realtime pcr is more sensitive than multiplex pcr for diagnosis and serotyping in children with culture negative pneumococcal invasive disease实时聚合酶链反应更敏感比多重pcr诊断和血清型的儿童文化-肺炎球菌侵入性疾病.pdfVIP

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realtime pcr is more sensitive than multiplex pcr for diagnosis and serotyping in children with culture negative pneumococcal invasive disease实时聚合酶链反应更敏感比多重pcr诊断和血清型的儿童文化-肺炎球菌侵入性疾病.pdf

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realtime pcr is more sensitive than multiplex pcr for diagnosis and serotyping in children with culture negative pneumococcal invasive disease实时聚合酶链反应更敏感比多重pcr诊断和血清型的儿童文化-肺炎球菌侵入性疾病

Realtime PCR Is More Sensitive than Multiplex PCR for Diagnosis and Serotyping in Children with Culture Negative Pneumococcal Invasive Disease Chiara Azzari*, Maria Moriondo, Giuseppe Indolfi, Martina Cortimiglia, Clementina Canessa, Laura Becciolini, Francesca Lippi, Maurizio de Martino, Massimo Resti Department of Pediatrics, Anna Meyer Children’s Hospital and University of Florence, Florence, Italy 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. Methodology/Principal Findings: 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 (res