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.