Evolution of the Clinical Microbiology Laboratory

• MALDI-TOF - matrix-assisted laser desorption/ionization time-of-flight
• MS - mass spectrometry
• PCR - polymerase chain reaction

• biochemical
• biomarkers
• diagnostic microbiology
• molecular testing

Infectious diseases have plagued humanity for millennia, yet it was only 140 years ago that the link between infection and microorganism was established. Since that time, various laboratory approaches and techniques have been developed and deployed to increase sensitivity and specificity in the identification of infectious agents. These approaches can be categorized into 3 separate areas based on identification: (1) biochemical or culture-based techniques requiring organism isolation, (2) molecular and genomic methods, and (3) detection of infectious-disease indicators and microbial markers detected in blood, which include serology that are often analyzed outside the microbiology department in the “core” laboratory.

The biochemical culture-based approach was the initial identification method established in the form of plated and tubed media containing various growth substrates, such as carbohydrates and proteins.^1,2 Later, it included the use of rapid (spot) enzyme testing,^3,4 which further established the biochemical methods as an accurate, adaptable, and versatile form of routine bacterial identification. By the 1950s, in the face of increasing population, health care demand, and private and public insurance availability,^5,6 the first significant advancement of the biochemical approach came in the form of miniaturized multitest media and kits,^7⇓-9 which simply took the existing principle of identification and shrunk it. The miniaturized multitest media and kits allowed for reduced labor, media, and waste disposal costs while providing accurate identification in a timely manner.¹⁰ The advantages of the miniaturized-kit approach were further realized with the incorporation of automated growth-based instrumentation,^11⇓-13 which remains the standard for bulk identification of bacteria and many yeast today.^14,15

Only within the last 10–15 years has the basic biochemical principle of growth dependency been replaced as the method for routine organism identification. Mass spectrometry (MS), an analytic chemistry technique used for decades, has been adapted for use as a powerful tool for the diagnostic microbiology laboratory.^16,17 Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, as it is specifically known in the diagnostic laboratory, produces a unique protein fragment “fingerprint” for many different microorganisms, bacterial and fungal alike.^18,19 The advantages of MALDI-TOF cannot be overstated; once an organism has been isolated, MALDI-TOF provides the most accurate and cost effective identification in the shortest amount of time of any technique.^20⇓-22 Of course, there are some limitations of current MS methods, chief among them are the initial cost of the instrument and the need to consistently update the proprietary databases.^23,24 However, the initial cost aside, MS is likely to become more common as laboratories continue to look for ways to handle increasing test volume while providing the fastest turn-around time possible.

Molecular diagnostics made its debut as polymerase chain reaction (PCR)-based testing, which sought to identify single specific organisms from patient samples.²⁵ The area of the microbiology laboratory that benefitted most from these early tests was the virology section, as virology testing typically required mammalian cultures to propagate the virus and took up to 2 weeks before identification was possible.²⁶ Since this first implementation of molecular diagnostics, new testing systems and modifications to PCR technologies have been developed. The technologies reduce hands-on time with test setup, decrease cost, decrease the time to obtain results, and enhance the sensitivity and specificity toward the target organism.²⁷ An example of a current advancement implemented in the microbiology laboratory is syndromic-panel testing, which may concomitantly detect several common infectious organisms associated with particular body system afflictions, such as the upper respiratory and gastrointestinal tract.²⁸

Traditional culture-based identification methods are still a cornerstone of clinical laboratory testing and will most likely remain so in the near future; however, molecular-based testing is becoming increasingly prevalent in microbiology laboratories. The prevalence is evident in the increasing number of molecular tests that receive and seek approval from the Food and Drug Administration for clinical use,²⁹ including next-generation sequencing and whole-genome sequencing of organisms found in patient samples.³⁰ With this potentially forthcoming paradigm shift in organism identification, comes numerous advantages and caveats that must be thoroughly considered before implementation of any of these systems. These considerations will need to be assessed based on an individual laboratory’s needs because there is not yet a “one size fits all” scheme for implementation. Nevertheless, molecular testing will continue to improve and remain a staple in microorganism identification and diagnostics.

Parallel to the biochemical culture-based and molecular-technology developments in the microbiology laboratory, there is a quest to find biomarkers with increased sensitivity and specificity for early identification of infection, especially for the diagnosis of sepsis. Traditional laboratory markers, such as the white blood cell count and erythrocyte sedimentation rate, are longstanding biomarkers of infection and inflammation.^31,32 Research efforts to identify emergent biomarkers for sepsis led to the inclusion of lactate and procalcitonin as effective biomarkers.³³ Several promising emergent biomarkers are being studied for sepsis, including pentraxin 3 and presepsin, which offer quicker diagnosis and prognosis.

Biomarker research expands beyond sepsis and includes promising emergent biomarkers for other bacterial and fungal infections. For example, the human neutrophil elastase and cathepsin G are being investigated as biomarkers for chronic wound infections, as early detection can prevent progression to systemic infection.^34,35 Timely treatment is critical in the case of bacterial pneumonia, and interleukin 6—a cytokine in the acute phase response—is being studied as a potential biomarker.³⁶ Currently, detection of galactomannan allows for rapid testing of infections caused by pathogenic species of Aspergillus while other potential biomarkers for invasive fungal infections are being investigated.³⁷

The technological advances seen throughout the history of diagnostic microbiology are spurred on by the necessity of immediate quality health care services for a growing population, and this will continue to factor in future developments. Yet, each laboratory must consider additional factors unique to their situation—such as their budget, patient population of service, and volume of testing—before deciding whether to implement the newest molecular syndromic testing panel, replace their current automated biochemical analyzer with MALDI-TOF, or validate a new biomarker for disease detection. Nevertheless, newer approaches and technology are becoming more commonly used and are likely to continue to be used as laboratories consolidate into fewer, but larger, facilities that look to maximize efficiency through automation.

This focus series presents a concise review with a brief history of these different approaches by describing their underlying methods, advantages, and disadvantages, while highlighting specific examples of each with the internal and external factors that influenced their development. This focus series is intended to be a basic review of the topic and is written for a general clinical audience.

• Received July 16, 2019.
• Accepted January 7, 2020.