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| [[File:US Navy 070905-N-0194K-029 Lt. Paul Graf, a microbiology officer aboard Military Sealift Command hospital ship USNS Comfort (T-AH 20), examines wound cultures in the ship's microbiology laboratory.jpg|right|380px]]
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| '''Title''': ''What types of testing occur within a medical microbiology laboratory?''
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| '''Author for citation''': Shawn E. Douglas
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| '''License for content''': [https://creativecommons.org/licenses/by-sa/4.0/ Creative Commons Attribution-ShareAlike 4.0 International]
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| '''Publication date''': April 2024
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| ==Introduction==
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| The medical [[microbiology]] [[laboratory]] has a variety of testing and workflow requirements that manage to separate it from other biomedical labs. These often complex requirements are in part due to the challenges of analyzing [[microorganism]]s at the microscopic level, as well as the vital role medical microbiology labs play in [[public health]]. As societal and economic pressures such as [[COVID-19]] and hiring challenges have forced these labs to adopt more automated methods to their work, the way these labs work and perform their tests have changed. Regardless, their mandate remains the same: detect, identify, and characterize microorganisms to improve patient outcomes and mitigate infectious agents from spreading across entire populations.
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| This brief topical article will examine the typical types of testing that occur in medical microbiology labs, while also touching upon a few elements of technology and automation, and how they have changed the way these labs perform their activities.
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| ==The medical microbiology lab in general==
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| A medical [[microbiology]] [[laboratory]] helps detect, identify, and characterize [[microorganism]]s for both individual patient treatment and broader population disease prevention and control. In the course of its work towards aiding in the diagnosis of individual patients' ailments, the lab may identify infectious agents of concern and trends in those infections as part of a greater [[public health]] effort. By extension, medical microbiology laboratories are also responsible for reporting those identifications and trends to various public health agencies (city, county, state, and federal). These reports are then used by [[Public health laboratory|public health laboratories]], in tandem with medical microbiology labs, to track incidences and attempt to identify outbreaks.<ref name="RhoadsClin14" /> In particular, the medical microbiology lab is uniquely suited to confirming infectious disease cases as part of outbreak investigations, with its analytical and interpretive "methods that are not commonly available in a routine laboratory setting."<ref name="ECDCCore10">{{cite web |url=https://www.ecdc.europa.eu/sites/default/files/media/en/publications/Publications/1006_TER_Core_functions_of_reference_labs.pdf |format=PDF |title=Core functions of microbiology reference laboratories for communicable diseases |author=European Centre for Disease Prevention and Control |date=June 2010 |publisher=European Centre for Disease Prevention and Control |isbn=9789291932115 |doi=10.2900/29017 |accessdate=24 April 2024}}</ref>
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| A standard consolidated medical microbiology laboratory will have the facilities for rapid microbiology, [[Microscope|microscopy]], [[Cell culture|cell culturing]], serology, molecular biology, parasitology, virology, communicable disease management (i.e., public health or reference activities<ref name="ECDCCore10" />) and more, and it also may have the facilities for environmental microbiology.<ref name="VandenbergConsol20">{{Cite journal |last=Vandenberg |first=Olivier |last2=Durand |first2=Géraldine |last3=Hallin |first3=Marie |last4=Diefenbach |first4=Andreas |last5=Gant |first5=Vanya |last6=Murray |first6=Patrick |last7=Kozlakidis |first7=Zisis |last8=van Belkum |first8=Alex |date=2020-03-18 |title=Consolidation of Clinical Microbiology Laboratories and Introduction of Transformative Technologies |url=https://journals.asm.org/doi/10.1128/CMR.00057-19 |journal=Clinical Microbiology Reviews |language=en |volume=33 |issue=2 |pages=e00057–19 |doi=10.1128/CMR.00057-19 |issn=0893-8512 |pmc=PMC7048017 |pmid=32102900}}</ref> A variety of specimen types will be tested, including urine, blood, stool, tissues, and precious fluids, as well as skin, mucosal, and genital swabs.<ref name="VandenbergConsol20" />
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| Culture-based and other microbiology test methods have largely been performed manually up until recently. As Antonios ''et al.'' noted at the end of 2021, "the introduction of automation in microbiology was considered difficult to apply for several reasons such as the complexity and variability of sample types, the variations of specimens processing, the doubtful cost-effectiveness especially for small and average-sized laboratories, and the perception that machines could not exercise the critical decision-making skills required to process microbiological samples."<ref name="AntoniosCurrent21">{{Cite journal |last=Antonios |first=Kritikos |last2=Croxatto |first2=Antony |last3=Culbreath |first3=Karissa |date=2021-12-30 |title=Current State of Laboratory Automation in Clinical Microbiology Laboratory |url=https://academic.oup.com/clinchem/article/68/1/99/6490228 |journal=Clinical Chemistry |language=en |volume=68 |issue=1 |pages=99–114 |doi=10.1093/clinchem/hvab242 |issn=0009-9147}}</ref> However, economic, employment, and other societal drivers have necessarily brought [[laboratory automation]] and [[large language model]]s (LLMs) more fully to the medical microbiology lab in recent years.<ref name="VandenbergConsol20" /><ref name="AntoniosCurrent21" /><ref name="SandleEnhanc21">{{cite web |url=https://www.europeanpharmaceuticalreview.com/article/166302/enhancing-rapid-microbiology-methods-how-ai-is-shaping-microbiology/ |title=Enhancing rapid microbiology methods: how AI is shaping microbiology |author=Sandle, T. |work=European Pharmaceutical Review |date=22 December 2021 |accessdate=17 April 2024}}</ref> This has allowed these labs to move from a traditional partial-day work schedule to a more 24-hour work schedule by, for example, the use of automated front-end plating systems.<ref name="AntoniosCurrent21" />
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| Whether manual or automated, successful medical microbiology workflows rely on specific quality controls, reporting, instruments, and test methods to achieve overall laboratory and healthcare objectives. The next section will specifically examine the types of testing that occur within a medical microbiology laboratory.
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| ==Medical microbiology testing==
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| Within the scope of detecting, identifying, and characterizing microorganisms, medical microbiology labs depend on a variety of scientific subspecialties (e.g., bacteriology, mycology, virology) and test methods to achieve their goals. What follows are examples of the more common detection, identification, and characterization activities conducted in these labs.
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| ===Detection of microbial growth===
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| By detecting the telltale signs of living microorganisms, such as growth (i.e., an increase in the number of cells), microbiologists can then make an initial diagnosis of microbiological infection and take a deeper dive into identifying the microorganism(s). (Note that measuring microbial growth is not a direct proxy for measuring microbial metabolism, however.<ref>{{Cite journal |last=Braissant |first=Olivier |last2=Astasov-Frauenhoffer |first2=Monika |last3=Waltimo |first3=Tuomas |last4=Bonkat |first4=Gernot |date=2020-11-17 |title=A Review of Methods to Determine Viability, Vitality, and Metabolic Rates in Microbiology |url=https://www.frontiersin.org/articles/10.3389/fmicb.2020.547458/full |journal=Frontiers in Microbiology |volume=11 |pages=547458 |doi=10.3389/fmicb.2020.547458 |issn=1664-302X |pmc=PMC7705206 |pmid=33281753}}</ref>) Growth can be demonstrated in multiple ways, including<ref name=":0">{{Cite book |last=Washington, J.A. |date=1996 |editor-last=Baron |editor-first=Samuel |title=Medical microbiology |chapter=Chapter 10: Principles of Diagnosis |edition=4th ed |publisher=University of Texas Medical Branch at Galveston |place=Galveston, Tex |isbn=978-0-9631172-1-2 |pmid=21413287}}</ref>:
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| *confirming turbidity, gas, or discrete colonies in broth;
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| *confirming discrete colonies on agar plates;
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| *confirming cytopathic effects or inclusions that distort the structures of cells in culture; and
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| *confirming "genus- or species-specific antigens or nucleotide sequences"<ref name=":0" /> in the specimen, culture medium, or culture system.
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| Cell culturing plays an important role, as hinted at above. Those cultures can occur in liquid broth, agar plates, or some other enhanced culture medium, as found with blood cultures in specialized bottles or tubes. Cultures are incubated to allow time for any microorganisms to multiply. Then signs of growth are sought out.<ref name=":0" /> However, detecting this growth is rarely straightforward and has its own set of complications.<ref>{{Cite journal |last=Zengler |first=Karsten |date=2009-12 |title=Central Role of the Cell in Microbial Ecology |url=https://journals.asm.org/doi/10.1128/MMBR.00027-09 |journal=Microbiology and Molecular Biology Reviews |language=en |volume=73 |issue=4 |pages=712–729 |doi=10.1128/MMBR.00027-09 |issn=1092-2172 |pmc=PMC2786577 |pmid=19946138}}</ref><ref name="ŹródłowskiClass20">{{Cite journal |last=Źródłowski |first=Tomasz |last2=Sobońska |first2=Joanna |last3=Salamon |first3=Dominika |last4=McFarlane |first4=Isabel M. |last5=Ziętkiewicz |first5=Mirosław |last6=Gosiewski |first6=Tomasz |date=2020-02-29 |title=Classical Microbiological Diagnostics of Bacteremia: Are the Negative Results Really Negative? What is the Laboratory Result Telling Us About the “Gold Standard”? |url=https://www.mdpi.com/2076-2607/8/3/346 |journal=Microorganisms |language=en |volume=8 |issue=3 |pages=346 |doi=10.3390/microorganisms8030346 |issn=2076-2607 |pmc=PMC7143506 |pmid=32121353}}</ref> This may necessitate other methods such as Gram staining or [[wikipedia:Fluorescence in situ hybridization|fluorescence ''in situ'' hybridization]] (FISH) for quicker and more accurate detection of growth.<ref name="ŹródłowskiClass20" />
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| ===Taxonomic identification and overall characterization===
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| As an extension of detecting microbial growth, microbiologists can examine the growth characteristics of the microorganism(s) in order to identify what type of bacteria or fungi is growing. The identification of viruses, on the other hand, is typically done by examining the cytopathic effects or inclusions that affect cells in culture, or through detection of antigens or nucleotides specific to a viral genus or species.<ref name=":0" /> Databases are commonly used as part of the identification process of microorganisms.<ref name="RhoadsClin14" /> The sources used for these databases highlights some of the identification techniques used. For example, a "biochemical reaction" database implies microorganisms identified with techniques such as [[polymerase chain reaction]] (PCR), [[ELISA|enzyme-linked immunosorbent assay]] (ELISA), fatty acid profiling (using [[gas chromatography]] [GC] and [[mass spectrometry]] [MS]), and metabolic/chemo profiling (using [[high-performance liquid chromatography]] [HPLC] and MS).<ref name="MooreBiochem21">{{cite web |url=https://www.news-medical.net/life-sciences/Biochemical-Tests-for-Microbial-Identification.aspx |title=Biochemical Tests for Microbial Identification |author=Moore, S. |work=News-Medical Life Sciences |date=14 January 2021 |accessdate=26 April 2024}}</ref> A "nucleic acid sequence" database implies microorganisms identified with PCR for a single pathogen<ref name="RhoadsClin14" />, or [[DNA microarray]]s, metagenomics analysis, and [[DNA sequencing#High-throughput methods|next-generation sequencing]] (NGS) for identifying multiple pathogens at the same time.<ref name="RhoadsClin14" /><ref>{{Cite journal |last=Yadav |first=Brijesh Singh |last2=Ronda |first2=Venkateswarlu |last3=Vashista |first3=Dinesh P. |last4=Sharma |first4=Bhaskar |date=2013-01 |title=Sequencing and Computational Approaches to Identification and Characterization of Microbial Organisms |url=http://journals.sagepub.com/doi/10.4137/BECB.S10886 |journal=Biomedical Engineering and Computational Biology |language=en |volume=5 |pages=BECB.S10886 |doi=10.4137/BECB.S10886 |issn=1179-5972 |pmc=PMC4147756 |pmid=25288901}}</ref>
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| All of these techniques have their place in the microbiology lab, with genotypic methods in particular proving useful "for assessing sterility test and media fill failures, and for tracking the route of contamination as part of a contamination control strategy."<ref name="SandleEnhanc21" /> This type of contamination tracking and tracing is enabled by genotypic methods that allow microorganisms to be "characterized," i.e., grouped together based upon the shared characteristics of their DNA fragment patterns or antigenic profiles.<ref>{{Cite journal |last=Kim |first=Young Ran |last2=Lee |first2=Shee Eun |last3=Kim |first3=Choon Mee |last4=Kim |first4=Soo Young |last5=Shin |first5=Eun Kyoung |last6=Shin |first6=Dong Hyeon |last7=Chung |first7=Sun Sik |last8=Choy |first8=Hyon E. |last9=Progulske-Fox |first9=Ann |last10=Hillman |first10=Jeffrey D. |last11=Handfield |first11=Martin |date=2003-10 |title=Characterization and Pathogenic Significance of Vibrio vulnificus Antigens Preferentially Expressed in Septicemic Patients |url=https://journals.asm.org/doi/10.1128/IAI.71.10.5461-5471.2003 |journal=Infection and Immunity |language=en |volume=71 |issue=10 |pages=5461–5471 |doi=10.1128/IAI.71.10.5461-5471.2003 |issn=0019-9567 |pmc=PMC201039 |pmid=14500463}}</ref> Other aspects of a culture may be characterized as well in order to provide a more accurate "description" of the microorganism for future identification efforts.<ref>{{Citation |last=Trüper |first=Hans G. |last2=Krämer |first2=Johannes |date=1981 |editor-last=Starr |editor-first=Mortimer P. |editor2-last=Stolp |editor2-first=Heinz |editor3-last=Trüper |editor3-first=Hans G. |editor4-last=Balows |editor4-first=Albert |editor5-last=Schlegel |editor5-first=Hans G. |title=Principles of Characterization and Identification of Prokaryotes |url=http://link.springer.com/10.1007/978-3-662-13187-9_6 |work=The Prokaryotes |language=en |publisher=Springer Berlin Heidelberg |place=Berlin, Heidelberg |pages=176–193 |doi=10.1007/978-3-662-13187-9_6 |isbn=978-3-662-13189-3 |accessdate=2024-04-26}}</ref>
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| ===Other analyses and techniques===
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| Medical microbiology labs will perform antibiogram and antimicrobial susceptibility testing (AST) as part of their public health function. An antibiogram is a cumulative summary or "overall profile of [''in vitro''] susceptibility testing results for a specific microorganism to an array of antimicrobial drugs," often given in a tabular form.<ref name="UnivMNHowTo20">{{cite web |url=https://arsi.umn.edu/sites/arsi.umn.edu/files/2020-02/How_to_Use_a_Clinical_Antibiogram_26Feb2020_Final.pdf |format=PDF |title=How to Use a Clinical Antibiogram |author=Antimicrobial Resistance and Stewardship Initiative, University of Minnesota |date=February 2020 |accessdate=17 April 2024}}</ref> Given that antibiotic resistance remains one of the primary challenges for global public health, determinations of how susceptible a microorganism is to certain antimicrobials before physician prescription or administration of an antibiotic is of significant value.<ref name="GajicAnti22">{{Cite journal |last=Gajic |first=Ina |last2=Kabic |first2=Jovana |last3=Kekic |first3=Dusan |last4=Jovicevic |first4=Milos |last5=Milenkovic |first5=Marina |last6=Mitic Culafic |first6=Dragana |last7=Trudic |first7=Anika |last8=Ranin |first8=Lazar |last9=Opavski |first9=Natasa |date=2022-03-23 |title=Antimicrobial Susceptibility Testing: A Comprehensive Review of Currently Used Methods |url=https://www.mdpi.com/2079-6382/11/4/427 |journal=Antibiotics |language=en |volume=11 |issue=4 |pages=427 |doi=10.3390/antibiotics11040427 |issn=2079-6382 |pmc=PMC9024665 |pmid=35453179}}</ref> There are multiple approaches to antibiograms for a wide variety of susceptibility testing, common to microbiology labs, including broth and agar dilution, gradient strip test, disk diffusion test, chromogenic and colorimetric test, PCR, DNA microarray, and other methods.<ref name="GajicAnti22" /> The nuances of antibiograms and susceptibility testing drive reporting requirements, particularly to the standard CLSI M39 ''Analysis and Presentation of Cumulative Antimicrobial Susceptibility Test Data''.<ref name="RhoadsClin14">{{Cite journal |last=Rhoads |first=Daniel D. |last2=Sintchenko |first2=Vitali |last3=Rauch |first3=Carol A. |last4=Pantanowitz |first4=Liron |date=2014-10 |title=Clinical Microbiology Informatics |url=https://journals.asm.org/doi/10.1128/CMR.00049-14 |journal=Clinical Microbiology Reviews |language=en |volume=27 |issue=4 |pages=1025–1047 |doi=10.1128/CMR.00049-14 |issn=0893-8512 |pmc=PMC4187636 |pmid=25278581}}</ref><ref>{{Cite journal |last=Simner |first=Patricia J. |last2=Hindler |first2=Janet A. |last3=Bhowmick |first3=Tanaya |last4=Das |first4=Sanchita |last5=Johnson |first5=J. Kristie |last6=Lubers |first6=Brian V. |last7=Redell |first7=Mark A. |last8=Stelling |first8=John |last9=Erdman |first9=Sharon M. |date=2022-10-19 |editor-last=Humphries |editor-first=Romney M. |title=What’s New in Antibiograms? Updating CLSI M39 Guidance with Current Trends |url=https://journals.asm.org/doi/10.1128/jcm.02210-21 |journal=Journal of Clinical Microbiology |language=en |volume=60 |issue=10 |pages=e02210–21 |doi=10.1128/jcm.02210-21 |issn=0095-1137 |pmc=PMC9580356 |pmid=35916520}}</ref> This highlights the importance of the lab not only accurately performing these analyses but also properly reporting the results for consistent and rapid interpretation.
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| Digital image analysis is another important technique used in the medical microbiology lab. This work has traditionally been performed with analog microscopy techniques to identify and characterize microorganisms, even into the early 2010s when digital imaging analysis was becoming more viable.<ref>{{Cite journal |last=Pasulka |first=Alexis L. |last2=Hood |first2=Jonathan F. |last3=Michels |first3=Dana E. |last4=Wright |first4=Mason D. |date=2023-01-19 |title=Flexible and open-source programs for quantitative image analysis in microbial ecology |url=https://www.frontiersin.org/articles/10.3389/fmars.2023.1052119/full |journal=Frontiers in Marine Science |volume=10 |pages=1052119 |doi=10.3389/fmars.2023.1052119 |issn=2296-7745}}</ref> In 2014, Rhoads ''et al.'' characterized automated or semi-automated methods in image interpretation as not being widely implemented in the medical microbiology lab, while at the same time recognizing those methods' potential for screening slides for identifications or characterizations, as well as improving standardization and turnaround time for analyzed specimens.<ref name="RhoadsClin14" /> Since then, laboratory automation, LLMs, and [[artificial intelligence]] (AI) tools—as well as the [[COVID-19]] [[pandemic]]—have pushed the microbiology imaging paradigm forward sufficiently to arguably make digital image analysis more mainstream.<ref name="AntoniosCurrent21" /><ref name="SandleEnhanc21" /><ref>{{Cite journal |last=Burns |first=Bethany L. |last2=Rhoads |first2=Daniel D. |last3=Misra |first3=Anisha |date=2023-09-21 |editor-last=Humphries |editor-first=Romney M. |title=The Use of Machine Learning for Image Analysis Artificial Intelligence in Clinical Microbiology |url=https://journals.asm.org/doi/10.1128/jcm.02336-21 |journal=Journal of Clinical Microbiology |language=en |volume=61 |issue=9 |pages=e02336–21 |doi=10.1128/jcm.02336-21 |issn=0095-1137 |pmc=PMC10575257 |pmid=37395657}}</ref><ref>{{Cite journal |last=Yakimovich |first=Artur |date=2024-02-28 |editor-last=Imperiale |editor-first=Michael J. |title=Toward the novel AI tasks in infection biology |url=https://journals.asm.org/doi/10.1128/msphere.00591-23 |journal=mSphere |language=en |volume=9 |issue=2 |pages=e00591–23 |doi=10.1128/msphere.00591-23 |issn=2379-5042 |pmc=PMC10900907 |pmid=38334404}}</ref> The introduction of automated microscopes "designed to collect high‑resolution image data from microscopic slides" and "high‑resolution image analysis systems that can detect small and mixed colonies, which a human eye cannot"<ref name="SandleEnhanc21" /> are examples of how modern medical microbiology labs are approaching their imaging work.
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| ==Conclusion==
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| With not only its goal of detecting, identifying, and characterizing [[microorganism]]s for improved patient outcomes, but also its public health component of infectious agent detection and trend analysis, the medical microbiology lab plays a pivotal role in disease detection and prevention. The technology, methods, and requirements associated with these efforts are in turn sophisticated, as one might expect when dealing with infectious agents at the micro scale. From cell cultures and digital imaging to genotypic analyses and AST, the complexities of this lab become obvious. We find numerous methods for detecting microbial growth, a precursor for detecting the presence of microorganisms in specimens. From there, identification using growth characteristics, cytopathic effects, and the detection of antigens or nucleotides can provide greater insight. And the characterization of microorganisms and their telltale signs—using numerous techniques like PCR and MS—further enhances the databased knowledge we have on them. We also find that antibiograms and AST are important components to responsible antibiotic use in the global population. Additionally, imaging methods are important and challenging to the medical microbiology lab, requiring more advanced automated systems to assist with identification and characterization in these often understaffed labs.
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| ==References==
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