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

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.

KEYWORDS:

• Carbapenem-resistant organism
• carbapenemase
• bloodstream infection
• rapid diagnostics
• cost-effective
• Multiplex-PCR

Acknowledgements

We thank the technical staffs of Dept. of Microbiology, Nil Ratan Sircar Medical College and Hospital, Kolkata for their assistance during the collection of study blood culture samples and bacterial isolates.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Authors’ contributions

AM: Formal analysis, validation and evaluation; AR: Formal analysis and validation; SS: Formal analysis, resources of blood cultures and isolates, and writing review and editing; KCM: Formal analysis, and writing review and editing; SD: Conceptualization, supervision, methodology, and writing original draft and editing.

All authors have read and approved the final version.

Additional information

Funding

The study was financially supported by the Indian Council of Medical Research (ICMR), New Delhi, India, including the junior research fellowship of AM (grant no. AMR/Adhoc/241/2020-ECD-II).