Customized molecular diagnostics of bacterial bloodstream infections for carbapenem resistance: A convenient and affordable approach

Abstract

ABSTRACT The acute crisis of carbapenem resistance impedes the empirical use of carbapenems in medical emergencies, especially, bloodstream infections. Carbapenemase-producing carbapenem-resistant organisms (CP-CROs) attribute high case-fatality, necessitating rapid diagnostics to initiate early targeted antibiotics. Expensive diagnostics are the major driver of antibiotic misuse, neglecting evidence-based treatment in India. One in-house molecular diagnostics assay was customized for rapid detection of CP-CROs using positive blood-culture (BC) broths at a low-cost. The assay was validated using a known-set of isolates and evaluated on positive BC broths. DNA was extracted from positive BC broths using a modified alkali-wash/heat-lysis method. One end-point multiplex-PCR was customized targeting five carbapenemases (KPC, NDM, VIM, OXA-48-, and OXA-23-type) with 16S-rDNA as internal extraction control. Carbapenem resistance due to other carbapenemases, efflux-pump activity, and loss of porins was not under the scope of the assay. Promising analytical performances (sensitivity and specificity, >90%; kappa = 0.87), encouraged to assess diagnostic value, qualified the assay for the WHO minimal requirements (both≥95%) for a multiplex-PCR. Higher LR+ (>10) and lower LR− (<0.1) indicate a good diagnostic tool for ruling in or ruling out CRO bloodstream infections. Inclusion of OXA-23-type improved assay positivity. Multiple carbapenemases were detected in>30% of samples. Good concordance was found (kappa = 0.91) with twenty-six discrepant results. The results were available in 3 hours. The running cost of the assay was US$10 per sample. Fast and reliable detection of carbapenemase(s) allows clinicians and infection-control practitioners to execute early-directed therapy and containment measures. This convenient approach facilitates implementing the assay in resource-limited healthcare settings.