The ability to rapidly diagnose bloodstream infection would have a large impact on the clinical care of patients, particularly in the hospital setting. In 2014, former President Obama outlined a national initiative directed at improving methods to combat antimicrobial resistance, which would benefit from an update in methodologies used to determine antimicrobial resistance. To that end, in this review we highlight some of the newer technologies aimed at rapidly diagnosing bloodstream infection, which are able to generate results in a time frame that is shorter than the 48-72 hour time frame usually required by conventional blood culture techniques. In the second part, we will discuss some of the new methods designed for measuring antimicrobial resistance. There are several opportunities for growth in these areas, and we will present a few.
The tools used for diagnostic clinical microbiology are due for a revolution.1 Excitingly, many researchers and engineers, especially in the field of microfluidics, strongly agree, and are actively working to usher in a new era of diagnostic clinical microbiology.2 Several recent reviews have highlighted the potential impact of microfluidics in diagnostic testing of infectious diseases more generally.3-6 Here we focus on one clinical syndrome that could greatly benefit from an upgrade in diagnostic techniques: bloodstream infection.
The empirical imperative
Given the morbidity and mortality associated with bloodstream infection, clinicians are quickly compelled to institute antimicrobial therapy, often empirically, at the first signs of systemic infection. This empiric antimicrobial therapy is given in an effort to minimize the morbidity and mortality that may result from untreated bloodstream infection. There is evidence that rapid antimicrobial therapy is extremely important in improving outcomes.7-9 What would be extremely valuable, however, would be the ability to rapidly give specific and directed antimicrobial therapy within hours of the first signs of systemic infection, rather than give an empiric choice of therapy based on the clinician’s most educated guess.
As astute as clinicians can be, empiric therapy based on even the best educated guess will usually need modification, once the causative pathogen and its antimicrobial susceptibility have been determined. Furthermore, empiric antimicrobial therapy choices should be modified when updated information about the microbial pathogen, such as species identification or antimicrobial susceptibility, is available for several reasons. These reasons include the need to reduce the propagation of resistant pathogens, to reduce specific patient exposure to unnecessary broad spectrum antimicrobial drugs, and to increase the likelihood that broad spectrum antimicrobials will retain their effectiveness against resistant pathogens. Therefore, rapidly obtaining pathogen-specific information in the diagnosis and treatment of bloodstream infection has the potential to revolutionize the practice of medicine in this area.
The current method of diagnosing bloodstream infection involves the use of blood culture.10-11 Blood culture is performed in the following way: for an adult, usually 10 milliliters of blood is collected from the patient, which is then used to inoculate a blood culture bottle. In general, blood culture bottles will contain growth media and additional additives meant to enhance the recovery of microorganisms that could be present within the collected patient sample. The inoculated blood culture bottle is then monitored using continuous-monitoring blood culture systems. These systems detect the growth of microorganisms by detecting a change in amount of carbon dioxide or oxygen present within the blood culture bottle; a change in the pressure within a blood culture bottle is another technique by which these systems monitor for the growth of microorganisms. Once the growth of microorganisms has been detected, additional steps are taken to more specifically identify the organism, which frequently includes further subculture steps. Alternatively, ribosomal DNA sequencing can be performed to specifically identify a microorganism.12 More recently, mass spectrometry has played an increasing role in microorganism identification with regard to diagnosing bloodstream infection. Notably, current methodologies and the majority of newer techniques being proposed focus on a key aspect of detection: amplifying the signal that corresponds to the presence of bloodstream infection.
Given that current methodologies require at least 48 to 72 hours for pathogen identification, there is certainly room for improvement. In this review, we present the progress in research and development that is ongoing to solve this important need for more rapid tests. We also highlight some of the progress that has been made in the area of test development for determining antimicrobial susceptibility.
Technologies in translation for clinical use
Outside of academic research, much of the focus has been on molecular analysis approaches such as PCR to enable the faster diagnosis of bloodstream infection.13-14 The ability to successfully directly detect pathogens present within patient blood samples without the need for incubation in culture would be a very significant addition. To that end, several technologies in this area have been undergoing commercial development.15
In one example, the developers have created a multiplexed probe PCR-based detection system that is based on work first reported by Lehman and colleagues in 2008.16,17 In this work, the authors analyze whole blood samples with a series of steps, which includes blood sample preparation, DNA amplification with real-time PCR and the use of specific hybridization probes to recognize PCR products, and computer software-mediated automated identification of PCR products generated from microorganism species and controls within the sample. Sample preparation is performed with mechanical lysis using ceramic beads. This is followed by DNA purification where a protease and a chaotrophic buffer18 are both used, to release nucleic acid material and protect released DNA from blood DNAses. Following this, after adding an internal control, DNA within the mixture is purified with a solid-phase extraction method using a spin-column with a glass fiber insert that is subjected to serial wash and centrifugation steps, to complete the sample preparation steps. DNA within the prepared sample is then amplified using real-time PCR, with universal and specific primers targeting a particular genomic region that has been shown to have a higher PCR amplification success rate.19 Finally, the authors use software designed to identify pathogen based on the kinetics of the PCR products generated from each pathogen.
This platform is run using a real-time PCR instrument.20 To date, this technique has been evaluated in 41 phase III clinical trials.21 While these studies were of variable quality, the platform has been shown to have higher specificity than sensitivity, with a specificity of 0.86 (95% CI 0.84-0.89) and a sensitivity of 0.68 (95% CI 0.63-0.73), when compared with conventional blood culture. In another study, authors suggest that the platform can be used to predict disease severity in the diagnosis of patients with sepsis.22 The developed platform is described as having the ability to detect twenty-five pathogen species present within 1.5 milliliters of whole blood in less than six hours.
In a second example, a platform employs a sample preparation step that reportedly enriches microbial nucleic acid material from human nucleic acid material, in an effort to enhance the signal that results from the presence of microbes in the sample.23-25 In this case, by utilizing chaotropic conditions generated within the collected sample, human cells are selectively lysed, after which, enzyme-mediated digestion of human DNA and extracellular pathogen DNA occurs, followed by sedimentation of intact pathogen cells with the aid of centrifugation.26 Intact pathogen cells then undergo enzymatic degradation for the purpose of pathogen DNA purification, which can be completed using a bind-wash-elute method, or any other DNA isolation kit. Purified pathogen DNA is then identified with a universal 16S rRNA gene-based PCR assay.
More advanced approaches
Another system has a similar approach, where microbial nucleic acid material is enriched from mammalian nucleic acid material, to improve test sensitivity.27,28 For this platform, the sample is subjected to a conventional DNA extraction method such as chemical lysis with a physical disruption technique. Enrichment of pathogen DNA from human DNA is achieved by affinity chromatography using a protein that will preferentially bind motifs characteristic of pathogen DNA.29 After these sample preparation steps, a multiplex PCR assay is run on the prepared sample, and species are identified via gel electrophoresis. With a similar approach, another platform is able to detect common causative pathogens of bloodstream infection, in addition to three drug resistance genes.30 In this prospective observational study, authors compared blood culture with three PCR- based assays available commercially.31 Whereas blood culture was positive in 26 percent of collected samples, the evaluated PCR assays were able to detect pathogens in 12 percent, 10 percent, and 14 percent of collected samples.
PCR can also be coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) in order to address the issue of quickly diagnosing bloodstream infection.32-34 The use of PCR/ESI-MS allows the mass of each PCR amplicon to be determined and the specific and clear calculation of the nucleotide base composition, so that organism identification can occur without the use of organism-specific PCR primers.35-40 A modification of the described technique involves using a higher volume of a collected blood sample to increase pathogen detection sensitivity and further optimization of the PCR conditions used for the assay. This platform can be used for the broad identification of fungal, candidal, and bacterial species.41,42
In a final example, superparamagnetic particles that have been functionalized with a pathogen-specific target allow detection of pathogens in bloodstream infections such as fungal candida species.43 This instrument uses a miniaturized, magnetic resonance-based diagnostic to detect pathogens that are bound to functionalized superparamagnetic particles.44-45 By applying the same technique that allows magnetic resonance imaging to be obtained for medical radiology imaging, the designers leverage the difference in signals generated in the presence of an applied magnetic field by aggregation of superparamagnetic particles bound to the pathogen of interest and water molecules present in whole blood.
Rapid determination of antimicrobial susceptibility would also be a key improvement in treated, diagnosed bloodstream infections. With regard to antimicrobial susceptibility testing, Choi and colleagues have developed a technique where images showing the morphological changes that bacteria exposed to various antimicrobials undergo are recorded. The recorded images are then analyzed to rapidly evaluate the antimicrobial susceptibility of tested microorganisms. This analysis is termed SCMA, or single cell morphological analysis. This is an improvement upon their prior reported technique, the microfluidic agarose channel (MAC) system, where, instead of obtaining single cell morphology data, the area occupied by bacteria is calculated, to determine whether or not bacteria are growing. In the SCMA design, morphology data is collected instead. After performing time-lapsed microscopy on bacteria within agarose, the images of bacteria that have been exposed to various antimicrobials are acquired and analyzed, to obtain morphological data corresponding to growth, which correlates with antimicrobial susceptibility testing performed using the broth microdilution method.46 This is in contrast to more conventional techniques, which depend on a more macro-level analysis such as optical density measurements of bacterial suspension turbidity, which reflects the number of organisms present in solution.
Moving into a new era
In summary, new strategies are being employed to bring microbiological diagnostics into a new era (Table 1). With regard to antimicrobial susceptibility testing, the ability to rapidly perform single cell analysis, and to correlate cell characteristics with the effect of an antimicrobial on each cell, has the potential to allow clinicians to more completely understand and quantify the presence of antimicrobial resistance at the single-cell level. This would be an improvement upon current measurements, which more reflect population dynamics at large. With regard to diagnosing bloodstream infection, by focusing on amplifying the signal present that corresponds with the presence of microorganisms in blood, such as microorganism nucleic acid material, the reported technologies are an improvement as compared with blood culture, and can hopefully increase our ability to detect bloodstream infection even in cases where microorganism viability is compromised. With the use of blood culture, there is a need for organisms to remain viable so that they adequately grow, thus allowing the amplification of the number of organisms present so as to facilitate their detection. With these new described molecular techniques, the emphasis is on enriching and amplifying microorganism nucleic acid material by PCR without requiring the organism to be viable. Thus, organism detection can still occur even when the organism is not viable.
In the highlighted technologies, the general approach is the same, although there is some variation in each component of the overall platform process. The exception is with PCR/ESI-MS, where mass spectrometry is now applied for the purpose of identifying nucleic acid amplification products, instead of for the purpose of identifying protein fragments, where mass spectrometry is customarily applied. While PCR is a valuable molecular diagnostic tool, the performance of this method is dependent on the capability of the primer employed for each particular assay. The choice of PCR primer typically requires prior knowledge of the nucleic acid sequence in the genomic region where amplification is desired. A platform that could amplify the presence of microorganisms without the need for culture or even the use of nucleic acid primers, such as with PCR, would be extremely valuable.
The authors would like to acknowledge Dr. Jeffrey Klausner, who provided comments and feedback for this article.
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Oladunni Adeyiga, MD, PhD, is an Assistant Clinical Professor in the Division of Infectious Diseases, Department of Medicine at UCLA. She plans to pursue a career in academic medicine developing diagnostics for use in clinical infectious diseases.
Dino Di Carlo, PhD, is a Professor in the Departments of Bioengineering and Mechanical Engineering in the Henry Samueli School of Engineering and Applied Science at UCLA.