Rapid detection and identification of pathogens in critically ill and immunocompromised hosts using molecular techniques

Introduction

Infectious complications in immunocompromised patients and critically ill patients represent a serious clinical problem. In these individuals, not only common nosocomial agents but also very unusual pathogens, pathogens with specific cultivation demands or fastidious pathogens can be identified. Timely information on the causative agents is highly important for rapid and accurate clinical decision-making and targeted pharmacotherapy.

Methods

Patients fulfilling the criteria for sepsis, severe sepsis and septic shock, as well as patients with hematological malignancies, patients undergoing chemotherapy, and transplant and post-transplant patients were included in our cohort. We developed a system employing pathogen-specific probes and real-time PCR technology to detect the 25 most frequent human pathogens causing severe nosocomial infections and five fastidious pathogens causing pulmonary infections. Each sample has also been tested using a pan-bacterial and pan-fungal broad-range PCR system coupled with direct sequencing of PCR products for precise identification of the causative agents.

Results

We have investigated 548 clinical samples (peripheral blood 236 samples, bronchoalveolar lavage (BAL) 186 samples, cerebrospinal fluid 32 samples, sputum 29 samples, aspirate from thorax cavity 19 samples, drainage fluids 12 samples, abscesses 11 samples, urine 10 samples, tissue 13 samples). In addition to common pathogens we have identified a set of unusual and fastidious pathogens: Chlamydiophilla pneumoniae (BAL), Mycoplasma pneumoniae (BAL), Peptostreptococcus micros (thorax cavity), Fusobacterium nucleatum, Listeria monocytogenes and Porphyromonas endodontalis (cerebrospinal fluid), Candidatus Neoehrlichia mikurensis (peripheral blood), Mycobacterium tuberculosis (tissue, cerebrospinal fluid), Aspergillus flavus (tissue, sputum), Malassezia pachydermatis (tissue), and Cryptococcus carnescens (BAL).

Conclusion

A pathogen-specific real-time PCR technique coupled with direct pan-bacterial and pan-fungal sequencing represents a very fast and useful tool to accelerate and refine the diagnostics of infections in critically ill patients.