Multiplex polymerase chain reaction amplification and differentiation of Entamoeba histolytica and Entamoeba dispar DNA from stool samples.

Núnez, Yury O.; Fernandez, Maria Aparecida; Torres-Nunez, Diamela; Silva, José A.; Montano, Ivón; Maestre, Jorge; Fonte, Luis

doi:10.4269/ajtmh.2001.64.293

Cited by 44 publications

(13 citation statements)

References 37 publications

(38 reference statements)

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“…Based on these results, PCR amplification was carried out on all 111 DNA samples at loci 1-2, 3-4, and 9-4 and at half locus [16][17][18][19]. In the final analysis, DNA from 11 of the 111 samples failed to amplify at any of these four loci.…”

Section: Resultsmentioning

confidence: 99%

“…Species-specific primers that amplify regions of several different genes have been used (1,7,14,17,18,19). Using trophozoites in culture, comparisons showed that PCR is more sensitive and specific than the enzyme-linked immunosorbent assay-based stool antigen detection kits, which employ monoclonal antibodies for the detection and differentiation of E. histolytica and E. dispar (16).…”

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confidence: 99%

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Simultaneous Differentiation and Typing of Entamoeba histolytica and Entamoeba dispar

et al. 2002

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Sequences corresponding to some of the polymorphic loci previously reported from Entamoeba histolytica have been detected in Entamoeba dispar. Comparison of nucleotide sequences of two loci between E. dispar strain SAW760 and E. histolytica strain HM-1:IMSS revealed significant differences in both repeat and flanking regions. The tandem repeat units varied not only in sequence but also in number and arrangement between the two species at both the loci. Using the sequences obtained, primer pairs aimed at amplifying species-specific products were designed and tested on a variety of E. histolytica and E. dispar samples. Amplification results were in complete agreement with the original species classification in all cases, and the PCR products displayed discernible size and pattern variations among the isolates.The realization that Entamoeba histolytica and Entamoeba dispar are two distinct but morphologically identical species (8) has had a major impact on all aspects of amebiasis research, most notably epidemiology (2, 3). The 10% prevalence estimate made by Walsh (21), though still widely quoted, was based on data collected before the formal redescription of the species and so does not distinguish between infections with E. histolytica, which can cause invasive intestinal and extraintestinal disease, and those with E. dispar, which do not. It is now known that even in areas where invasive amebiasis is common E. dispar is by far the more prevalent species (11).Tools that allowed accurate differentiation of the two species were clearly needed, and in the past decade differentiation based on DNA amplification has been a research focus of many groups. Species-specific primers that amplify regions of several different genes have been used (1,7,14,17,18,19). Using trophozoites in culture, comparisons showed that PCR is more sensitive and specific than the enzyme-linked immunosorbent assay-based stool antigen detection kits, which employ monoclonal antibodies for the detection and differentiation of E. histolytica and E. dispar (16). A significant percentage of individuals in areas of high endemicity could be simultaneously infected with both E. histolytica and E. dispar (16). However, the major drawback of using culture is that mixed infections are overlooked and were only detected after PCR was used on DNA extracted directly from stool (12). Field studies that compared PCR and antigen detection methods directly on stool samples suggest that these methods perform equally well (13).But is E. dispar really nonpathogenic, and should it on this basis be completely dismissed as a subject for further investigation? It has been shown to be capable of producing variable focal intestinal lesions in animals (4, 9, 20) and of destroying epithelial cell monolayers in vitro (10). There is also some evidence that pathological changes may occur in some humans (15), though invasive lesions and symptomatic infections have to date not been reported. Whether these characteristics are variable among strains is unknown. None of the above s...

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Simultaneous Differentiation and Typing of Entamoeba histolytica and Entamoeba dispar

et al. 2002

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“…Several PCR assays designed to differentiate E. histolytica for E. dispar have been described [5,6,21,[22][23][24][25]32,33]. Most of them targeted either the small subunit ribosomal RNA gene or specific episomal repeats species.…”

Section: Discussionmentioning

confidence: 99%

“…In reference laboratories, PCR is the method of choice for differentiation between the pathogenic specie (E. histolytica) from the non-pathogenic (E. dispar). Many investigations have reported successful application of PCR to the diagnosis of amoebiasis as a tool for final confirmatory identification of intestinal amoebiasis [5,6,[20][21][22][23][24][25][26].…”

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Comparison of multiplex-PCR and antigen detection for differential diagnosis of Entamoeba histolytica

Santos¹,

Peralta²,

Macedo³

et al. 2007

Braz J Infect Dis

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Amebiasis is an infection caused by Entamoeba histolytica. However, differentiation between E. histolytica and Entamoeba dispar, which are morphologically identical species, is essential for treatment decision, precaution of the invasive disease and public health. The purpose of the present study was to evaluate a Multiplex -PCR for detection and differentiation of E. histolytica from E. dispar from fresh stool samples in comparison with the coproantigen commercial ELISA. Microscopic examination of stools using the Coprotest method, detection of stool antigen by enzyme-linked immunosorbent assay kit and a home made Multiplex-PCR, were used for the diagnosis of amoebiasis infection. Analysis of the 127 stools samples by microscopy examination demonstrated that only 27 (21%) samples were positive for E. histolytica/E. dispar complex. Among these stool samples, 11 were positive by Multiplex-PCR, with nine presenting the diagnostic fragment characteristic of E. dispar (96 bp) and two presenting diagnostic fragment of E. histolytica (132 bp). Among negative samples detected by microscopic examination, three positive samples for E. dispar and one positive for E. histolytica by Multiplex-PCR was observed. This denotes a low sensibility of microscopic examination when a single stool sample is analyzed. Assay for detection of E. histolytica antigen was concordant with multiplex-PCR in relation to E. histolytica. Statistical analysis comparing the sensibility tests was not done because of the low number of E. histolytica cases. The results demonstrate the importance of the specific techniques use for the differentiation between E. histolytica and E. dispar.

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“…No reduction in the ability to perform PCR amplifications of E. histolytica DNA fixed in 1 to 10% formalin was noted for 7 days (169). Núñez et al (141) described multiplex PCR amplification for the detection and characterization of both E. histolytica and E. dispar in stool samples by using two pairs of specific primers combined in a single reaction mixture. This novel approach had 94% sensitivity and 100% specificity.…”

Section: Molecular Biology-based Diagnostic Tests and Pcrmentioning

confidence: 99%

Laboratory Diagnosis of Amebiasis

2003

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The detection of Entamoeba histolytica, the causative agent of amebiasis, is an important goal of the clinical microbiology laboratory. To assess the scope of E. histolytica infection, it is necessary to utilize accurate diagnostic tools. As more is discovered about the molecular and cell biology of E. histolytica, there is great potential for further understanding the pathogenesis of amebiasis. Molecular biology-based diagnosis may become the technique of choice in the future because establishment of these protozoa in culture is still not a routine clinical laboratory process. In all cases, combination of serologic tests with detection of the parasite (by antigen detection or PCR) offers the best approach to diagnosis, while PCR techniques remain impractical in many developing country settings. The detection of amebic markers in serum in patients with amebic colitis and liver abscess appears promising but is still only a research tool. On the other hand, stool antigen detection tests offer a practical, sensitive, and specific way for the clinical laboratory to detect intestinal E. histolytica. All the current tests suffer from the fact that the antigens detected are denatured by fixation of the stool specimen, limiting testing to fresh or frozen samples

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Multiplex polymerase chain reaction amplification and differentiation of Entamoeba histolytica and Entamoeba dispar DNA from stool samples.

Cited by 44 publications

References 37 publications

Simultaneous Differentiation and Typing of Entamoeba histolytica and Entamoeba dispar

Simultaneous Differentiation and Typing of Entamoeba histolytica and Entamoeba dispar

Comparison of multiplex-PCR and antigen detection for differential diagnosis of Entamoeba histolytica

Laboratory Diagnosis of Amebiasis

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