title: The performance of four molecular methods for the laboratory diagnosis of congenital toxoplasmosis in amniotic fluid samples.
abstract: BACKGROUND
  Toxoplasmosis may be life-threatening in fetuses and in immune-deficient patients. Conventional laboratory diagnosis of toxoplasmosis is based on the presence of IgM and IgG anti-Toxoplasma gondii antibodies; however, molecular techniques have emerged as alternative tools due to their increased sensitivity. The aim of this study was to compare the performance of 4 PCR-based methods for the laboratory diagnosis of toxoplasmosis. One hundred pregnant women who seroconverted during pregnancy were included in the study. The definition of cases was based on a 12-month follow-up of the infants.
  
  
  METHODS
  Amniotic fluid samples were submitted to DNA extraction and amplification by the following 4 Toxoplasma techniques performed with parasite B1 gene primers: conventional PCR, nested-PCR, multiplex-nested-PCR, and real-time PCR. Seven parameters were analyzed, sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (PLR), negative likelihood ratio (NLR) and efficiency (Ef).
  
  
  RESULTS
  Fifty-nine of the 100 infants had toxoplasmosis; 42 (71.2%) had IgM antibodies at birth but were asymptomatic, and the remaining 17 cases had non-detectable IgM antibodies but high IgG antibody titers that were associated with retinochoroiditis in 8 (13.5%) cases, abnormal cranial ultrasound in 5 (8.5%) cases, and signs/symptoms suggestive of infection in 4 (6.8%) cases. The conventional PCR assay detected 50 cases (9 false-negatives), nested-PCR detected 58 cases (1 false-negative and 4 false-positives), multiplex-nested-PCR detected 57 cases (2 false-negatives), and real-time-PCR detected 58 cases (1 false-negative).
  
  
  CONCLUSIONS
  The real-time PCR assay was the best-performing technique based on the parameters of Se (98.3%), Sp (100%), PPV (100%), NPV (97.6%), PLR (âˆž), NLR (0.017), and Ef (99%).
authors.forename: Leandro Emídio
authors.initials: LE
authors.lastname: Teixeira
authors.forename: Kelly Aparecida
authors.initials: KA
authors.lastname: Kanunfre
authors.forename: Paulo Tadashi
authors.initials: PT
authors.lastname: Shimokawa
authors.forename: Lília Spaleta
authors.initials: LS
authors.lastname: Targa
authors.forename: Jonatas Cristian
authors.initials: JC
authors.lastname: Rodrigues
authors.forename: Wilson
authors.initials: W
authors.lastname: Domingues
authors.forename: Lidia
authors.initials: L
authors.lastname: Yamamoto
authors.forename: Thelma Suely
authors.initials: TS
authors.lastname: Okay
mesh_terms: D000653:Amniotic Fluid / Q000737:chemistry / Q000469:parasitology*; D000913:Antibodies, Protozoan / Q000032:analysis; D017931:DNA Primers; D016054:DNA, Protozoan / Q000032:analysis; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis; D007075:Immunoglobulin M / Q000032:analysis; D007231:Infant, Newborn; D016133:Polymerase Chain Reaction / Q000379:methods; D011237:Predictive Value of Tests; D011247:Pregnancy; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014122:Toxoplasma / Q000235:genetics / Q000276:immunology; D014125:Toxoplasmosis, Congenital / Q000175:diagnosis*
chemical_list: D000913:Antibodies, Protozoan; D017931:DNA Primers; D016054:DNA, Protozoan; D007074:Immunoglobulin G; D007075:Immunoglobulin M