Standardization of inhouse developed real time polymerase chain reaction (PCR) for universal carbapenemase resistant genes amongst Enterobacterales isolated from septicemic patients in intensive care settings

Abstract

Objectives: To develop inhouse real time polymerase chain reaction (PCR) for carbapenemase anti-microbial resistant genes comprising of imipenem (IMP), Klebsiella pneumoniae carbapenemase (KPC), oxacillinase-48(OXA-48), New Delhi metallo-beta-lactamase (NDM) and verona integron-encoded metallo-beta-lactamase (VIM), keeping multiplex PCR via biofire filmarray (BFA) as the gold standard, and to calculate its diagnostic accuracy.

Method: The cross-sectional, interdisciplinary, diagnostic accuracy (DA) study was conducted from May 9^, 2024, to June 30^, 2025, at the Microbiology Department of the Armed Forces Institute of Pathology, National University of Medical Sciences, Rawalpindi, Pakistan. The sample comprised septicemic inpatients fulfilling Sepsis 3 guidelines. Blood cultures found positive by best available control technology, showing Gram-negative rods on direct staining, were selected for cultures proceedings along with BFA and real time PCR processing. Data was analysed using SPSS 27.

Results: Of the 116 patients, 45(38.8%) were aged <12 years with a mean of 5+3.1years, and 71(61.2%) were aged >12 years with a mean of 48+26.3years. Males were 77(66.4%) and females were 39(33.6%). Of the 116 samples, 102(87.9%) blood culture were found positive. The insilico-designed primers for IMP, KPC, OXA-48 and NDM were successfully optimised and validated. No isolate was found positive for VIM, so its primers cannot be optimised. The inhouse real time PCR demonstrated a DA of 98.9%, sensitivity of 100% and specificity of 98.4% on comparison with blood culture identification panel of BFA.

Conclusion: The optimisation and validation of primers via real time PCR for IMP, KPC, OXA-48 and NDM were successfully completed, showing a high diagnostic accuracy.

Key Words: Septicaemia, Biofire filmarray, Real time PCR, IMP, KPC, NDM, OXA-48.