title: A 10-year retrospective comparison of two target sequences, REP-529 and B1, for Toxoplasma gondii detection by quantitative PCR.
abstract: This study aimed to evaluate the repeated sequence REP-529 compared to that of the B1 gene in the molecular diagnosis of toxoplasmosis by quantitative PCR (qPCR) in routine diagnosis. Over a 10-year period (2003 to 2013), all patients prospectively diagnosed with a positive REP-529 qPCR result for toxoplasmosis were included. All DNA samples (76 samples from 56 patients) were simultaneously tested using the two qPCR methods (REP-529 and B1). The mean cycle threshold (CT) obtained with the B1 qPCR was significantly higher (+4.71 cycles) than that obtained with REP-529 qPCR (P<0.0001). Thirty-one out of 69 extracts (45.6%) positive with REP-529 qPCR were not amplified with the B1 qPCR (relative sensitivity of 54.4% compared to that with REP-529), yielding false-negative results with 15/28 placenta, 5 cord blood, 2 amniotic fluid, 4 cerebrospinal fluid, 1 aqueous humor, 2 lymph node puncture, and 1 abortion product sample. This defect in sensitivity would have left 20/56 patients undiagnosed, distributed as follows: 12/40 congenital toxoplasmosis, 4/5 cerebral toxoplasmosis, 2/8 patients with retinochoroiditis, and 2 patients with chronic lymphadenopathy. This poor performance of B1 qPCR might be related to low parasite loads, since the mean Toxoplasma quantification in extracts with B1 false-negative results was 0.4 parasite/reaction. These results clearly show the superiority of the REP-529 sequence in the diagnosis of toxoplasmosis by PCR and suggest that this target should be adopted as part of the standardization of the PCR assay.
authors: INSERM U1085, Institut de Recherche en Santé Environnement et Travail, Université Rennes 1, Rennes, France Laboratoire de Parasitologie et Mycologie, Centre Hospitalier Universitaire de Rennes, Rennes, France.
authors: Sorya
authors: S
authors: Belaz
authors: INSERM U1085, Institut de Recherche en Santé Environnement et Travail, Université Rennes 1, Rennes, France Laboratoire de Parasitologie et Mycologie, Centre Hospitalier Universitaire de Rennes, Rennes, France.
authors: Jean-Pierre
authors: JP
authors: Gangneux
authors: Laboratoire de Parasitologie et Mycologie, Centre Hospitalier Universitaire de Rennes, Rennes, France.
authors: Peggy
authors: P
authors: Dupretz
authors: Laboratoire de Parasitologie et Mycologie, Centre Hospitalier Universitaire de Rennes, Rennes, France.
authors: Claude
authors: C
authors: Guiguen
authors: INSERM U1085, Institut de Recherche en Santé Environnement et Travail, Université Rennes 1, Rennes, France Laboratoire de Parasitologie et Mycologie, Centre Hospitalier Universitaire de Rennes, Rennes, France florence.robert-gangneux@univ-rennes1.fr.
authors: Florence
authors: F
authors: Robert-Gangneux
mesh_terms: D000328:Adult; D016054:DNA, Protozoan / Q000235:genetics*; D005260:Female; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D011247:Pregnancy; D011446:Prospective Studies; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012189:Retrospective Studies; D012680:Sensitivity and Specificity; D014122:Toxoplasma / Q000235:genetics* / Q000302:isolation & purification; D014123:Toxoplasmosis / Q000175:diagnosis* / Q000469:parasitology
chemical_list: D016054:DNA, Protozoan
references: Diagn Microbiol Infect Dis. 2011 Oct;71(2):174-6
references: 21856107
references: J Clin Microbiol. 2004 Apr;42(4):1719-22
references: 15071031
references: J Clin Microbiol. 2012 Dec;50(12):3944-51
references: 23035201
references: Diagn Microbiol Infect Dis. 2013 Jun;76(2):244-7
references: 23518182
references: BMC Infect Dis. 2003 May 2;3:7
references: 12729464
references: Lancet. 2004 Jun 12;363(9425):1965-76
references: 15194258
references: J Clin Microbiol. 1989 Aug;27(8):1787-92
references: 2768467
references: Prenat Diagn. 1993 Jun;13(6):543
references: 8372082
references: Presse Med. 1996 Mar 30;25(11):541-5
references: 8731798
references: J Clin Microbiol. 1999 Sep;37(9):2893-8
references: 10449471
references: J Clin Microbiol. 2006 Mar;44(3):720-4
references: 16517845
references: Pediatr Infect Dis J. 2007 Sep;26(9):845-6
references: 17721384
references: J Clin Microbiol. 2008 Jun;46(6):1965-7
references: 18400917
references: Diagn Microbiol Infect Dis. 2009 Jan;63(1):10-5
references: 18990529
references: Clinics (Sao Paulo). 2009;64(3):171-6
references: 19330240
references: Pediatr Infect Dis J. 2010 Jan;29(1):33-8
references: 19858771
references: Rev Soc Bras Med Trop. 2013 Sep-Oct;46(5):584-8
references: 24409481
references: Int J Parasitol. 2000 Jan;30(1):69-75
references: 10675747
references: Obstet Gynecol. 2001 Feb;97(2):296-300
references: 11165598
references: Trans R Soc Trop Med Hyg. 2002 Apr;96 Suppl 1:S205-15
references: 12055840
references: J Clin Microbiol. 2003 Oct;41(10):4826-8
references: 14532233
references: Am J Obstet Gynecol. 2004 Mar;190(3):797-802
references: 15042017
references: Clin Microbiol Rev. 2012 Apr;25(2):264-96
references: 22491772