Introduction
Bacterial infections and sepsis cause major morbidity and death in patients hospitalised to the intensive care unit, and their management is complicated by the rise in antibiotic resistance worldwide. In this context, novel diagnostic approaches capable of surpassing standard microbiology’s limitations in terms of turnaround time and accuracy are urgently needed.
While, in some respects, clinical microbiology laboratories have relied upon techniques that have evolved little for many decades, there are a number of emerging technologies that are set to revolutionise how microbial diagnostics may be performed in the near future. Optimisation of existing culture-based approaches, quick antigen detection, nucleic acid detection (including multiplex PCR assays and microarrays), sepsis biomarkers, innovative pathogen detection methods (e.g. T2 magnetic resonance), susceptibility testing and the application of direct metagenomics are all discussed.
This article aims to review the current state of the art, and emerging technologies, that may improve our capacity for rapid and accurate microbiological diagnosis in patients with significant bacterial infections and sepsis.
Established Rapid Diagnostic Methods
Currently, automated Blood Culture (BC) systems are the gold standard for bloodstream infection detection. Most automated BC systems have an internal sensor that detects carbon dioxide or pH as an indicator of microbial growth. Automated image acquisition and machine learning–based approaches for automated Gram stain classifications have been explored, showing promising accuracy although still far from being able to perform as fully automated systems.
Direct antigen testing of clinical samples has aided in the rapid species identification. Despite a shortened TAT, antigen testing suffers from poor sensitivity and specificity. Rapid antigen detection from other clinical samples such as blood, throat swab, synovial fluid, pleural fluid and cerebrospinal fluid (CSF) has been examined previously but is not as commonly used in practice.
Nucleic acid amplification testing (NAAT) or polymerase chain reaction (PCR) tests are a reliable non-culture microbial detection method, frequently used in laboratories around the globe for the diagnosis of a wide array of microbial pathogens. Numerous biomarkers have been explored to assist in the rapid diagnosis of serious infections in ICU. Along with the leucocyte count, the most established of these is C-reactive protein (CRP).
New and Emerging Methods
Emerging Diagnostics for Meningitis and Severe Respiratory Infections
Multiplex PCR are increasingly used in clinical practice for the diagnosis of central nervous system infections and pneumonia in the ICU setting. The BioFire FilmArray Meningitis/Encephalitis panel (bioMerieux) is an FDA-cleared, multiplex PCR detecting 14 pathogens from CSF in 1 h.
Similarly, the BioFire FilmArray Pneumonia plus Panel can detect 27 microorganisms and 7 resistance markers on respiratory specimens, including nosocomial pathogens associated to hospital-acquired or ventilator-acquired pneumonia, and its role in improving antimicrobial stewardship (AMS) in critically ill patients with coronavirus disease 2019 has been suggested.
Nucleic Acid Detection from Blood Cultures
The BioFire FilmArray BC identification panel (BCID) is a multiplex PCR which detects 24 pathogens and 3 resistance genes from positive cultures with good analytical performance. In the specific setting of S. aureus bacteraemia, the Xpert MRSA/SA BC Assay (Cepheid) can detect methicillin-susceptible and methiillin-resistant S. aureus (MRSA) from positive BC. This test is associated with high sensitivity and specificity. Verigene system (Luminex) uses multiplex PCRs and subsequent microarray hybridisation for detection of 22 bacteria and their resistance determinants from positive BC.
Pathogen Detection Direct from Blood
New technologies are emerging that may be used directly on whole blood. The T2 magnetic resonance (T2MR) can identify microorganisms from whole blood with a brand-new methodology. It uses pathogen-specific PCR and miniaturised magnetic resonance to detect microorganisms and resistance genes.
Among NAAT-based methods, Lightcycler SeptiFast Test (Roche) and Magicplex Sepsis Real-Time test (Seegene) are real-time PCR assays detecting several microorganisms and some markers of resistance from whole blood. Despite having broad panels, their low sensitivity makes recommendations about their clinical use difficult.
Direct Metagenomics
Metagenomics-based assays are among the most promising emerging tools in clinical microbiology as they can potentially identify any microorganisms in a given sample. SepsiTest (Molzym) is a semiautomated assay based on pathogen detection from blood. Despite being able to detect polymicrobial infections and fastidious organisms, its role in informing clinical decisions is limited as it suffers of low sensitivity and does not provide AST. Compared to shotgun metagenomics, next-generation sequencing (NGS) reads complete bacterial genomes by parallel sequencing. The ability of shotgun-metagenomics to perform a comprehensive analysis of the microbial genetic material in a biological sample holds great promise. However, limitations exist that make the implementation of these assays complex and a limited real-life clinical impact for diagnosis of infection has been reported by some studies .
New Rapid AST Methods
The detection of resistance genes is not always reliable to reflect the actual susceptibility pattern of the identified pathogen. The FDA-approved Accelerate Pheno system (Accelerate Diagnostics) can detect 16 microorganisms from positive BC based on FISH technology as well as perform phenotypic AST by morphokinetic cellular analysis.
Advances in microfluidics, electronics, optic and biosensor techniques are promising approaches for next-generation rapid AST and at the early stages of translation into practice. Evidences on their role to address point of care testing (POCT) needs, however, are still scarce, and studies are still in progress to achieve FDA approval and CE mark.
Host Response and Transcriptomics
There is growing interest in the role of omics technologies to interrogate the proteome, metabolome, epigenome or transcriptome to more comprehensively characterise infection phenotypes. Transcript-based classifiers have the potential to characterise patients not only by pathogen (bacterial, viral or fungal) but by inflammatory phenotype thereby offering the possibility of successful personalised immunomodulation in sepsis. Establishing the role of transcript-based disease classifiers in infections on ICU will require an understanding of how such assays can be performed in a timely way, and a demonstration of their impact, including cost-effectiveness, in clinical trials.
How Should the Clinical Utility of Novel Rapid Diagnostics Be Evaluated?
Reduced turnaround times (TAT) are not sufficient to show the improved usefulness of a tester in identifying or susceptibility information, even if they are essential components. Sensitivity, specificity, the result kind, and trust from the respective physician acting on the outcome are other parameters. A thorough impact and value assessment of quick diagnostic microbiological technologies will probably be more than assessing the results of TATs and AMS.
We need controlled trials to evaluate several major clinical and process outcomes, like death, acute renal injury, lifespan and readmittion. We require interrupted time series analyses over longer durations. This would ideally be combined with a comprehensive cost-effectiveness analysis, assessing not only hospital admission costs, but value of quality-adjusted-life-year (QALY) saved, costs of laboratory implementation of RDT programs and adjunct AMS programs.
Conclusion
A variety of novel microbiological technologies are anticipated to improve our ability to quickly and reliably identify pathogens in critically ill patients. To define their role in improving the management of severe infections, well-designed trials examining critical clinical outcomes are required.
Source: Peri, A.M., Stewart, A., Hume, A. et al. New Microbiological Techniques for the Diagnosis of Bacterial Infections and Sepsis in ICU Including Point of Care. Curr Infect Dis Rep 23, 12 (2021). https://doi.org/10.1007/s11908-021-00755-0
