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Differentiating Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii using nested polymerase chain reaction (PCR) in rural. Background Entamoeba histolytica, E. dispar and E. moshkovskii are the most frequent species described in human infection where E. This study investigated the presence of Entamoeba histolytica, Entamoeba dispar , and Entamoeba moshkovskii in stool samples from a patient population in.

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Entamoeba moshkovskii is prevalent in developing countries and morphologically indistinguishable from pathogenic Entamoeba histolytica and nonpathogenic Entamoeba dispar. It is not known if E. Mice were intracecally challenged with the trophozoites of each Entamoeba spp. In Mirpur, Dhaka, Bangladesh, E. Diarrhea occurred temporally with acquisition of a new E. In children, the acquisition of E.

These data are consistent with E. Entamoeba histolytica causes extensive mortality and morbidity worldwide through diarrheal disease and abscess formation in parenchymal tissues such as liver, lung, and brain. In contrast, other amoebae that infect humans include Entamoeba disparEntamoeba moshkovskii, Entamoeba coli, Entamoeba hartmanni, and Endolimax nanawhich have been considered nonpathogenic commensals of the human gut [ 1—3 ].

Dientamoeba fragilis and Entamoeba polecki have been associated with diarrhea and Entamoeba gingivalis with periodontal entamoebz [ 45 ]. This species mosjkovskii Entamoeba was first entamoeb in sewage in Moscow by Tshalaria in [ 7 ] and was initially thought to be a free-living common protozoan species in anoxic sediments and in environments such as brackish coastal pools. The first human isolate was obtained from a resident of Laredo, Texas, who suffered from diarrhea, weight loss, and epigastric pain moshkvskii [ 8 ].

At first, this isolate was named E. Both the Laredo strain and E. Subsequent molecular studies revealed that E. These studies for the most part tested stool samples submitted to clinical microbiology laboratories from patients with gastrointestinal symptoms, suggesting that E.

Thus, the ability of E. Here we tested the ability of E. In addition, we tested in a longitudinal study of children in Bangladesh not only if E. Animals moshlovskii maintained under specific pathogen-free conditions at Animal Research Center for Tropical Infectious Diseases, Nagasaki University, and were challenged when they were 5—8 weeks old.

Trophozoites of the E. Trophozoites under log phase of growth were used in the experiments. Trophozoites were harvested from culture tubes of E. Then, the trophozites were collected, and the number of trophozoites was determined. We anesthetized mice with domitor medetomidine hydrochloride: Then the cecum was blotted and the peritoneum and the skin were sutured. The study was approved by the animal ethical review board of Nagasaki Entamoeha. The primer sequences used for polymerase chain reaction PCR were described elsewhere [ 22 ].

The study was conducted in Mirpur, an urban slum in Dhaka. Infants were enrolled in the first week after birth and followed until one year of age, beginning in January Field research assistants FRAs visited each study house every other day and collected information related to child morbidity, especially for diarrheal illness, through a structured questionnaire.


If the FRA found any child with an acute illness, then she referred the child to the study clinic for further management by the medical officer. Parents or guardians were also encouraged to visit the study clinic for medical assistance if the study child became sick. FRAs collected nondiarrheal monthly stool specimens as well as diarrheal stool specimens from the home or in the study field clinic.

All stool specimens were transported from the field to the clinic mosbkovskii a cold box. In the field clinic an aliquot of the diarrheal stool specimens was placed into Carry-Blair medium. All specimens were transported from the field clinic to the ICDDR,B Parasitology laboratory within 3 hours of collection, with a cold chain maintained.

A diarrheal episode was defined as being separated from another episode by at least 3 diarrhea-free days. Informed written consent was obtained from the parents or guardians for the participation of their child in the study. Entamoeba histolytica, Cryptosporidium, and Giardia were identified by real-time PCR as described elsewhere [ 24 ].

Multiplex RT- PCR ebtamoeba probe-based detection with Luminex beads for conceivable diarrhea-causative microbes was performed as described elsewhere in the literature [ 25—27 ]. After RT- PCR was performed with the conditions described elsewhere, samples were analyzed on the BioPlex system using bead on which fntamoeba and hybridization were performed according to published protocols [ 28 ]. Trophozoites of either E.

These data demonstrated that in contrast to nonpathogenic E. Furthermore, obvious weight loss was observed during the course of E. Together these data indicated that E. After infection, diarrhea, colitis, and weight loss were monitored.

Normal Aloose Band bloody feces C were observed as was indicated in the results. Amoebae were observed in the lumen of the ceca in successfully infected mice D. Macroscopic and histopathological observations of ceca in mice exhibited bloody diarrhea were shown in panels E and F.

The study was repeated 3 times with similar results.

The time course of each Entamoeba spp. As was reported [ 20 ], E. Time course of each Entamoeba spp.

Entamoeba moshkovskii infections in children, Bangladesh.

The association between diarrheal episodes and infection with each Entamoeba spp. These studies were part of a prospective cohort study on diarrheal diseases [ 29 ]. Newborn children were enrolled in the Mirpur community of Dhaka, Bangladesh, and prospectively followed for diarrheal illness by every other day home visits. A total of diarrheal episodes were recorded during the first 12 months of life in children.

PCR analyses of the diarrheal samples revealed that 66 episodes were positive for E. As such, in diarrheal samples, the detection rates of either E.

Two episodes moshkovskui found to be mixed infections with E. In order to attempt to discern if the E. The preceding 2 months of surveillance stool samples collected when the child did not have diarrhea were tested for the presence of E. This study design therefore temporally controlled for E.


Entamoeba moshkovskii infections in children, Bangladesh.

In the 1 and 2 months preceding E. This supported the hypothesis that temporal acquisition of a new E. The diarrheal severity score was comparable among episodes associated with E. The duration of diarrhea was also comparable among these episodes positive for E. The mean age of the onset of diarrheal episodes associated with E. Thus, moshkovskki diarrhea related to E. As there are many microbes that can potentially induce diarrhea, the presence of other diarrheagenic microbes was tested in the 42 diarrheal samples that were associated with E.

In the 42 diarrheal stool samples with E. The application of these state-of-the-art diagnostic techniques in this cohort has on average identified a minimum of 2 different enteropathogens in every diarrheal modhkovskii sample E.

Taniuchi, personal communication, It was therefore not surprising that the diarrheal episodes associated with E. In order to investigate the genetic diversity in E. However, PCR did not reveal any obvious product size differences among these samples data not shown. Because same size PCR products do not necessarily mean identical DNA sequences, we sequenced PCR products directly without cloning them into any vectors in order to minimize the chances of any sequence selection to detect sequence variation.

Sequencing did reveal that the E. The sequence alignment at locus R-R revealed that the E. The SNPs detected in this study moshovskii distributed randomly across the locus R-R sequences, and as a result, these SNPs could not be used to differentiate asymptomatic and diarrheal strains of E.

However, we noticed from the sequence traces that the 2 asymptomatic strains IDs: The significance of this remains unknown at present. All positions are based on the consensus sequence in the alignment.

This work draws into question the paradigm that E. In the murine model of intestinal amebiasis, E. In this way, E. In children in Bangladesh, the new acquisition of E.

In contrast, the nonpathogenic parasite E. The finding that E. Mouse strain-dependent resistance to E. In humans, one important means of innate resistance of entmoeba epithelial cells to amebiasis is leptin, which acts via STAT3 signaling to protect intestinal epithelial cells from parasite killing [ 3334 ]. In this context, it will be interesting to entqmoeba whether this observation is also true in E.

If the mechanism of resistance to E. Weight loss was also observed during the course of infection, which was more severe in mice moshkovakii with E. The observation that E. However, it is unclear what kinds of differences among Entamoeba spp. Entamoeba histolytica possesses molecules such as pathogen-associated molecular patterns PAMPs on its surface that stimulate proinflammatory cytokines production from antigen-presenting cells [ 35 ].

It will be important in future studies of the potential pathogenicity of E.