Combining Gram stain and 16S qPCR improved diagnostic accuracy for suspected pneumonia and could become a new metric in the rapid diagnosis of lower respiratory tract infections

<b>Introduction.</b> The frequency of multidrug-resistant organisms (MDROs) in hospitals and the risk of delaying effective treatment result in the culture of respiratory secretions for nearly all patients with suspected pneumonia. Culture delays contribute to over prescribing and use of broader spectrum antibiotics.<b>Gap statement.</b> The need for improved rapid diagnostics for early assessment of suspected hospital pneumonia.<b>Aim.</b> To validate a new metric, enhanced Gram stain (EGS), to provide a rapid diagnostic test of high diagnostic accuracy that could be assessed in clinical trials of the use of antibiotics in suspected pneumonia.<b>Methodology.</b> Ninety-two residual lower respiratory samples previously tested by culture and Gram stain were re-tested by 16S ribosomal DNA real-time polymerase chain reaction (16S qPCR) and reported as a combined metric with Gram stain termed EGS. The EGS was assessed for diagnostic accuracy, standard performance measurements and correlation against culture. For samples with discordance between culture and EGS, 16S ribosomal DNA whole operon sequencing (16S rDNA WOS) was used for test resolution. An amended EGS (A-EGS was reassessed against culture.<b>Results.</b> Gram stain, 16S qPCR, EGS and A-EGS had respective diagnostic accuracies of 77.01 %, 82.76 %, 84.04 % and 94.19 %. The same platforms had respective correlation with culture of <i>r</i> = 0.67, <i>r</i> = 0.71, <i>r</i> = 0.81 and <i>r</i> = 0.89. EGS had the highest negative predictive value (NPV) of 93.18 % (81.99 %-97.62 %). Adding an 16S qPCR result is achievable in most routine laboratories and, combined with Gram stain, could improve early decision-making in patients with suspected hospital pneumonia.<b>Conclusion.</b> EGS could improve early decision-making in patients with suspected hospital pneumonia and could be assessed in clinical trials. The 16S rDNA WOS results in the A-EGS also supported the use of pathogen genomic sequencing in early decision making of suspected pneumonia.