Emerging Options for the Diagnosis of Bacterial Infections and the Characterization of Antimicrobial Resistance

Abstract

Precise and rapid identification and characterization of pathogens and antimicrobial resistance patterns are critical for the adequate treatment of infections, which represent an increasing problem in intensive care medicine. The current situation remains far from satisfactory in terms of turnaround times and overall efficacy. Application of an ineffective antimicrobial agent or the unnecessary use of broad-spectrum antibiotics worsens the patient prognosis and further accelerates the generation of resistant mutants. Here, we provide an overview that includes an evaluation and comparison of existing tools used to diagnose bacterial infections, together with a consideration of the underlying molecular principles and technologies. Special emphasis is placed on emerging developments that may lead to significant improvements in point of care detection and diagnosis of multi-resistant pathogens, and new directions that may be used to guide antibiotic therapy.

Keywords: antimicrobial susceptibility testing; bacterial infections; infectious disease; pathogen identification; point-of-care diagnosis; precision medicine; resistance profiling.

Figure 1

Typical timeframes required for techniques in current use for the diagnosis of bacterial infections. The classical cultivation of biological specimens, in combination with biochemical characterization, requires ~42 h as a minimum estimate. Replacement of biochemical methods with MALDI-TOF mass spectrometry (MS) reduces this timeframe significantly. The use of nucleic acid testing (NAT) bypasses the initial cultivation of clinical specimens, and thus reduces the timeframes to fewer than 4 h. However, NAT methods are sequence-dependent and involve only a limited number of primer-combinations; as such, these methods require a priori knowledge of the suspected pathogen(s). The use of NGS-based methods eliminates the need for any a priori knowledge of a suspected pathogen, although typical timeframes are increased.

Figure 2

Possible shortcuts for bacterial pathogen identification from patient specimens, using MALDI-TOF MS. Typically, specimens are cultivated and identified via biochemical approaches. By MALDI-TOF-based analysis of single colonies obtained during the first round of cultivation, long turnaround times obligatory for biochemical characterization, can be bypassed. Furthermore, patient specimens can be directly analyzed by purification and concentration of bacterial cells, further shortening turnaround time. However, in case of polymicrobial infections, single bacteria need to be further separated, increasing the time to result. As polymicrobial infections cannot be ruled out in most clinical cases, a further separation of single species should be integrated into routine workflows.

Figure 3

Illustration of different technologies for pathogen identification, suitable for point-of-care (POC) application. General approaches can be divided into antigen detection (top) and nucleic acid testing (bottom). Lateral Flow Immunoassays, for example, are readily applicable for detection of pathogen specific antigens, multiplexing-approaches for identification of several different species can be realized by incorporating different fluorescence labels (e.g., quantum dots). Furthermore, Plasmonic Biosensors show a great potential for POC applications, as sensitivity can be drastically increased and sample treatment can be avoided [105]. Another emerging technology for POC application is the Whispering Gallery Mode sensor technology, attracting much attention over the past decade. Here, the binding of molecules to the resonators surface can be detected as a change of the effective refractive index. Although the WGM technology displays a promising candidate, there are currently still several challenges hindering transformation into the clinical environment [78]. In case of approaches for POC applicable nucleic acid testing, several variable approaches are present. In general, shorter time periods during the amplification step can be achieved via implementation of paper-based (e.g., isothermal amplification [19]) or micro-fluidic- based (e.g., micro-fluidic PCR [106]) approaches. For detection of the amplicon, several different technologies can readily be used, including Nucleic Acid Lateral Flow Assays or intercalating dyes.

Figure 4

Overview of current and promising technologies suitable for antimicrobial resistance profiling. Beside classical culture-based approaches (not included), currently available commercial solutions include PCR multiplex panels (e.g., Biofire^®-FilmArray® panels (biomérieux)), MALDI-TOF MS (MTB-STAR Assays (Bruker Daltonics, Inc.)), biochemical tests (e.g., RAPIDEC® CARBA NP test (biomérieux)) and protein marker tests (e.g., RESIST-3 O.K.N. K-SeT (Coris BioConcept, Gembloux, Belgium)). Further promising technological advances have been made in the field of Next-generation Sequencing (e.g., [174]) adaption of biochemical tests to faster electrochemical formats (e.g., [167]) or development of electrochemical sensors for bacterial growth (e.g., [154]). Several of these approaches are suspected to result in commercially available solutions for antimicrobial resistance profiling in the future.