Application Study Of Target Enriched Multiplex Polymerase Chain Reaction For Pneumonic Bacterial Etiologic Diagnosis In Children

ObjectiveTo approach the clinic value of Target enriched multiplex PCR for the pneumonic bacterial etiologic diagnosis in children, estabalish the rapid,sensitive,specific diagnostic method for common bacterial pathogen, direct etiologic diagnosis and antibiotic rational use, and understand the distribution of bacterial etiology.MethodsFrom Feb 2006 to Jun 2006,188 hospitalized children in Shenzhen children' s hospital, were collected deep tracheal aspirate at the time of hospitalization. The respiratory tract secretions were immediately sent for bacterial culture with 3 kinds of medium:ordinary medium, Hemophilus influenzae selective medium, Streptococcus penumoniae selective medium. Then we extracted the total nucleic acids from secretions, and detected Mycoplasma pneumoniae by single fluorescent quantitation PCR. Simultaneously, 14 respiratory tract pathogenic bacterium and Mycoplasma pneumoniae were detected by Target Enriched multiplex PCR(Tem-PCR). Amplification products were identified by the Luminex100 suspension array. Take the culture and single FQ-PCR as the referenced standard, the sensitivity and specificity of Tem-PCR for the bacteria and Mycoplasma pneumoniae were evaluated. The affect for pneumonic bacterial etiologic distribution was analysised by the combined detection with bacterial culture and Tem-PCR assay.Results(1) Of the 188 respiratory tract specimens, 111 were positive by bacteria culture, including 3 were simultaneously detected 2 kinds of bacterium, the positive strain were : Hemophilus influenzae 24, Escherichia coli 16, Serratia marcescens 14, Klebsiella pneumoniae 12, Hemophilus parainfluenzae 11, Staphylococcus aureus 9, Streptococcus penumoniae 7, Pseudomonas aeruginosa 3, Acinetobacter baumannii 2, Enterobactor cloacae 2, the other bacterium was 9 kinds, 14 strains. The positive rate of culture was 59.1% (111/188) .(2) 93 target gene were detected by Tem-PCR from 75 specimen, they were: Hemophilus influenzae 40, Streptococcus penumoniae 36, Acinetobacter baumannii 10, Pseudomonas aeruginosa 4, Staphylococcus aureus 3, the other 9 kinds of bacterium including Escherichia coli,Klebsiella pneumoniae and Enterobactor cloacae were not detected by Tem-PCR, the positive rate of Tem-PCR was 39.9% (75/188) .(3) For the 14 kinds of bacterium designed by Tem-PCR, compared with the culture, the sensitivity,specificity and coincidence of Tem-PCR is 51.0%, 68.0%, 58.3% respectively. For Streptococcus penumoniae,Hemophilus in0fluenzae,Staphylococcus aureus,Acinetobacter baumannii,Pseudomonas aeruginosa,the sensitivety of Tem-PCR was 100%,41.7 %,11.1%,50.0 %,66.7 % respectively; the specificity was 84.0%,81.7%,98.9%,95.2%,98.9% respectively. Tem-PCR was more sensitive than culture in the detection of Streptococcus penumoniae (36/19.1% vs. 7/3.7%, p<0.01), and there is no significant difference between Tem-PCR and culture for the detection of Hemophilus influenzae (40/21.3% vs. 24/12.8%, p>0.05 ).(4) The total positive rate by combined detection of culture and Tem-PCR assay was 78.2%(147/188), of which 36.7% (54/147) were Hemophilus influenzae , 24.5% (36/147) were Streptococcus pneumoniae, 10.9% (16/147) were Escherichia coli, 8.2% (12/147) were Pseudomonas aeruginosa, 7.5% (11/147) were Staphylococcus aureus, 7.5% (11/147) were Acinetobacter baumannii, 3.4% (5/147) were Pseudomonas aeruginosa, and 1.4% (2/147) were Enterobacter cloacae. The combined detection may increase the positive case of Staphylococcus aureus, Hemophilus influenzae, Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii. (5) Of the 188 respiratory tract specimens, 42.6% (80/188) were positive by single fluorescent quantitation PCR for Mycoplasma pneumoniae, 27.1% (51/188) was positive by Tem-PCR. There is no significant difference between Tem-PCR and FQ-PCR(P>0.05 ). Take the single FQ-PCR as the reference standard, the sensitivity,specificity and coincidence of Tem-PCR was 30.0%,74.3%,55.9%, respectively.(6) The mixed infection with Mycoplasma pneumoniae and bacterium was 16 by Tem-PCR. Of which, the most common mixed infection was Mycoplasma pneumoniae and Hemophilis influenzae, it was 50.0%(8/16).ConclusionsTem-PCR is highly sensitive for detection of Streptococcus penumoniae causing pneumonia in children. While sensitivities for the other bacterium and Mycoplasma pneumonia need improvement. The combined detection of bacterial culture and Tem-PCR assay may increase the positive rate of bacterial pathogens in hospitalized children with CAP and can really demonstrate the distribution of bacterial etiology. Tem-PCR can timely indicate the mixed infection with Mycoplasma pneumoniae and bacterium in etiologic agent causing CAP.