title: 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: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.forename: K P
authors.initials: KP
authors.lastname: Dinoop
authors.affiliation: Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education & Research, Puducherry, India.
authors.forename: Subhash Chandra
authors.initials: SC
authors.lastname: Parija
authors.forename: Jharna
authors.initials: J
authors.lastname: Mandal
authors.forename: R P
authors.initials: RP
authors.lastname: Swaminathan
authors.forename: P
authors.initials: P
authors.lastname: Narayanan
mesh_terms: D000293:Adolescent; D000328:Adult; D002648:Child; D004748:Entamoeba histolytica / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D005243:Feces / Q000469:parasitology; D005260:Female; D006801:Humans; D007194:India; D008101:Liver Abscess, Amebic / Q000175:diagnosis* / Q000235:genetics* / Q000469:parasitology; D008297:Male; D008875:Middle Aged; D060888:Real-Time Polymerase Chain Reaction; D063128:Tertiary Health Care