Introduction

Clinicians are at the forefront of the post-antibiotic era where antimicrobial resistance (AMR) in bacterial infections are yielding a global mortality and morbidity burden on par with major infectious diseases such as HIV and malaria [1••, 2, 3••]. In the USA alone, over 2.8 million AMR infections and 35,000 associated deaths were estimated in 2019 [1••]. Novel and concerning forms of AMR have been observed across the globe and continue to emerge [4,5,6]. The economic burden is substantial with estimates of $4.6 billion in US healthcare costs for treatment of hospitalized patients with AMR bacterial infections in 2017 [7]. The COVID-19 pandemic has exacerbated an already difficult-to-contain global health threat. In the USA, years of progress to reduce the burden of AMR infections and related deaths were reversed during the SARS-CoV-2 pandemic [8•].

Rapid diagnostic tests (RDTs) represent a core tool in the effort against AMR and, in turn, patient care optimization. Time to effective antibiotic therapy is a key mediator of clinical outcomes in septic patients as a delay in effective antibiotics is associated with decreasing survival [9]. Rapid identification of pathogens to tailor therapy is crucial but it is also paramount to have rapid detection of resistance genes or rapid phenotypic susceptibility to escalate therapy as needed [10••]. This is an unfortunate reality in the setting of growing AMR globally even to novel agents slated to treat AMR bacteria [11]. In contrast, RDTs can also facilitate de-escalation or discontinuation of unnecessary antibiotics within hours as opposed to days when using conventional identification and susceptibility testing methods [12•]. For these reasons, antimicrobial stewardship guidelines advocate for the use of RDTs to optimize antibiotic therapy, all in the context of a multidisciplinary antimicrobial stewardship team (physicians, pharmacists, clinical microbiologists) to interpret, apply, and optimize RDTs [13,14,15]. In this review, we will critically evaluate recent literature in this area and provide insight into the findings.

Rapid Diagnostic Tests for Bacterial Infections

Traditional growth-based phenotypic methods have been the main tools for bacterial identification and susceptibility testing in clinical microbiology laboratories [16,17,18]. While conventional phenotypic antimicrobial susceptibility test (AST) methods like broth microdilution (BMD) and disk diffusion are the reference AST methods, they are limited by long turnaround time (TAT) and significant hands-on time to perform the test by a well-trained microbiology laboratory technologist.

The evolution of RDTs that use genotypic or rapid phenotypic methods have revolutionized the field of clinical microbiology. Genotypic methods like multiplex polymerase chain reaction (PCR)-based syndromic panels have allowed for identification of organisms and resistance determinants within 2–4 h. Rapid phenotypic methods combined with automated fluorescent in situ hybridization (FISH) technology have allowed for rapid bacterial identification and antibiotic susceptibility results [19].

There are various Food and Drug Administration (FDA)-cleared assays that allow for rapid detection of microorganisms from blood, stool, and respiratory specimens. This section provides an overview of notable rapid diagnostic technologies currently available to detect pathogens using various clinical specimens. Tables 1 and 2 list available FDA-cleared RDTs used for bacterial infections and their key features, including the targeted bacteria, resistance determinants, and turnaround times.

Table 1 US FDA-cleared rapid diagnostic assays for the identification of bacteria and/or bacterial resistance determinants from blood specimens
Table 2 US FDA-cleared rapid diagnostic assays for the identification of bacteria and/or bacterial resistance determinants from non-blood specimens1

Fluorescent In Situ Hybridization/Rapid Phenotypic AST

Combining FISH with time-lapse microscopy allows the Accelerate Pheno® system (Accelerate Diagnostics, Tucson, AZ, USA) to identify microorganisms through FISH probes and provide rapid phenotypic AST in a fully automated manner directly from positive blood cultures [19]. The assay is capable of detecting a number of bacteria (both Gram-positive and -negative) and yeast. In a study evaluating the use of the Accelerate Pheno® system in positive blood cultures for Gram-negative bacteria, this system correctly detected organisms in 88.7% of all bacteremia episodes, and 97.1% of the on-panel isolates were correctly identified using the system’s panel; in addition, 91.3% of specimens containing on-panel organisms yielded AST results [19]. Compared to culture-based methods, the Accelerate Pheno® system decreased time to pathogen identification by 28 h, and for antimicrobial susceptibility by almost 41 h [19].

Multiplex Array Polymerase Chain Reaction

RDTs known as ‘syndromic panels’ are utilized in clinical practice to identify infections with pathogen(s) (e.g., meningoencephalitis; respiratory tract infections; gastroenteritis). For diagnostics, some panels utilize multiplex PCR technology to help identify multiple pathogens in a single test. In 2015, the FDA approved the FilmArray® Meningitis/Encephalitis panel (Biofire® Diagnostics, Salt Lake City, UT, USA) for use in cerebrospinal fluid (CSF) samples obtained from patients with meningitis or encephalitis, with a TAT of approximately 60 min. This test detects 6 bacteria, 7 viruses, and Cryptococcus neoformans/gattii. The FilmArray® Gastrointestinal (GI) panel (Biofire® Diagnostics, Salt Lake City, UT, USA) can be used on stool samples from patients with a diarrheal illness, with a TAT of 60 min. This test can detect 22 targets (bacterial, viral, and parasitic), including species of Campylobacter, Vibrio, Escherichia coli, Yersinia, Salmonella, and Plesiomonas (Table 2). As for respiratory syndromes, the FilmArray® Respiratory panel (Biofire® Diagnostics, Salt Lake City, UT, USA) detects up to 22 targets, including 4 bacterial species (Bordetella parapertussis, B. pertussis, Chlamydia pneumoniae, and Mycoplasma pneumoniae), with a TAT of 45–60 min (Table 2). Another similar panel is GenMark ePlex® RP or RP2 panel (GenMark Diagnostics, Inc., Carlsbad, CA, USA) which utilizes a nasopharyngeal swab sample to detect 2 bacterial species and a multitude of viruses. The Unyvero LRT panel (Curetis GmbH, Germany) utilizes multiplex PCR to detect 20 bacterial species and 10 resistance determinants in lower respiratory tract samples (Table 2).

Multiplex array PCR tests can also be utilized in positive blood culture results to reduce the time to identification of Gram-positive or Gram-negative bacteria, as well as testing for resistance genes. The FDA-cleared FilmArray® Blood Culture Identification (BCID) panel (Biofire® Diagnostics, Salt Lake City, UT, USA) can identify bacterial pathogens from positive blood cultures including Staphylococcus spp., Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii; moreover, it identifies resistance genes such as Klebsiella pneumoniae carbapenemase (KPC)-encoding genes, mecA/C, and vanA/B (Table 1). Other tests utilizing multiplex PCR methods in positive blood cultures include Cepheid® Xpert MRSA/SA panel (Cepheid®, Sunnyvale, CA, USA) and Xpert Carba-R, which can detect genes encoding carbapenemases (Table 1). Uniquely, the Cepheid® Xpert MRSA/SA SSTI test provides rapid identification of S. aureus and MRSA resistance determinants from skin and soft tissue swab specimens in approximately 1 h.

Nanosphere’s FDA-approved Verigene® Gram-Positive Blood Culture Nucleic Acid Test (BC-GP) and Verigene® Gram-Negative Blood Culture Nucleic Acid Test (BC-GN) (Nanosphere, Northbrook, IL) detect multiple bacteria in positive blood cultures along with a multitude of resistance genes in 150 min (Table 1).

The FDA-cleared ePlex® BCID panels (GenMark Diagnostics, Carlsbad, CA, USA) use multiplex PCR platforms to identify pathogens and resistance genes from positive blood cultures. The ePlex BCID-GP panel detects 20 Gram-positive bacteria and 4 resistance genes (Table 1). The BCID-GN panel detects 21 Gram-negative bacteria and 7 resistance genes that confer either extended-spectrum beta-lactamases (ESBLs) or carbapenemases (Table 1). Both panels have a TAT of 90 min.

Nuclear Magnetic Resonance

There are currently 2 FDA-cleared, culture-independent tests that utilize nuclear magnetic resonance technology to detect bacterial (T2Bacteria®) and Candida (T2Candida®) species in whole blood (T2 Biosystems®, Lexington, MA, USA) [20]. The T2Bacteria® Panel can detect 5 common bacterial species implicated in bloodstream infections: Staphylococcus aureus, Enterococcus faecium, Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae. This test has a sensitivity of 83% and specificity of 98% for diagnosing bacteremia with the aforementioned species, with a TAT of 3–5 h [21].

Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry

Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) Mass Spectrometry (MS) technology has significantly helped to reduce time to accurate identification of bacteria, mycobacteria, and fungi with a rapid TAT of <15 min from bacterial growth isolated on plated culture media. There are 2 leading manufacturers in the field of diagnostic microbiology: Vitek MS and Bruker Microflex LT MALDI-TOF MS [22]. Bruker and Vitek have both FDA-cleared and research-use only databases that are regularly updated to incorporate new organisms and relevant taxonomic changes for identification of bacteria, mycobacteria, and fungal pathogens. Additionally, the MBT Sepsityper® Kit US IVD utilizes MALDI-TOF MS technology to identify microorganisms directly from signal positive blood cultures [23].

Clinical Applications of RDTs

Bloodstream Infections

Bloodstream infection (BSI), the primary etiology of sepsis, is the leading cause of inpatient mortality in the USA, with the prevalence rising over the last decade. It is responsible for over 270,000 deaths annually [24] and is the most expensive reason for hospitalization, with over $20 billion in annual cost [25]. In the setting of sepsis, early initiation of appropriate but broad antimicrobial therapy after blood culture collection is critical for improving patient’s morbidity and survival [26] and is recommended by the international Surviving Sepsis Campaign Guidelines [27]. Swift de-escalation of antibiotics to targeted therapy is strongly encouraged; however, this can often be limited by the TAT of conventional testing as it can typically take 12 h to 5 days before an organism is detected [25, 28]. Additionally, patients initially diagnosed with sepsis but later found not to have an infection are often treated with unnecessary broad-spectrum antibiotics for long periods [29].

Advancement in rapid diagnostic tests for detection of organisms from positive blood cultures have shown to improve clinical outcomes. With organism identification being readily available to clinicians within 90 min and the AST results within 7 h, prompt de-escalation or escalation of antibiotics is possible. A randomized controlled trial evaluating outcomes associated with use of RDT for identification of microorganisms and resistance genes demonstrated significant reduction in the time to appropriate antimicrobial de-escalation and escalation and reduced the use of broad-spectrum antimicrobials [12•]. In an observational study performed by Satlin et al. looking at impact of RDT for KPC-encoding gene (blaKPC) and the use of ceftazidime-avibactam on outcomes in patients with carbapenem-resistant Enterobacterales bacteremia, patients who had the rapid blaKPC testing had a shorter time to active antibiotic (median: 24 vs 50 h; p = 0.009) and decreased 14-day (16% vs 37%; p = .007) and 30-day mortality (24% vs 47%; p = 0.007) when compared to patients without blaKPC testing [30]. MALDI-TOF MS, for example, has become the most widely adapted RDT over the recent years because it has shown to overall improve patient outcomes by reduction in time to antibiotic optimization and effective therapy, decreased ICU length of stay (LOS), decreased hospital LOS, and decreased 30-day all-cause mortality [24, 31, 32].

Clinical outcomes in BSI are augmented when paired with antimicrobial stewardship programs (ASP). O’Donnell et al. demonstrated that when early ASP intervention was paired with MALDI-TOF, the time to definitive therapy was shortened by 30.3 h (71.6 vs 41.3 h, p = 0.01) with shorter hospital LOS following the first positive blood cultures (8.7 vs 11.2 days, p = 0.049) when compared to standard of care [33]. Moreover, Verroken et al. demonstrated a drastic reduction in time to optimal antibiotic from 14 h on standard management with MALDI-TOF MS to 4 h when FilmArray® BCID panel was additionally performed [34]. Studies have shown that limited target PCR panels, in combination with ASP, reduced time to optimal therapy as well as decreased hospital costs and shorter LOS in patients with Staphylococcus aureus bacteremia [31, 35]. Additionally, the use of microarray-based early identification and resistance marker detection system (such as Verigene BC-GN) have shown a significant reduction in time to effective antibiotic therapy in ESBL-producing Enterobacterales and vancomycin-resistant Enterococcus BSI as well as high rate of antibiotic optimization in several studies [36,37,38]. This was particularly shown by Kunz Coyne et al., where the time to effective antimicrobial therapy was reduced (15.9 h [IQR 1.9 to 25.7 h] vs 28.0 h [IQR 9.5 to 56.7 h], p < 0.001) with the addition of Verigene BC-GN molecular RDT to existing ASP measures [39]. No statistically significant mortality benefit was seen [39]. Lastly, the utility of Accelerate Pheno® with ASP on clinical outcomes was also assessed in an observational study by Elliott et al., where they noted a potential impact to reduce the time to antibiotic optimization (estimated 18-h reduction from 54.7 to 36.6 h in a simulation-based analysis) [40].

Respiratory Infections

Respiratory tract infections (RTIs) are one of the leading reasons for urgent care and emergency department (ED) visits and account for approximately 4 million deaths per year in children and adults worldwide [41]. It is also one of the most common causes of antibiotic misuse. Though the majority of these infections are viral where treatment with antibiotics is not indicated, over half of acute RTIs are inappropriately treated with antibiotics [42,43,44] leading to secondary adverse effects, complications, and AMR. Thus, RDTs play a critical role not only in limiting unnecessary antibiotic use but also in improving patient care, aiding in therapeutic management, and preventing infection.

In the setting of suspected group A Streptococcus (GAS) infections, for example, the use of highly sensitive RDTs could prevent unnecessary antibiotic use. Some pediatric studies have shown that the use of rapid antigen detection test (RADT) with confirmatory culture was less specific and sensitive which led to increased rates of inappropriate antibiotic use. However, point-of-care (POC) PCR test, compared to RADT, for detection of GAS was highly sensitive and specific with a rapid TAT and led to higher use of targeted antibiotic therapy (97.1% vs 87.5%; p = .0065) [45]. Similarly, a prospective study by Ralph et al. showed that the use of molecular testing compared to traditional throat swab cultures was more sensitive and specific (100% and 79.3%, respectively) with positive and negative predictive values of 48.8% and 100%, respectively. The authors of the study felt that the use of molecular testing could improve antibiotic use [46]. In pediatric patients presenting to the ED with suspicion for streptococcal pharyngitis, it has also been shown that the use of RDTs is associated with decreased antibiotic use [47].

A retrospective analysis of ambulatory, urgent care, and ED encounters found that the use of RDTs aimed for primary diagnosis of upper or lower RTIs were infrequently used in the outpatient setting. Rapid GAS test was predominantly ordered to diagnose pharyngitis with direct implication of ordering antibiotics if the test was positive. However, antibiotics were also often inappropriately prescribed in the setting of a negative result. Similarly, Li et al. describe that of the 323 patients discharged from the ED with a viral diagnosis, 21.1% were inappropriately prescribed antibiotics [48]. The infrequent use of RDTs in the outpatient setting highlights the importance of clinician education on the utility of RDTs and implementation in settings of high volume of patient encounters.

For suspected community-acquired pneumonia (CAP), hospital-acquired pneumonia (HAP), or ventilator-associated pneumonia (VAP), prompt initiation of empirical antibiotics based on the patient’s risk factors is recommended, followed by de-escalation to targeted therapy based on microbiology data [49, 50]. However, this traditional method often causes delays in appropriate therapy given the long TAT. The use of RDTs with reverse transcription (RT)-PCR has played a remarkable role in improving patient care with its quick TAT. Several studies have highlighted that the use of RDTs compared to traditional methods directly correlates with increased documentation of a microbiological etiology [51]. The use of multiplex RDTs in respiratory samples has become particularly helpful during the SARS-CoV-2 pandemic as it can report significantly more viral pathogens than traditional culture techniques when trying to distinguish between a bacterial versus viral etiology for CAP. A prospective study of adult patients with CAP in Taiwan showed that bacterial pathogens were detected in 106 (50%) patients, viruses in 77 (36.3%) patients, and fungal pathogen in 1 patient (0.5%) with overall detection rate being 70.7% (n = 150) in culture and molecular testing method versus 36.7% (n = 78) in traditional microbial culture alone. The most common pathogens were influenza virus (16.1%), Klebsiella pneumoniae (14.1%), P. aeruginosa (13.6%), human rhinovirus (11.8%), and Streptococcus pneumoniae (9.9%) [52]. In addition, several studies have shown not only de-escalation or modification of empirical treatment for CAP/HAP/VAP, but reduction in LOS and hospital costs with the use of RDTs [53,54,55,56,57,58,59,60].

The development and implementation of multiplex RDTs [61] in respiratory infections play a significant role in prompt diagnosis that can affect patient care, possibly limiting the use of antibiotics in the setting of a viral infection. Thus, clinician education and targeted implementation of RDTs are important to enhance and optimize the management of both bacterial and non-bacterial respiratory infections.

Central Nervous System Infections

Accurate diagnosis of central nervous system (CNS) infections is often complicated and challenging due to the overlapping clinical manifestations of infectious and non-infectious etiologies, limited number of RDTs available, and rapid initiation of empiric antimicrobial therapy prior to adequate sample collection for testing, which may decrease the likelihood of organism detection. Greater than 50% of acute meningitis and encephalitis cases do not have an etiology documented, demonstrating the ongoing challenges associated with accurate diagnosis of CNS infections [62, 63]. Syndromic RDTs may help with prompt diagnosis of CNS infections and provide opportunities for selecting a targeted therapy or alternatively allow early discontinuation of unnecessary antimicrobials [63].

The FilmArray® Meningitis/Encephalitis (ME) panel utilizes multiplex PCR technology to detect an array of common pathogens including 6 bacteria: Escherichia coli K1, Haemophilus influenzae, Listeria monocytogenes, Neisseria meningitidis, Streptococcus agalactiae, and Streptococcus pneumoniae. A recent systematic review and meta-analysis compared the diagnostic accuracy of the FilmArray® ME to both traditional culture results (CSF or blood) and clinician adjudicated diagnosis based on clinical presentation, CSF analysis, and/or alternative molecular test results compatible with bacterial infection [64]. This recent review specifically reported on the accuracy of the FilmArray® ME for bacterial pathogens, separate from viral and fungal pathogens also captured by the panel [64]. Across 16 studies (n = 6183), the reported sensitivity and specificity were 89.5% (95% CI 81.1–94.4), and 97.4% (95% CI 94–98.9), respectively, when compared to traditional culture-based diagnosis. When using clinician adjudicated diagnosis, sensitivity and specificity slightly increased to 92.1% (95% CI 86.8–95.3) and 99.2% (95% CI 98.3–99.6) across 15 studies (n = 5524). Moderate sensitivities (range: 64.9–87.5) and high specificities (range: 98.5–99.6) were reported for each individual bacteria, with lowest sensitivities reported for L. monocytogenes, H. influenzae, and E. coli. Therefore, the authors concluded that the FilmArray® ME can be a great tool for ruling in bacterial CNS infections but less reliable for ruling them out; the varying degrees of accuracy per pathogen type is an important consideration in result interpretation [64].

The impact of the FilmArray® ME panel on important clinical outcomes, such as reducing antibiotic use or shortening hospital LOS, remains uncertain. A prospective cohort study of pediatric patients by Posnakoglou et al. reported significant reductions in duration of antimicrobials (4 vs 7 days, p < .001) and the median hospital LOS (5 vs 8 days, p < .001) in patients randomized to the FilmArray® ME panel versus standard of care [65]. However, other studies have shown negligible differences in length of hospitalization and inconsistent effect on reducing antimicrobial use [66]. One study reported that 78% of patients with negative results continued on empiric therapy, demonstrating the reluctance of clinicians to use negative results to rule out an infectious source as contributory to CNS manifestations [67]. Such outcome is in line with the limited utility of the FilmArray® ME to rule out an infection as aforementioned study suggests [64]. Most importantly, all patients should undergo standard diagnostic testing (Gram stain, culture, etc.) for CNS infections as the FilmArray® ME is an adjunct tool that screens a very limited number of bacterial species.

Gastrointestinal Infections

Acute diarrheal illness accounts for approximately 500,000 hospitalizations annually and may be a manifestation of a gastrointestinal (GI) infection [68]. Healthcare costs due to an acute diarrheal illness can be substantial given the need for ED visits, hospitalizations, and multiple diagnostic tests associated with diagnostic workup [69]. Standard diagnostic tests include stool cultures and/or pathogen-directed molecular panels specific to bacteria or viruses or toxins produced by a pathogen. A major challenge in diagnosing infectious diarrhea includes the low sensitivity of stool cultures. Furthermore, clinician knowledge and selection of an appropriate rapid molecular test is essential to minimize delay to accurate etiological diagnosis and treatment of GI infections [70, 71].

There are 11 commercially available multiplex panels for GI infections [72]. The currently available multiplex panels can target varying numbers of pathogens including bacteria, viruses, and parasites. In particular, the BioFire FilmArray® GI panel has the broadest target (13 bacterial, 5 viral, 4 parasitic) and provides a result within 2 h of testing [73]. With high sensitivity and specificity and significant reduction in TAT compared to culture-dependent methods (which may take several days to result), these panels can potentially reduce the number of diagnostics such as imaging studies and additional stool tests [73]. Ultimately, they may affect clinically important patient outcomes such as antibiotic duration, isolation needs, and hospital LOS. A study comparing the FilmArray® GI panel to conventional stool cultures showed that patients tested using the panel had a lower number of stool tests (0.58 vs 3.02 tests per patient, p = .0001) and imaging studies (0.18 vs 0.39, p = .0002) compared to the control group [74]. A cost analysis showed healthcare cost savings of $293.61 per patient [74]. Similarly, Machiels et al. tested the FilmArray® GI panel by running it in conjunction with routine pathogen-directed molecular panels of providers’ choice (n = 182) [70]. The FilmArray® GI panel had a shorter TAT (16 h) compared to standard of care (53 h) and a higher likelihood of a positive finding (39.6% vs 28.6%) in 20 additional patients that had a negative result on standard testing. It was estimated that the FilmArray® GI panel could have resulted in earlier discontinuation of isolation orders by a median of 29 h for 26 patients, 3.6 antibiotic-days saved, and additional imaging prevented in 5 patients. However, these potential benefits may be an overestimate as providers may not be able to review and act upon the results immediately in the real-world setting. Several studies have shown a positive impact of multiplex GI PCR panels on judicious antimicrobial use [68, 71, 75, 76]. A retrospective review of antibiotic use pre- and post-implementation of syndromic GI panel (n = 300) found decreased time to appropriate therapy (1 vs 2 days, p < .0001), increased discharges without antibiotics (14.0% vs 4.5%, p < .001), and shorter hospital LOS (3 vs 7.5 days, p = .0002) compared to a historical cohort that utilized standard stool cultures [68]. Other studies reported an increase in appropriate antibiotic use in the setting of positive result on syndromic GI panel, which is best explained by the higher diagnostic yield compared to that of stool cultures [71, 75]. A study evaluating diagnostic yield and agreement of results of a multiplex GI panel in pediatric patients (n = 125) demonstrated that the use of the panel increased likelihood of microbiological diagnosis compared to conventional microbiology methods (68.8% vs 35.2%, p < .001) [75]. Furthermore, multiplex panel results led to a change in antimicrobial agent for 18 patients with a positive finding (11 new starts, 5 switches to targeted antimicrobial, 2 discontinuations of empiric antimicrobial). Authors concluded that use of the multiplex GI panel increased the judicious use of antimicrobials in hospitalized children [75]. Similarly, a prospective, single-center, randomized controlled trial in the ED (n = 74) showed that the use of multiplex GI panel increased appropriate antibiotic use for bacterial and protozoal diarrheal illnesses compared to culture-dependent testing [71].

Overall, recent studies have similarly shown that multiplex GI panels can shorten time to diagnosis, improve antimicrobial therapy optimization, lead to possible cost-savings from shortened hospitalization, reduce the number of diagnostic tests, and result in early discontinuation of isolation orders. However, like any other PCR-based molecular assays, multiplex GI panels are culture-independent tests and are unable to provide susceptibility data. In addition, clinician interpretation is crucial in the setting of a positive result as detected enteric organisms may not necessarily represent an infection (e.g., C. difficile or E. coli colonization). Lastly, cost-effectiveness data are limited and the use of less broad, pathogen-directed tests should be considered over multiplex assays in the setting of high pretest probability for specific infection such as C. difficile infection.

Interplay Between Rapid Diagnostic Tests and Antimicrobial Stewardship

Between 2014 and 2019, national resistance trends in a cohort of 890 hospitals in the USA demonstrated increasing incidences of ESBL- or carbapenemase-producing Escherichia coli and Klebsiella pneumoniae phenotypes [33]. Using only conventional methods for pathogen detection and identification may result in delayed detection of the resistant phenotype and consequently delay effective therapy compared to RDTs [39]. In the setting of Gram-negative bacteremia, ineffective antimicrobial therapy is a predictor for mortality, second only to intensive care unit (ICU) status [33]. RDTs have demonstrated the potential to reduce time to microbiological data, increase diagnostic yield, and subsequently allow for timely de-escalation or escalation of empiric regimens and lead to improved clinical outcomes [77].

RDTs have demonstrated faster identification of certain pathogens and resistance genes resulting in shorter time to effective treatment and reduction in antibiotic misuse [78•]. However, the majority of studies evaluate the impact of RDTs in conjunction with effective ASP measures. To date, no RDT has been shown to be effective and accurate as the sole method in the diagnosis of bacterial infections [77]. ASPs involve interdisciplinary practices which design and implement strategies to promote the timely evaluation of microbiology data with the objective to optimize antimicrobial use and, subsequently, decrease the risk of unintended adverse effects of suboptimal or ineffective therapy. Because RDTs shorten the time to microbiology results, ASP interventions can be implemented earlier in therapy and allow for earlier optimization of therapeutics. A single-center, prospective cohort study demonstrated that while the use of MALDI-TOF decreased the time to pathogen identification by up to 24 h, only the study cohort with additional ASP measure, in the form of prospective review and feedback by pharmacists, had a significantly decreased time to definitive therapy (41.3 vs 71.6 h, p = 0.01) and decreased hospital LOS (8.7 vs 11.2 days, p = 0.049) [33]. Numerous studies continue to support ASP interventions in conjunction with RDTs for reducing inappropriate antibiotic use and antibiotic-related adverse effects, resulting in lower healthcare costs [24, 30, 33, 39, 667778•].

RDT results may not be as effective in improving patient outcomes without ASPs, which may be related to incorrect interpretation of test results by providers [24]. Interpretation of novel RDTs may be complex and requires educational interventions [78•]. For example, the FilmArray® ME panel was not as effective in reducing inappropriate treatment when diagnostic guidance was not available. This was attributed to the lack of provider knowledge regarding test availability and interpretation of the results [66]. Another consideration is that despite the benefits of RDTs, conventional BMD for AST continues to be the gold standard. For RDTs to be appropriately utilized, providers must understand that results may still require confirmation by conventional methods [24]. In the case of molecular RDTs, genotype detection may not equate to phenotypic susceptibility (i.e., minimum inhibitory concentration [MIC] results), especially in cases of more complex resistance mechanisms. For example, the lack of detection of beta-lactamase genes in Gram-negative bacteria may not exclude phenotypic resistance given other possible resistance mechanisms not included on the panel (e.g., porin mutations, efflux upregulation) [78•]. A mixed method study by Burrowes et al. investigated trends in provider knowledge, behavior, and attitudes towards RDTs, which showed that most clinicians often rely on their clinical judgment over prescribing guidelines. Their survey also found that many clinicians had limited understanding or familiarity with comprehensive respiratory PCR panel and procalcitonin which can help differentiate RTIs of viral or bacterial etiology [79]. In order to maximize the impact of an RDT, the institution must emphasize clinician buy-in and education before, during, and after implementation. The continued education and assessment of provider knowledge can be achieved with an effective ASP in place.

Role of Procalcitonin in the Diagnosis of Bacterial Infections

ASPs are increasingly imperative to ensure optimal utilization of antimicrobials and minimize AMR and healthcare costs. Diagnostic uncertainty and provider reluctance to discontinue antibiotics hinder improvement of ASP outcomes. Procalcitonin (PCT) is a serum biomarker that has shown promise in distinguishing between viral and bacterial infections [80]. PCT is undetectable in healthy conditions but increases rapidly in response to proinflammatory mediators and endotoxins; it has been shown to correlate with the extent and severity of bacterial infections [81]. PCT demonstrates favorable kinetics; levels increase within 3 to 6 h of exposure to bacteria or bacterial endotoxin and peak at 6 to 13 h with a half-life of approximately 22 to 36 h [80, 82,83,84]. Normal PCT serum levels are <0.1 ng/L, whereas PCT levels >0.25 ng/mL may be indicative of bacterial infection; PCT levels decline with clinical improvement [85]. Owing to these characteristics, PCT can be considered a reliable marker of bacterial infections, with a possible role in trending clinical improvement and de-escalation of antibiotics [86]. As such, PCT has been evaluated as a diagnostic tool for bacterial infections to reduce antibiotic use; however, the results have been conflicting [81, 87,88,89].

PCT has been evaluated in various populations and infections, but RTIs have been represented in most of the randomized controlled trials. As a result, in 2017, the FDA cleared the use of the PCT assay to guide the initiation and discontinuation of antimicrobials for suspected lower RTIs and discontinuation in patients with sepsis [90]. Furthermore, studies to date have demonstrated that PCT-based algorithms for antibiotic discontinuation are safe and offer a cost-effective means of reducing antibiotic exposure [91, 92]. More recently, PCT has gained recognition in clinical guidelines to help guide the discontinuation of antimicrobials and initiate antimicrobials independent of PCT level [27, 49].

Although PCT assays have shown promising results over the years, several limitations require consideration before implementing in everyday clinical practice. For example, PCT reliability for guiding antimicrobials has been questioned in various conditions, such as renal dysfunction, cardiac compromise, or immunosuppression. In these situations, PCT levels are elevated at baseline with a further increase in the presence of infection. Additionally, PCT production is particularly elevated in nonbacterial inflammatory processes such as trauma, burns, carcinomas, and immunomodulatory therapy, which can all increase proinflammatory cytokines, making PCT interpretation challenging. While PCT testing can produce rapid results within 1 to 2 h if performed on-site, factors such as intermittent batching or sending to an outside laboratory can delay the availability of results to several days. Such delay can render ineffective the clinical utility of PCT in guiding timely de-escalation of antibiotics.

As with any diagnostic tool, PCT could be part of a clinical algorithm to assist clinicians in evaluating potential infection. However, clinical decisions should not be made exclusively on PCT but in concert with clinical context and evaluation of other diagnostic results. Definitive recommendations remain elusive owing to conflicting data, but PCT’s usefulness in guiding antibiotic discontinuation in the absence of bacterial infections appear promising.

Challenges and Limitations of RDTs

Despite the advances in rapid diagnostic platforms and enhancements in patient care, currently available RDTs are not without challenges and limitations. When introducing new technology into practice, clinician education should be a key component of the implementation process as inadequate dissemination of knowledge of the RDT can lead to incorrect or suboptimal use and interpretation of the test results [79, 93, 94]. While syndromic tests may be useful in quickly identifying a pathogen for early diagnosis and transition to appropriate antibiotic treatment, these platforms are limited to a specific set of common organisms and thus infections by less common organisms cannot be ruled out. This is especially important to consider in patients with different infectious disease risk factors, such as those with compromised immunity. Thus, a negative result may not necessarily rule out an infection. Conversely, false positives as a result of detecting colonizers or contaminants can occur, which is an inherent limitation of molecular-based RDTs. Clinicians are responsible for interpretation of the results and must distinguish colonization (or contamination) from true infection. Overuse of RDTs in cases of low pretest probability not only inappropriately deplete resources, but can also increase likelihood of false negative or positive results; false positive results may mislead clinicians to overtreat which can lead to AMR, antibiotic-associated adverse events, and increased healthcare costs [66, 93,94,95]. For these reasons and more, diagnostic stewardship, ordering the right tests for the right patient at the right time to inform optimal clinical care, continues to emerge as a core part of ASPs to optimize antibiotic use and improve patient outcomes [96•].

In an era of AMR, rapid diagnostics have shown to be useful in providing essential information on drug resistance by capturing genes that encode for certain resistance mechanisms. However, it is prudent to remember the continued evolution of AMR and the development of new and complex resistance mechanisms as this can further challenge treatment decisions [78•, 97]. The utility of a molecular panel to rapidly rule out potential AMR can be limited, especially in areas with high baseline resistance rates, for drug and bacteria combinations with heterogenous resistance mechanisms, and in patients susceptible to resistant organisms (e.g., bone marrow transplant recipients). In contrast, confounding factors such as polymicrobial samples or specific bacterial species may trigger RDT panels to inaccurately report drug resistance [98]. Such errors can misinform clinicians and prompt the use of broad-spectrum antibiotics and delay the opportunity to use a targeted therapy for a given infection. Lastly, there are still limited data to demonstrate the cost-effectiveness of the available RDT platforms. RDTs can be expensive and providing an accurate cost analysis remains challenging as cost savings from improved outcomes and decreased overall healthcare costs are difficult to capture [74, 94, 99].

Emerging Technologies

While there are a number of rapid diagnostic technologies currently available, there remain gaps in which novel or expanded diagnostics would yield benefits in clinical practice. This includes technologies that address novel infection sites or specimen types, detection of clinically important bacteria, and broader susceptibility testing or resistance detection. Such advancements would enhance the impact of RDTs on therapeutic optimization. We note select emerging technologies that address these gaps.

Rapid phenotypic assays have been a welcome advancement in rapid diagnostics given the complexity of resistance mechanisms and associated phenotypic susceptibility prediction. However, expansion in the number of identified organisms and tested antibiotics has the potential to improve patient care. An example is the Next-Generation Phenotyping platform (Selux Diagnostics Inc., Charlestown, MA, USA), which can provide rapid susceptibility results within 5 h. This is a novel method that uses signal amplification of bacterial surfaces to measure bacterial surface area and allows for the differentiation of bacterial morphologies in response to antibiotics (e.g., filamentation versus septation) to delineate susceptible versus resistant isolates by predicting phenotypic MICs. Bacterial surface area-determined MICs had high essential agreement with MICs by Clinical and Laboratory Standards Institute (CLSI) reference method [100]. Most notably, it provides testing capacity for 5 times as many antibiotics in parallel as compared to current automated AST systems [101]. Various other rapid phenotypic AST methods have been developed with other types of technologies [102,103,104].

Bone and joint infections are an often encountered infectious syndrome in clinical practice; however, the yield of conventional culture from synovial fluid to make a microbiologic diagnosis is suboptimal [105]. The BioFire® Joint Infection Panel (BioFire® Diagnostics, Salt Lake City, UT, USA) is a rapid molecular test (1 h) that provides a syndromic panel of 38 target organisms, including fastidious bacteria, and 8 AMR genes using synovial fluid specimen. The broad panel includes targets for aerobic and anaerobic Gram-positive and -negative bacterial pathogens associated with joint infections. The performance characteristics (>90% sensitivity and >99% specificity) also provide a high negative predictive value allowing for potential discontinuation of empiric antibiotics [106]. Clinical data are currently limited and will be necessary to understand real-world utility and impact.

Conclusion

When treating patients for bacterial infections, time to effective therapy influences clinical outcomes. The advancement and implementation of RDTs, in conjunction with antimicrobial stewardship, to enhance treatment selection for bacterial infections should be regarded as a core element to improve clinical outcomes for patients in various healthcare settings. Furthermore, it is crucial to use RDTs in the appropriate clinical context to maximize benefit while ensuring cost-effectiveness. Data suggests that RDTs, particularly combined with ASPs, are cost-effective and can reduce healthcare costs [99]. Although challenges exist in the use of rapid diagnostics, there is a need for continued innovation in technology, implementation science, and collaboration across clinical professions — microbiologists, physicians, pharmacists, nursing, and many others — to optimize care for patients suffering from bacterial infections. High-quality studies will continue to be necessary for current and emerging technologies to understand their clinical role and impact.