Direct Screening of Blood by PCR and Pyrosequencing for a 16S rRNA Gene Target from Emergency Department and Intensive Care Unit Patients Being Evaluated for Bloodstream Infection.
Here we compared the results of PCR/pyrosequencing to those of culture for detecting bacteria directly from blood. DNA was extracted from 1,130 blood samples from 913 patients suspected of bacteremia (enrollment criteria were physician-ordered blood culture and complete blood count [CBC]), and 102 controls (healthy blood donors). Real-time PCR assays for beta-globin and Universal 16S rRNA gene targets were performed on all 1,232 extracts. Specimens identified by Universal 16S rRNA gene PCR/pyrosequencing as containing staphylococci, streptococci, or enteric Gram-negative rods had target-specific PCR/pyrosequencing performed. Amplifiable beta-globin (melting temperature [Tm], 87.2°C ± 0.2°C) occurred in 99.1% (1,120/1,130) of patient extracts and 100% (102/102) of controls. Concordance between PCR/pyrosequencing and culture was 96.9% (1,085/1,120) for Universal 16S rRNA gene targets, with positivity rates of 9.4% (105/1,120) and 11.3% (126/1,120), respectively. Bacteria cultured included staphylococci (59/126, 46.8%), Gram-negative rods (34/126, 27%), streptococci (32/126, 25.4%), and a Gram-positive rod (1/126, 0.8%). All controls screened negative by PCR/pyrosequencing. Clinical performance characteristics (95% confidence interval [CI]) for Universal 16S rRNA gene PCR/pyrosequencing included sensitivity of 77.8% (69.5 to 84.7), specificity of 99.3% (98.6 to 99.7), positive predictive value (PPV) of 93.3% (86.8 to 97.3), and negative predictive value (NPV) of 97.2% (96.0 to 98.2). Bacteria were accurately identified in 77.8% (98/126) of culture-confirmed sepsis samples with Universal 16S PCR/pyrosequencing and in 76.4% (96/126) with follow-up target-specific PCR/pyrosequencing. The initial PCR/pyrosequencing took ∼5.5 h to complete or ∼7.5 h when including target-specific PCR/pyrosequencing compared to 27.9 ± 13.6 h for Gram stain or 81.6 ± 24.0 h for phenotypic identification. In summary, this molecular approach detected the causative bacteria in over three-quarters of all culture-confirmed cases of bacteremia directly from blood in significantly less time than standard culture but cannot be used to rule out infection.
The George Washington University, School of Public Health, Department of Epidemiology and Biostatistics, Washington, DC, USA.
M S
MS
Moore
The George Washington University, School of Public Health, Department of Epidemiology and Biostatistics, Washington, DC, USA.
M G
MG
McCarroll
The George Washington University, School of Public Health, Department of Epidemiology and Biostatistics, Washington, DC, USA.
C D
CD
McCann
The George Washington University, Medical Faculty Associates, Department of Emergency Medicine, Washington, DC, USA.
L
L
May
The George Washington University, School of Public Health, Department of Epidemiology and Biostatistics, Washington, DC, USA.
N
N
Younes
The George Washington University, School of Public Health, Department of Epidemiology and Biostatistics, Washington, DC, USA jajordan@gwu.edu.
J A
JA
Jordan
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D016470:Bacteremia / Q000175:diagnosis*; D001419:Bacteria / Q000145:classification / Q000302:isolation & purification*; D001431:Bacteriological Techniques / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D004636:Emergency Service, Hospital; D005260:Female; D006801:Humans; D007362:Intensive Care Units; D008297:Male; D008403:Mass Screening / Q000379:methods*; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D011237:Predictive Value of Tests; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D055815:Young Adult
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S