Differential identification of Entamoeba spp. based on the analysis of 18S rRNA

Santos, Helena Lúcia Carneiro; Bandea, Rebecca; Martins, Luci Ana Fernandes; Macedo, Heloísa Werneck de; Peralta, Regina Helena Saramago; Peralta, José Mauro; Ndubuisi, MacKevin; Silva, Alexandre J. da

doi:10.1007/s00436-010-1728-y

Cited by 30 publications

(24 citation statements)

References 18 publications

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“…Most molecular diagnostic tools detect only E. histolytica and do not detect other morphologically identical or similar species, giving rise to several questions. A previous study has already demonstrated a situation in which samples that were positive by microscopic examination for the E. histolytica / E. dispar complex were identified as E. hartmanni after analysis by two different molecular methods [20]. To avoid this potential confusion and provide complete epidemiologic data on parasitic infections, it is important to develop a reliable system for the identification and differentiation of pathogenic and nonpathogenic parasites.…”

Section: Discussionmentioning

confidence: 99%

“…For E. hartmanni , however, a cloning procedure was necessary to obtain a positive control because a protozoan culture of E. hartmanni was not available. For this process, positive DNA samples characterized in a previous study were used [20]. The specific E. hartmanni sequence was amplified with primers EhartF and EhartR2; this fragment was then cloned using the pCR 2.1-TOPO vector as described in the protocol with the pCR 2.1-TOPO TA Cloning Kit (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

“…Previously, data determined by microscopic examination have demonstrated a prevalence of E. hartmanni of 13.3% in HIV patients and 2.5% in children in Brazilian groups [18, 19]. Identification of Entamoeba species other than the complex using molecular techniques has been conducted in only a single study in Brazil, in which the evaluation of Brazilian clinical samples previously identified as positive for the complex by microscopic examination demonstrated the presence of E. hartmanni DNA in more than one sample, while E. moshkovskii DNA was not detected among the samples analyzed [20]. These results showed the presence of E. hartmanni in Brazil but yielded no data about E. moshkovskii .…”

Section: Introductionmentioning

confidence: 99%

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Differential Diagnosis ofEntamoebaspp. in Clinical Stool Samples Using SYBR Green Real-Time Polymerase Chain Reaction

Gomes

Garcia

Cunha

et al. 2014

The Scientific World Journal

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Amoebiasis, a disease caused by Entamoeba histolytica, is usually diagnosed by microscopic examination, which does not differentiate the morphologically identical species of the E. histolytica/E. dispar complex. Furthermore, morphologically similar species such as Entamoeba hartmanni contribute to misidentification. Therefore, there is a need for more sensitive and specific methods. This study standardized a multiplex real-time PCR system for E. histolytica and E. dispar and a single real-time PCR for E. hartmanni. The multiplex protocol detected up to 0.0143 pg of E. histolytica DNA and 0.5156 pg of E. dispar DNA, and the average melting temperature (T m) was 73°C and 70°C, respectively. For E. hartmanni, the T m was 73°C and the amplification was successful down to 0.03 fg of plasmid DNA. Negative controls and other intestinal parasites presented no amplification. Among the 48 samples tested, E. dispar DNA was detected in 37; none exhibited E. histolytica DNA and 11 were negative in the multiplex protocol. In 4 of these 11 samples, however, E. hartmanni DNA was amplified. SYBR Green is demonstrated to be an interesting option and these combined PCR reactions can improve laboratory diagnosis of amoebiasis in developing countries.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Differential Diagnosis ofEntamoebaspp. in Clinical Stool Samples Using SYBR Green Real-Time Polymerase Chain Reaction

Gomes

Garcia

Cunha

et al. 2014

The Scientific World Journal

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“…Among these, E. histolytica is the only pathogenic species (201,236,351). Improvements in the understanding of the biochemical, immunological, and genetic differences of the members of this genus have resulted in the confirmation of three species, E. histolytica, E. dispar, and E. moshkovskii, that are morphologically identical in both their cyst and trophozoite stages (41,169).…”

Section: Entamoeba Speciesmentioning

confidence: 99%

Enteric Protozoa in the Developed World: a Public Health Perspective

et al. 2012

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SUMMARY Several enteric protozoa cause severe morbidity and mortality in both humans and animals worldwide. In developed settings, enteric protozoa are often ignored as a cause of diarrheal illness due to better hygiene conditions, and as such, very little effort is used toward laboratory diagnosis. Although these protozoa contribute to the high burden of infectious diseases, estimates of their true prevalence are sometimes affected by the lack of sensitive diagnostic techniques to detect them in clinical and environmental specimens. Despite recent advances in the epidemiology, molecular biology, and treatment of protozoan illnesses, gaps in knowledge still exist, requiring further research. There is evidence that climate-related changes will contribute to their burden due to displacement of ecosystems and human and animal populations, increases in atmospheric temperature, flooding and other environmental conditions suitable for transmission, and the need for the reuse of alternative water sources to meet growing population needs. This review discusses the common enteric protozoa from a public health perspective, highlighting their epidemiology, modes of transmission, prevention, and control. It also discusses the potential impact of climate changes on their epidemiology and the issues surrounding waterborne transmission and suggests a multidisciplinary approach to their prevention and control.

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“…Finally, the identification of Entamoeba spp. was done as previously reported [15]. All PCRs were carried out in a 25 μL volume containing 12.5 μL of PCR master mix 2X (Promega Italia, Milan, Italy), 100-200 ng of template DNA, and 0.6 mM of each primer.…”

Section: Fecal Dna Extractionmentioning

confidence: 99%

Gut microbiota related to Giardia duodenalis, Entamoeba spp. and Blastocystis hominis infections in humans from Côte d’Ivoire

Iebba

Santangelo

Totino

et al. 2016

J Infect Dev Ctries

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Introduction: Literature data provide little information about protozoa infections and gut microbiota compositional shifts in humans. This preliminary study aimed to describe the fecal bacterial community composition of people from Côte d'Ivoire harboring Giardia duodenalis, Entamoeba spp., and Blastocystis hominis, in trying to discover possible alterations in their fecal microbiota structure related to the presence of such parasites. Methodology: Twenty fecal samples were collected from people inhabiting three different localities of Côte d'Ivoire for copromicroscopic analysis and molecular identification of G. duodenalis, Entamoeba spp., and B. hominis. Temporal temperature gradient gel electrophoresis (TTGE) was used to obtain a fingerprint of the overall bacterial community; quantitative polymerase chain reaction (qPCR) was used to define the relative abundances of selected bacterial species/group, and multivariate statistical analyses were employed to correlate all data. Results: Cluster analysis revealed a significant separation of TTGE profiles into four clusters (p < 0.0001), with a marked difference for G. duodenalis-positive samples in relation to the others (p = 5.4×10 -6 ). Interestingly, qPCR data showed how G. duodenalis-positive samples were related to a dysbiotic condition that favors potentially harmful species (such as Escherichia coli), while Entamoeba spp./B. hominis-positive subjects were linked to a eubiotic condition, as shown by a significantly higher Faecalibacterium prausnitzii-Escherichia coli ratio. Conclusions: This preliminary investigation demonstrates a differential fecal microbiota structure in subjects infected with G. duodenalis or Entamoeba spp./B. hominis, paving the way for using further next-generation DNA technologies to better understand host-parasite-bacteria interactions, aimed at identifying potential indicators of microbiota changes.

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Differential identification of Entamoeba spp. based on the analysis of 18S rRNA

Cited by 30 publications

References 18 publications

Differential Diagnosis ofEntamoebaspp. in Clinical Stool Samples Using SYBR Green Real-Time Polymerase Chain Reaction

Differential Diagnosis ofEntamoebaspp. in Clinical Stool Samples Using SYBR Green Real-Time Polymerase Chain Reaction

Enteric Protozoa in the Developed World: a Public Health Perspective

Gut microbiota related to Giardia duodenalis, Entamoeba spp. and Blastocystis hominis infections in humans from Côte d’Ivoire

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