# Multiplex PCR assay for the rapid detection of Klebsiella pneumoniae pathotypes

## TL;DR

This paper introduces a new PCR test that can quickly and accurately detect different types of Klebsiella pneumoniae, including drug-resistant and highly virulent strains.

## Contribution

The study presents a novel multiplex PCR assay that simultaneously detects multiple pathotype markers in Klebsiella pneumoniae with high specificity and sensitivity.

## Key findings

- The m-PCR assay achieved 100% specificity when compared to whole-genome sequencing data.
- The assay successfully detected all target genes without cross-amplification in control strains.
- It demonstrated high sensitivity with efficient amplification from minimal DNA input.

## Abstract

Introduction.
Klebsiella pneumoniae (Kp) is a major cause of nosocomial infections, with its evolving pathotypes including multidrug-resistant, hypervirulent (hvKp) and convergent strains posing significant diagnostic and treatment challenges due to combined antimicrobial resistance and virulence.

Gap Statement. While there is a pressing requirement for thorough detection of Kp pathotypes, current assays in resource-limited environments are unable to effectively focus on essential carbapenemase and hypervirulence genes with the necessary reliability and precision.

Aim. To develop and validate a multiplex PCR (m-PCR) assay capable of simultaneously detecting Kp isolates including those carrying partial or full virulence markers, alongside antimicrobial resistance.

Methodology. In this study, an m-PCR assay was designed and optimized for the simultaneous detection of key biomarkers associated with hypervirulent (rmpA, rmpA2, iucA, peg344 and iroB), carbapenem-resistant (blaNDM, blaOXA-48-like and blaKPC) and convergent Kp pathotypes in clinical isolates. The assay was evaluated on clinical isolates and validated against whole-genome sequencing (WGS) data for accuracy, specificity and sensitivity.

Results. The developed m-PCR assay exhibited 100% specificity when compared to WGS data, successfully detecting all target genes without cross-amplification in ATCC control strains. The assay demonstrated high sensitivity, efficiently amplifying bacterial genomes from minimal DNA input as low as 1 ng µl−1. Additionally, validation through sequencing confirmed the accuracy of detected amplicons.

Conclusion. This m-PCR assay offers a rapid, sensitive and specific diagnostic tool for differentiating Kp pathotypes in clinical settings, aiding in timely intervention and improved infection control measures.