Direct Amplification and Differentiation of Pathogenic and Nonpathogenic Entamoeba histolytica DNA from Stool Specimens

Katzwinkel-Wladarsch, S.; Löscher, Thomas; Rinder, Heinz

doi:10.4269/ajtmh.1994.51.115

Cited by 72 publications

(47 citation statements)

References 3 publications

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“…It was found to be dependent on the particular stool sample, presumably due to differences in the kind andlor the quantity of DNA polymerase inhibitors present. The stool quantity of 0.1 g had previously been shown to be the optimum between a maximum amount of stool and a minimum of inhibition for this method (Katzwinkel-Wladarsch et al 1994 (Vossbrinck et al 1993;Weiss et al 1994;Zhu et al 1994). Gibco in…”

Section: Detection Limitmentioning

confidence: 99%

“…In an adaptation of a method for the isolation of Entamoeba histolytica-DNA from stool specimens (Katzwinkel-Wladarsch et al 1994), 0.1 g of stool was transferred to a I.S-ml microcentrifuge tube; 33.3 p1 of I M KOH and 9.3 p1 of, I M DTT were added and mixed thoroughly by stirring with the pipette tip. After incubation at 65°C for 15 minutes, the samples were neutralized with 4.3 pl of 2 5 % HCI, buffered with 80 p1 of z M Tris-HCI (pH 8.3), and the suspension was mixed again.…”

Section: Dna Isolationmentioning

confidence: 99%

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Direct amplification and species determination of microsporidian DNA from stool specimens

Katzwinkel-Wladarsch

Lieb

Heise

et al. 1996

Tropical Med Int Health

199

121

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SummaryMicrosporidia are recognized as a major aetiological agent in chronic diarrhoea of immunocompromised patients. Their detection by light microscopy is hampered by the small size of the spores. A simple and rapid DNA extraction method has been developed for the detection of microsporidian DNA by PCR directly from stool specimens. It can be performed at room temperature in a I.g-ml microcentrifuge tube format in less than I hour. The subsequent nested polymerase chain reaction permits the detection of 3-100 spores in a 0.1-g stool sample. The amplification products can be verified and the species Enterocytozoon bieneusi, Encephalitozoon cuniculi and Encephalitozoon (Septata) intestinalis distinguished by a simple restriction endonuclease digest.keywords microsporidia, DNA, PCR

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Section: Detection Limitmentioning

confidence: 99%

Section: Dna Isolationmentioning

confidence: 99%

Direct amplification and species determination of microsporidian DNA from stool specimens

Katzwinkel-Wladarsch

Lieb

Heise

et al. 1996

Tropical Med Int Health

199

121

View full text Add to dashboard Cite

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“…Amplification of ameba DNA fragments by PCR has been proven to constitute a sensitive and specific method to detect E. histolytica or E. dispar from human feces (1,5,14,21,30). The PCR protocols reported so far, however, require further processing of the amplicon, which is time-consuming and prone to false-positive results due to possible cross-contamination.…”

mentioning

confidence: 99%

“…However, the recent identification of Entamoeba dispar as a separate but nonpathogenic species which is morphologically indistinguishable from E. histolytica and does not require treatment has indicated the need of alternative detection methods which are able to differentiate between the two organisms (8). A number of assays have been developed during recent years, such as protein and DNA detection systems, which are able to distinguish E. histolytica from E. dispar (1,2,5,7,10,14,18,21,22,24,28,29,30). However, most of these assays are not suitable for a rapid diagnosis directly from stool samples, in particular when large numbers of samples have to be processed.…”

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confidence: 99%

Real-Time PCR for Detection and Differentiation ofEntamoeba histolyticaandEntamoeba disparin Fecal Samples

et al. 2002

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A closed-tube, real-time PCR assay was developed for sensitive and specific detection and differentiation of the two closely related intestinal protozoan parasites Entamoeba histolytica and Entamoeba dispar directly from human feces. The assay is performed with the LightCycler system using fluorescence-labeled detection probes and primers amplifying a 310-bp fragment from the high-copy-number, ribosomal DNA-containing ameba episome. The assay was able to detect as little as 0.1 parasite per g of feces. The two pairs of primers used were specific for the respective ameba species, and results were not influenced by the presence of other Entamoeba species even when present in exceeding amounts. PCR was evaluated using several hundred stool samples from areas of amebiasis endemicity in Vietnam and South Africa, and results were compared with those of microscopy and ameba culture. PCR was found to be significantly more sensitive than microscopy or culture, as all samples positive by microscopy and 22 out of 25 (88%) samples positive by culture were also positive by PCR, but PCR revealed a considerable number of additional E. histolytica-or E. dispar-positive samples. Compared to culture and subsequent ameba differentiation by isoenzyme analysis, PCR was 100% specific for each of the two Entamoeba species. Interestingly, the comparison with PCR revealed that culture, in particular, underestimates E. histolytica infections. Given the high sensitivity and specificity of the developed PCR assay, the inability of microscopy to distinguish between the two ameba species, and the time it takes to culture and subsequently differentiate entamoebae by isoenzyme analysis, this assay is more suitable than microscopy or culture to correctly diagnose intestinal E. histolytica or E. dispar infection.

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“…An antigen detection kit for the specific identification of E. dispar and E. moshkovskii is yet to be developed. Several PCR-based techniques that amplify and detect E. histolytica DNA are currently used for the clinical and epidemiological studies in non-endemic rich countries (Acuna-Soto et al, 1993;Katzwinkel-Wladarsch et al, 1994;Calderaro et al, 2006;Hamzah et al, 2006). The sensitivity and specificity of PCR-based methods for the diagnosis of E. histolytica infection approach those of stool culture followed by isoenzyme analysis.…”

Section: Diagnosis Of Amoebiasismentioning

confidence: 99%

Amoebiasis in the Tropics: Epidemiology and Pathogenesis

Samie¹,

ElBakri²,

AbuOdeh³

2012

Current Topics in Tropical Medicine

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Direct Amplification and Differentiation of Pathogenic and Nonpathogenic Entamoeba histolytica DNA from Stool Specimens

Cited by 72 publications

References 3 publications

Direct amplification and species determination of microsporidian DNA from stool specimens

Direct amplification and species determination of microsporidian DNA from stool specimens

Real-Time PCR for Detection and Differentiation ofEntamoeba histolyticaandEntamoeba disparin Fecal Samples

Amoebiasis in the Tropics: Epidemiology and Pathogenesis

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