Dataset Information

Comparison of nested-multiplex, Taqman & SYBR Green real-time PCR in diagnosis of amoebic liver abscess in a tertiary health care institute in India.

ABSTRACT: Amoebiasis is a common parasitic infection caused by Entamoeba histolytica and amoebic liver abscess (ALA) is the most common extraintestinal manifestation of amoebiasis. The aim of this study was to standardise real-time PCR assays (Taqman and SYBR Green) to detect E. histolytica from liver abscess pus and stool samples and compare its results with nested-multiplex PCR.Liver abscess pus specimens were subjected to DNA extraction. The extracted DNA samples were subjected to amplification by nested-multiplex PCR, Taqman (18S rRNA) and SYBR Green real-time PCR (16S-like rRNA assays to detect E. histolytica/E. dispar/E. moshkovskii). The amplification products were further confirmed by DNA sequence analysis. Receiver operator characteristic (ROC) curve analysis was done for nested-multiplex and SYBR Green real-time PCR and the area under the curve was calculated for evaluating the accuracy of the tests to dignose ALA.In all, 17, 19 and 25 liver abscess samples were positive for E. histolytica by nested-multiplex PCR, SYBR Green and Taqman real-time PCR assays, respectively. Significant differences in detection of E. histolytica were noted in the real-time PCR assays evaluated ( P<0.0001). The nested-multiplex PCR, SYBR Green real-time PCR and Taqman real-time PCR evaluated showed a positivity rate of 34, 38 and 50 per cent, respectively. Based on ROC curve analysis (considering Taqman real-time PCR as the gold standard), it was observed that SYBR Green real-time PCR was better than conventional nested-multiplex PCR for the diagnosis of ALA.Taqman real-time PCR targeting the 18S rRNA had the highest positivity rate evaluated in this study. Both nested multiplex and SYBR Green real-time PCR assays utilized were evaluated to give accurate results. Real-time PCR assays can be used as the gold standard in rapid and reliable diagnosis, and appropriate management of amoebiasis, replacing the conventional molecular methods.

SUBMITTER: Dinoop KP

PROVIDER: S-EPMC4822369 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Comparison of nested-multiplex, Taqman & SYBR Green real-time PCR in diagnosis of amoebic liver abscess in a tertiary health care institute in India.

Dinoop K P KP Parija Subhash Chandra SC Mandal Jharna J Swaminathan R P RP Narayanan P P

The Indian journal of medical research 20160101 1

<h4>Background & objectives</h4>Amoebiasis is a common parasitic infection caused by Entamoeba histolytica and amoebic liver abscess (ALA) is the most common extraintestinal manifestation of amoebiasis. The aim of this study was to standardise real-time PCR assays (Taqman and SYBR Green) to detect E. histolytica from liver abscess pus and stool samples and compare its results with nested-multiplex PCR.<h4>Methods</h4>Liver abscess pus specimens were subjected to DNA extraction. The extracted DNA ...[more]

PMID: 26997014

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Project description:BackgroundInfection with Entamoeba histolytica and associated complications are relatively rare in developed countries. The overall low prevalence in the Western world as well as the possibly prolonged latency period between infection with the causing pathogen and onset of clinical symptoms may delay diagnosis of and adequate treatment for amoebiasis. Amoebic liver abscess (ALA) is the most common extraintestinal manifestation of invasive amoebiasis. Pregnancy has been described as a risk factor for development of invasive amoebiasis and management of these patients is especially complex.Case presentationA 30-year-old Caucasian woman in early pregnancy presented to our emergency department with abdominal pain alongside elevated inflammatory markers and liver function tests. Travel history revealed multiple journeys to tropic and subtropic regions during the past decade and a prolonged episode of intermittently bloody diarrhea during a five month stay in Indonesia seven years prior to admission. Sonographic and magnetic resonance imaging revealed a 5?×?4 cm hepatic abscess. After ultrasound-guided transcutaneous liver drainage, both abscess fluids and blood cultures showed neither bacterial growth nor microscopic signs of parasitic disease. Serological testing confirmed an infection with Entamoeba histolytica, which was treated with metronidazole, followed by eradication therapy with paromomycin. Subsequent clinical, laboratory and imaging follow-up exams showed regression of the ALA. In addition, the pregnancy completed without complications and a healthy baby boy was born 7 months after termination of treatment.ConclusionsThis case of invasive amoebiasis in early pregnancy outside of endemic regions and several years after exposure demonstrates the importance of broad differential diagnostics in the context of liver abscesses. The complex interdisciplinary decisions regarding the choice of imaging techniques as well as interventional and antibiotic therapy in the context of pregnancy are discussed. Furthermore, we present possible explanations for pregnancy as a risk factor for an invasive course of amoebiasis.

Project description:The nodule-forming actinobacterial genus Frankia can generally be divided into 4 taxonomic clusters, with clusters 1, 2, and 3 representing nitrogen-fixing strains of different host infection groups and cluster 4 representing atypical, generally non-nitrogen-fixing strains. Recently, quantitative PCR (qPCR)-based quantification methods have been developed for frankiae of clusters 1 and 3; however, similar approaches for clusters 2 and 4 were missing. We amended a database of partial 23S rRNA gene sequences of Frankia strains belonging to clusters 1 and 3 with sequences of frankiae representing clusters 2 and 4. The alignment allowed us to design primers and probes for the specific detection and quantification of these Frankia clusters by either Sybr Green- or TaqMan-based qPCR. Analyses of frankiae in different soils, all obtained from the same region in Illinois, USA, provided similar results, independent of the qPCR method applied, with abundance estimates of 10 × 105 to 15 × 105 cells (g soil)-1 depending on the soil. Diversity was higher in prairie soils (native, restored, and cultivated), with frankiae of all 4 clusters detected and those of cluster 4 dominating, while diversity in soils under Alnus glutinosa, a host plant for cluster 1 frankiae, or Betula nigra, a related nonhost plant, was restricted to cluster 1 and 3 frankiae and generally members of subgroup 1b were dominating. These results indicate that vegetation affects the basic composition of frankiae in soils, with higher diversity in prairie soils compared to much more restricted diversity under some host and nonhost trees.IMPORTANCE Root nodule formation by the actinobacterium Frankia is host plant specific and largely, but not exclusively, correlates with assignments of strains to specific clusters within the genus. Due to the lack of adequate detection and quantification tools, studies on Frankia have been limited to clusters 1 and 3 and generally excluded clusters 2 and 4. We have developed tools for the detection and quantification of clusters 2 and 4, which can now be used in combination with those developed for clusters 1 and 3 to retrieve information on the ecology of all clusters delineated within the genus Frankia Our initial results indicate that vegetation affects the basic composition of frankiae in soils, with higher diversity in prairie soils compared to much more restricted diversity under some host and nonhost trees.

Dataset Information

Comparison of nested-multiplex, Taqman & SYBR Green real-time PCR in diagnosis of amoebic liver abscess in a tertiary health care institute in India.

Publications

Comparison of nested-multiplex, Taqman & SYBR Green real-time PCR in diagnosis of amoebic liver abscess in a tertiary health care institute in India.

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