Automation and Molecular Diagnostics: a New Era in Clinical Microbiology

INTRODUCTION

The clinical microbiology laboratory has experienced rapid advancements in technology and automation in recent years. Culture-based testing methods remain the “gold standard” for most laboratories, but there have been several technology advancements incorporated that decrease result turnaround time and subsequently enhance patient care. The previous article for this focus series discusses at length the biochemical and traditional testing that is still a large component of organism identification as well as the increased incorporation of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) technology. For this article, these methods will be referred to as “conventional” testing. The term “conventional” encompasses all culture-based methods that require a pure isolated colony for testing to occur. Although not yet a conventional method, MALDI-TOF technology still relies on pure isolates and will only be briefly discussed.

In this review, we will focus on recent advances in molecular diagnostics that have entered the microbiology laboratory, which include single-target polymerase chain reaction (PCR) based- and nucleic acid hybridization-based assays to the newest syndromic panels that are rapidly receiving Food and Drug Administration (FDA) approval. We will also discuss how this technology is being incorporated into point-of-care (POC) testing, classified as waived by the Clinical Laboratory Improvement Amendments (CLIA), and the associated advantages and pitfalls. Lastly, we will look into the future of the clinical microbiology laboratory with the potential incorporation of next-generation sequencing (NGS), metagenomics for organism identification, and advancements in microbiology total laboratory automation (TLA). With all of the recent advances, there are many corporations developing new testing platforms that are rapidly receiving FDA-approval or will receive FDA-approval in the near future. Literature searches render many comprehensive reviews that discuss these new technologies, give detailed comparisons among platforms, and make compelling arguments for the pros and cons of each. The scope of this review is limited and geared toward a general audience and, therefore, cannot acknowledge every advancement or piece of literature. Any mention of specific technology, scientific studies, or reviews is in no way an endorsement of one over another.

SINGLE-TARGET MOLECULAR DIAGNOSTICS

The development of PCR by Cary Mullis¹ in 1985 is arguably one of the biggest contributions to science and modern medicine. The principles behind this technique have allowed science to decipher the genomes of many species and serve as the basis for the field of molecular diagnostics.² Some of the first molecular tests to become FDA-approved and introduced into the clinical microbiology laboratory were single-target tests that use PCR-based amplification techniques. These assays were followed by technique modifications such as transcription-mediated amplification, loop-mediated isothermal amplification, and helicase-dependent amplification, to name a few.³ These single-target molecular tests use signal amplification and hybridization techniques.⁴ One of the first areas of the clinical laboratory to greatly benefit from these advancements was the virology section. Molecular testing for viral pathogens in patient samples made way for the elimination of tedious and contamination-prone viral cultures using mammalian-cell systems. These culture methods can take weeks for a positive result, if any viable virus can be propagated at all.⁵ Culture-based assays also require the clinical microbiology laboratory to have a designated biosafety cabinet, incubators, and reagents specific for mammalian-cell culture.⁶ The introduction of molecular diagnostics eliminated this need and allowed for determination of viral presence directly from patient specimens with results determined in hours instead of days or weeks.⁵ Arguably one of the largest impacts molecular testing has had on patient and public health is detecting HIV. The rapidness and sensitivity of molecular tests allow for early detection of the virus so that proper treatment regimens may be started sooner and—from the public health standpoint—provide the patient with the knowledge of their HIV status, which helps in the prevention of the spread of the disease. ⁵ Another example is the introduction of PCR-based testing for influenza A and B.⁷ These influenza tests give very rapid results to the ordering physician so that patients may be started on appropriate antiviral therapies. The influenza tests also provide valuable epidemiologic information about the prevalence of the virus during yearly influenza seasons. As can be seen on the FDA website,⁸ there are multiple testing platforms and kits that allow for the direct detection of viral presence. Of the PCR-based technologies listed all have their specific pros and cons, mainly surrounding variability in sensitivity and specificity,² but they collectively improve patient care. Selection of the ideal platform is a laboratory-specific determination that must be based on technical expertise as well as space and budget constraints.

Another area of microbiology testing that has benefitted from molecular advancements is the identification of sexually-transmitted infections (STIs).² STIs are noted public health issues⁹ and some of the causative organisms are notoriously difficult or impossible to culture in standard laboratory settings (ie, Chlamydia trachomatis and Trichomonas vaginalis). Others, such as Neisseria gonorrhoeae, can cause asymptomatic infection—especially in women—leading to missed diagnoses. Using molecular-based testing for suspected STIs or STI screening, especially for individuals at higher risk or who engage in high-risk behaviors, greatly benefits public health because of more rapid testing and result reporting.^2,10

These single-target assays have also been of great use to patient treatment and/or hospital management in terms of identification of drug-resistant organisms (methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococci, etc) and causes of nosocomial infections, such as Clostridioides difficile, which typically leads to patients being placed in isolation.¹¹ Recognition of these pathogens in a timely manner helps prevent the spread of these organisms throughout patient-care areas. Conversely, it also helps prevent unnecessary isolation procedures, which leads to reduced hospital costs.¹²

A summary of the number of available FDA-approved molecular tests for single organisms or organism groups (ie, same genus or subtypes) can be found in Table 1.

POINT-OF-CARE MOLECULAR DIAGNOSTICS

As seen with other methods employed in clinical microbiology laboratories (and other laboratory sections), once an assay or technique has been thoroughly vetted and validated the move to simplify the assay or technique and reduce laboratory hands-on time and overall costs is possible. The majority of POC testing in the past has been of the immunochromatography variety,¹³ but the introduction of POC-molecular testing is coming to the forefront owing to “PCR-in-a-box” technology that uses a closed system, thus greatly reducing the potential for contamination and aberrant or incorrect results.⁴ This type of testing has already been employed in the form of influenza A and B and Group A Streptococcus testing.¹³ It is easy to see the utility of these types of tests in a POC setting, such as a walk-in clinic. Patients may receive their results while still in the examination room (in most instances), allowing rapid progression from diagnosis to prescribed treatment. A major benefit of this rapid testing is the minimization of an afflicted individual’s contact with other people. Another benefit is that POC tests are designed to be foolproof by being simplistic in design and procedure; they are unlikely to give false results, thus allowing nonlaboratory personnel to run tests with success.¹⁴ This, however, does give cause for concern and requires a watchful eye as more CLIA-waived tests come to the forefront because they often lack accredited laboratory professionals performing regular quality control and assurance.

SYNDROMIC PANELS

Syndromic panels are exciting new laboratory tools that are actively being used in many clinical microbiology laboratories. These multiplexed panels are a hot topic in clinical microbiology, and extensive reviews and studies have been published describing their design and clinical performance.^{15⇓⇓⇓⇓-20} The highlights and capabilities of these systems will briefly be covered.

Multiplexed panels are referred to as “syndromic” because they are designed to test for a battery of organisms (viral, bacterial, fungal, and parasitic) that are commonly associated with a specific set of symptoms exhibited by the patient. A major advantage of these systems, which is attributed to their rapid introduction into the clinical laboratory, is that testing is performed directly from patient samples without the need for culturing isolated organisms. To date, panels for sepsis, respiratory tract infections, gastrointestinal (GI) tract infections, and central nervous system (CNS) (meningitis/encephalitis) infections are approved for clinical use.¹⁵ BioFire Diagnostics’s FilmArray (or FilmArray Torch) and Luminex Corporation’s Verigene systems have panels for syndromes listed as previously mentioned. GenMark Diagnostics Inc’s eSensor and ePlex systems offer a respiratory panel.

NEXT-GENERATION SEQUENCING

One of the newest technologies developed is NGS, which is accompanied by subsequent metagenomics analyses. NGS is promising in that it can examine every organism in a patient specimen without requiring specific probes or primer sets.⁵² NGS technology sequences small segments of the entire microbial genome and then compares the sequences with established databases for identification. Pyrosequencing, one of the first NGS technologies developed, allows entire sequencing of 16S ribosomal RNA as well as whole genomes of microbes.⁵² Since then, other methods have been introduced that enhance NGS diagnostics.⁵²

The massive amount of information collected from whole-genome sequencing or the tailored identification of every organism in a biofilm or the gut biome is staggering, but exciting, in terms of the diagnostic information that can be obtained and used for patient care. This technology can deliver significantly more information on an isolate or disease state than any current identification method in the clinical microbiology laboratory, including multiplexed nucleic-acid detection and MALDI-TOF. NGS techniques can identify and differentiate organism serotypes and antibiotic resistance genes, and they can provide additional genomic detail about an organism that may be useful.^{52⇓⇓⇓⇓-57} The FDA is in the process of creating the Food and Drug Administration Database for Regulatory-Grade Microbial Sequences (FDA-ARGOS), which is a regulatory-grade database for microbial sequences that would be the reference standard to which NGS-generated diagnostic sequences would be compared.⁵⁸

Despite the powerful analytic capabilities of NGS, there are many caveats that act as a hindrance to its incorporation into clinical microbiology laboratories. First, the lack of FDA-approval is a significant hurdle; however, as just discussed, this should be alleviated in the near future once the FDA-ARGOS database is put into clinical use. Second, NGS in its current state is expensive and time-consuming compared with already available technology, such as multiplexed PCR testing.⁵⁹ Additionally, because NGS tests patient samples directly, therein lies the challenge of determining which organisms are colonizers/average flora or which are present in a pathologic state. Determination would be even more challenging in the setting of an immunocompromised patient, where the expected colonizer can very well be the source of disease. There is also the obstacle of how laboratories can implement and validate an instrument that can pick up any and all known pathogens, whereas current molecular testing uses preestablished groups of target organisms.⁶⁰ As technology improves and becomes more streamlined, associated costs become more justified, regulatory standards are set, and the ability to incorporate NGS in clinical laboratories will start to become more of a reality.^{52,58⇓⇓⇓⇓-63}

TOTAL LABORATORY AUTOMATION

TLA and robotics have been a part of the clinical laboratory (especially chemistry and hematology) for decades; however, the microbiology laboratory has not progressed very much in this area. There is stand-alone automation in the microbiology laboratory, such as blood culture and identification systems (discussed in the previous focus series article); However, the idea of unified total automation, from specimen processing to release of patient results, is a new concept. A large reason for this is the variety of specimen types processed in the clinical microbiology laboratory. The variety has prevented a “one size fits all” approach to sample processing and distribution for culture, which would be a cornerstone for automation.

Liquid transport media has been suggested as a solution to specimen standardization⁶⁴ for total automation that also works well with the newest identification techniques in the microbiology laboratory, such as MALDI-TOF and multiplexed PCR.⁶⁵ The push for TLA is also driven by the often understaffed laboratory that is handling increased volumes of patient specimens sent in for testing.⁶⁵ Collectively, this makes automation very appealing because it can free up valuable time from what some may consider mundane or repetitive tasks such as media inoculation.

Currently, there are 2 systems for TLA in clinical microbiology: the Becton Dickinson (BD) Kiestra and the COPAN Diagnostics Inc Walk-Away Specimen Processor (WASP) Lab system. Both systems can store, label, and inoculate multiple types of media with samples and include a track system that will transport the inoculated plates to smart incubators.⁶⁶ Multiple studies have demonstrated that automating specimen inoculation alone can dramatically increase the quality and numbers of individually isolated colonies.^{67⇓⇓⇓-71} Additionally, automation may enhance the number of fastidious organisms isolated from urine samples.⁷²

The aptly named smart incubators continuously monitor agar plate media for organism growth by using high-resolution imaging at regularly-timed intervals. These images can then be viewed by laboratorians at a workbench display where decisions can be made regarding further workup. The BD Kiestra is also able to deliver the plates, upon request, to the workbenches should the laboratory scientist want to inspect them manually.⁶⁶

The huge advantage of this type of automated analysis is that colony images can be shared with other laboratories, colleagues, and specialists around the globe. High-resolution imaging also leads to earlier single-colony detection, which may be sufficient for identification via MALDI-TOF systems.⁶⁶ A thorough review by Croxatto et al⁶⁶ describes in detail the similarities and differences in technical specifications between the BD Kiestra and COPAN WASPLab systems.

There are a number of obstacles in the way concerning the ubiquitous adoption of total automation in diagnostic laboratories, primarily the cost and size of the units. These automated laboratories are expensive and require space that may not be feasible to obtain with many current hospital infrastructures.⁶⁵ Another issue is that they are not inclusive of every culture and specimen type. Also, in the instance of a system or software crash or malfunction, backup processes and tests must still be completed. Overall, it is exciting to imagine this technology being incorporated into everyday clinical microbiology laboratories, but the reality is that it may not be ready for widespread use yet. It will take much deliberation to determine the best course of action for any particular laboratory or laboratory network to assess the feasibility of incorporating TLA.

DISCUSSION

Clinical microbiology laboratories are going through very exciting changes. New technology and vast improvements to existing technology are rapidly improving patient care and decreasing patient costs through reduction of the time it takes to identify infectious agents causing disease. Currently, diagnostic microbiology seems to be at a crossroad of conventional testing, implementation of advanced testing platforms, and full-scale automation. Admittedly, there are concerns surrounding this explosion of advanced technology. Laboratory directors and managers must scrutinize and decide what works best for their laboratory environment because a small rural hospital, if they still maintain a fully functioning microbiology laboratory at all, is not likely to see the same traffic as a larger hospital in an urban center. Therefore, the rural locale would likely not benefit as greatly by moving to a fully automated laboratory or incorporating the bulk of current and future advanced technology.

There is also concern that the new testing panels, with all of their advantages, will lure physicians into ordering tests that are unnecessary and potentially increase patient and laboratory costs.¹⁵ On top of this, there is the issue of so much information being produced from these tests that interpretation of the actual meaning of the results could be made more difficult or altogether lost in translation. This could potentially lead to poorer patient outcomes as well as reversion to conventional methods to provide answers the panel could not provide. These are issues to take into consideration when incorporating new technology into existing laboratory workflows.

From an educational standpoint, these advances pose a conundrum in both the workforce and in the classroom. It has been estimated that the average age of a clinical microbiologist is over 50 years old.⁶⁵ This indicates that much of the current workforce has been taught, trained, and certified in conventional testing methodologies. The onus is placed on existing microbiologists to become competent and demonstrate expert judgement with technology rather than assume the “set it and forget it” style of newer technology platforms is error-free and, therefore, take all results at face value. In the classroom setting, the challenge lies with balancing the curriculum to match what is happening in the clinical laboratories. This leads to nearly double the amount of educational material because clinical laboratory educators are still currently held to the standard of teaching conventional methods as well as the principles behind the new technology that our graduates may encounter early on in their careers. Educators are now faced with the decision of whether to shift their curriculum further toward PCR-based and MALDI-TOF identification over conventional identification methods or to double their course content. Regardless of the path taken, the wheels of technology are spinning fast and watching the effects they have on clinical microbiology laboratories will be filled with a healthy mix of skepticism and wonder.

The technologic landscape of the clinical microbiology laboratory is rapidly evolving and does not appear to be slowing. In just a few decades, clinical microbiologists have witnessed the advances from solely culture-based manual-biochemical testing to automated-biochemical testing to the incorporation of MALDI-TOF organism identification. In addition to these impactful advances, PCR-based testing has already been deeply rooted into clinical microbiology testing. A key advancement associated with molecular testing is that it can be performed directly on patients’ samples without the need for isolation of pure bacterial colonies, enhancing time to result, and—subsequently—patient care. Even now, current FDA-approved molecular assays continue to be improved in terms of sensitivity and number of targets in conjunction with the development of new assays. The excitement brought on by these changes is further compounded by the diagnostic potential for the next era of technologic progress, such as NGS and TLA. It will be thrilling to see what the future holds for the clinical microbiology laboratory.