title: Realtime PCR is more sensitive than multiplex PCR for diagnosis and serotyping in children with culture negative pneumococcal invasive disease.
abstract: BACKGROUND
  Pneumococcal serotyping is usually performed by Quellung reaction, considered the gold standard test. However the method cannot be used on culture-negative samples. Molecular methods can be a useful alternative. The aim of the study was to evaluate the use of Multiplex-sequential-PCR (MS-PCR) or Realtime-PCR on blood samples for diagnosis and serotyping of invasive pneumococcal disease (IPD) in a pediatric clinical setting.
  
  
  RESULTS
  Sensitivity and specificity of MS-PCR and Realtime-PCR have been evaluated both on 46 well characterized pneumococcal isolates and on 67 clinical samples from children with culture-negative IPD. No difference in sensitivity and specificity between MS-PCR and Realtime PCR was found when the methods were used on isolates: both methods could type 100% isolates and the results were always consistent with culture-based methods. On the contrary, when used on clinical samples 43/67 (64.2%) were typeable by MS-PCR and 61/67 (91.0%) by Realtime-PCR (p = 0.0004,K Cohen 0.3, McNemar's p<0.001). Non-typeability by MS-PCR was associated in 18/20 cases (90.0%) with low bacterial load. The difference between the two methods was present both when they were used on normally sterile fluids (respectively 31/33 (93.9%) typeable samples for Realtime-PCR and 24/33 (72.7%) for MS-PCR, p = 0.047, 95%CL 0.03-0.98; K Cohen 0.3; McNemar's p = 0.0016) and when they were used on nasopharyngeal swabs (respectively 30/34 (88.2%) typeable samples for Realtime-PCR and 19/34 (55.9%) for MS-PCR, p = 0.007, 95%CL 0.04-0.66); the presence of multiple pneumococcal serotypes in nasopharyngeal swabs was found more frequently by Realtime PCR (19/30; 63.3%) than by Multiplex-sequential PCR (3/19; 15.8%; p = 0.003;95%CL 1.87-39.97).
  
  
  CONCLUSIONS
  In conclusion, both MS-PCR and Realtime PCR can be used for pneumococcal serotyping of most serotypes/serogroups directly on clinical samples from culture-negative patients but Realtime-PCR appears more sensitive.
authors.affiliation: Department of Pediatrics, Anna Meyer Children's Hospital and University of Florence, Florence, Italy. c.azzari@meyer.it
authors.forename: Chiara
authors.initials: C
authors.lastname: Azzari
authors.forename: Maria
authors.initials: M
authors.lastname: Moriondo
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Indolfi
authors.forename: Martina
authors.initials: M
authors.lastname: Cortimiglia
authors.forename: Clementina
authors.initials: C
authors.lastname: Canessa
authors.forename: Laura
authors.initials: L
authors.lastname: Becciolini
authors.forename: Francesca
authors.initials: F
authors.lastname: Lippi
authors.forename: Maurizio
authors.initials: M
authors.lastname: de Martino
authors.forename: Massimo
authors.initials: M
authors.lastname: Resti
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D002648:Child; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods; D011008:Pneumococcal Infections / Q000175:diagnosis* / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012703:Serotyping / Q000379:methods; D013296:Streptococcus pneumoniae / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; C500439:CpsA protein, Streptococcus pneumoniae; D017931:DNA Primers; D004269:DNA, Bacterial