Rapid Identification of Bacterial Antibiotic Resistance by qPCR in Infants with Gram-Negative Septicaemia: A Proof-of-Concept Study

Abstract

Background: Neonatal sepsis remains an important cause of neonatal morbidity and mortality. Tools to rapidly predict antibiotic resistance in neonatal sepsis would be extremely valuable. Objectives: To develop quantitative polymerase chain reaction (qPCR) primer/probe sets that can rapidly detect antibiotic resistance genes common to a neonatal unit, and to investigate the feasibility of direct detection of antibiotic resistance genes in whole blood of infants with Gram-negative septicaemia without first isolating the organism. Methods: Primer/probe sets were designed to detect genes that produce aminoglycoside-modifying enzymes or extended-spectrum β-lactamase. In phase 1, Gram-negative organisms isolated from neonatal clinical specimens within a 12-month period were analysed by qPCR to detect preselected genes. In phase 2, blood specimens of infants with Gram-negative septicaemia were subjected to qPCR analysis to detect antibiotic resistance genes for comparison against conventional antibiotic resistance profile results. Results: Two primer/probe sets showed promising diagnostic utilities for the prediction of antibiotic resistance; the diagnostic utilities (sensitivity, specificity, positive predictive value and negative predictive value) were 90.9, 96.4, 92.6 and 95.5%, respectively, for AAC3-2 [aac(3ʼ)-IIa/aacC3/aacC2, aac(3ʼ)-IIc/aacC2] to detect gentamicin resistance, and 59.3, 99.3, 94.1 and 92.6%, respectively, for BLA-C1 (bla_CTX-M-9, bla_CTX-M-14, bla_CTX-M-24, bla_CTX-M-27) to detect cephalosporin resistance. Twenty-six infants were tested in phase 2, and both gentamicin and cephalosporin resistance patterns were predicted with 100% sensitivity and 100% specificity by AAC3-2 and BLA-C1, respectively. Conclusions: qPCR with appropriately designed primer/probe sets can predict antibiotic resistance directly from neonatal blood, and it can substantially reduce the turnaround time for antibiotic resistance results.

Practice Summary of Antimicrobial Therapy for Commonly Encountered Conditions in the Neonatal Intensive Care Unit: A Canadian Perspective

“…Real-time PCR has the advantage of detecting organisms present in low concentrations using small volumes of blood and providing rapid results. However, the sensitivity and specificity for diagnosis of EOS and detection of antibiotic resistance remains under investigation (53,84).…”