry, India.

The prevalence of Laredo strain--Entamoeba moshkovskii--and non-pathogenic E. dispar in patients attending the Jawaharlal Institute of Postgraduate Medical Education and Research hospital, Pondicherry, India, is reported here. E. moshkovskii is reported for the first time in India. The species are morphologically indistinguishable from pathogenic E. histolytica. Of 746 stool samples screened, 68 showing cyst or trophozoite stage of E. histolytica, E. dispar, or E. moshkovskii were subjected to small subunit (SSU) rRNA gene-based polymerase chain reaction, which revealed a higher prevalence of E. dispar (8.8%) and E. moshkovskii (2.2%) compared to E. histolytica (1.7%) in patients. Only 19% of the 68 stool samples, resembling E. histolytica by microscopy, were actually E. histolytica, implying that 81% of suspected infections were misdiagnosed and would have been treated unnecessarily with anti-amoebic drugs.

Animals↗
PubMed2004

Comparative evaluation of hexokinase isoenzyme typing and antigen detection for differentiation of Entamoeba histolytica and Entamoeba dispar.

Entamoeba histolytica, the causative organism of invasive amebiasis is a potential pathogen, while asymptomatic infection is caused by E. dispar. Differentiation of the species is not possible on the basis of morphological characters by microscopic examination. In the present study an attempt has been made to differentiate E. histolytica from E. dispar in 45 isolates obtained from culture and direct stool samples respectively on the basis of hexokinase isoenzyme analysis and Tech Lab ELISA. A 100% correlation was found between these two techniques. However, Tech Lab E. histolytica antigen detection test was found to be both rapid and technically simple. Its use in diagnosis and epidemiological studies is recommended.

Animals↗
PubMed2006

Use of molecular tools to distinguish Entamoeba histolytica and Entamoeba dispar infection among the aborigines in Cameron Highlands.

Amoebiasis is an infectious diseased caused by parasitic one-celled protozoan called Entamoeba histolytica. Numerous protozoa also can inhabit the gastro-intestinal tract of human. Majority of these protozoa are non-pathogenic commensals or only causes disease under certain circumstances. Morphologically, E. histolytica, the invasive form, share the same characteristic with the nonpathogenic form, E. dispar. Both strains can be distinguished by using DNA identification. Many previous researches in Malaysia only reported infection with E. histolytica infection. Therefore in this study we tried to classify infection among the aborigines in Cameron Highland as true E. histolytica or E. dispar by Nested Polymerase Chain Reaction (Nested PCR) and Restriction enzyme (RE) digestion. Results showed that 31 samples were positive by microscopic examination, however of these 28 (13.2%) samples were positive for E. histolytica and 12 (5.6%) samples were positive for E. dispar by molecular tools.

Adolescent↗
PubMed2005

Entamoeba histolytica and Entamoeba dispar infections in cynomolgus monkeys imported into Japan for research.

Three hundred and three stool samples of cynomolgus monkeys (Macaca fascicularis) imported from China and the Philippines were examined for Entamoeba histolytica/Entamoeba dispar infections. Microscopy detected E. histolytica/E. dispar cysts in 41 samples. Positive rates were higher in the monkeys from China (37.5%) than in the monkeys from the Philippines (3.7%). PCR analysis of 25 samples successfully cultured from the cysts demonstrated that 24 were E. dispar, one of the samples from China was E. histolytica. The one sample was also identified as E. histolytica by an antigen detection kit, although the monkey was asymptomatic and serology was negative. To our knowledge, this is the first report of E. histolytica isolation from cynomolgus monkeys based on the discrimination between E. histolytica and E. dispar.

Animals↗
PubMed2000

Asymptomatic cyst passers of Entamoeba histolytica but not Entamoeba dispar in institutions for the mentally retarded in Japan.

Entamoeba histolytica/Entamoeba dispar was isolated from 50 asymptomatic amebic cyst passers in three institutions for the mentally retarded in Kanagawa Prefecture, Japan. To distinguish between E. histolytica and E. dispar, the isolates were analyzed by PCR, reactivity to monoclonal antibodies, and zymodemes. All isolates were identified as E. histolytica. The results lead us to conceive that, in Japan, E. histolytica is predominant even in asymptomatic cyst passers.

Animals↗