Direct detection of Pseudomonas aeruginosa from patients with healthcare associated pneumonia by real time PCR.
Using oprL sequences, a TaqMan real time PCR was developed and used for quantitative detection of Pseudomonas aeruginosa from 99 broncoalveolar lavage and 11 sputum specimens collected from patients with health care associated pneumonia. All specimens were cultured on appropriate media to isolate bacteria. Twenty five specimens were positive by both methods. Polymicrobial infections were found in 13 specimens. Amplification of oprL in serial dilutions ranged from 10(9)CFU/ml to 10(2)CFU/ml. Standard curve of duplicated every dilution had slope 3.25±0.1 and R(2)>0.99 with SD 0.1. Our real time PCR assay showed high sensitivity (100%) and specificity (98.85%). This technique could detect and enumerate 100 bacteria directly from clinical specimens and showed that the threshold is 10(3)CFU/ml in cases with clinical symptoms. Our method can be used for quantitative detection of P. aeruginosa from BAL and sputum specimens in 1h and 10min.
Feizabadi
Mohammad Mehdi
MM
Department of Microbiology, School of Medicine, Tehran University of Medical Sciences, Iran. mfeizabadi@sina.tums.ac.ir
Majnooni
Araz
A
Nomanpour
Bizhan
B
Fatolahzadeh
Bahram
B
Raji
Nafiseh
N
Delfani
Somayeh
S
Habibi
Minoo
M
Asadi
Soroor
S
Parvin
Mahmood
M
D001992:Bronchoalveolar Lavage Fluid / Q000382:microbiology; D003428:Cross Infection / Q000175:diagnosis* / Q000382:microbiology; D004269:DNA, Bacterial / Q000032:analysis; D006801:Humans; D057230:Limit of Detection; D018410:Pneumonia, Bacterial / Q000175:diagnosis* / Q000382:microbiology; D016133:Polymerase Chain Reaction; D011552:Pseudomonas Infections / Q000175:diagnosis* / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000302:isolation & purification*; D011957:Receptors, Opioid / Q000235:genetics; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology; D000094942:Nociceptin Receptor
D004269:DNA, Bacterial; D011957:Receptors, Opioid; D000094942:Nociceptin Receptor