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Febrile #59 – Hide & Seq: An ID Fellow Primer on Molecular Diagnostics

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Summary

Drs. Pratik Patel, Kevin Messacar, and Robin Patel provide a primer on molecular diagnostics, from the basics of DNA and RNA to navigating PCR and metagenomic next generation sequencing.

Table of Contents

Credits

Hosts: Pratik Patel, Sara Dong

Guests: Robin Patel, Kevin Messacar

Writing: Pratik Patel, Sara Dong

Producing/Editing/Cover Art/Infographics: Sara Dong

Our Guests

Guest Co-Host

Pratik “Tik” Patel, MD

Dr. Tik Patel is a 2nd year pediatric ID fellow at Emory University and Children’s Healthcare of Atlanta. He also completed a pediatric hematology/oncology fellowship at the same institution. He wishes to leverage his training in both fields to advance the infectious disease care of immunocompromised children with a focus on those undergoing treatment of cancer and hematopoietic stem cell transplant. He has a burgeoning research interest in introduction and implementation of novel diagnostics for improved stewardship and clinical care.

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Guest Consultant

Kevin Messacar, MD, PhD

Kevin Messacar, MD PhD, is an Associate Professor of Pediatrics at the University of Colorado School of Medicine. He is an attending pediatric hospitalist and infectious disease consultant at Children’s Hospital Colorado. Dr. Messacar obtained a BS with honors in biochemistry at the University of Michigan, MD at the University of Michigan Medical School, and PhD in Clinical Investigations at the University of Colorado Graduate School. Dr. Messacar did his pediatric residency and infectious disease fellowship training at the University of Colorado where he received numerous teaching awards.

Dr. Messacar’s research seeks to improve diagnostic tests and surveillance for central nervous system infections with a focus on enteroviruses and other emerging infectious diseases. He is interested in the process of selecting, implementing, and evaluating newly rapid diagnostic technologies using concepts of diagnostic and antimicrobial stewardship. In 2014, Dr. Messacar received the Colorado Department of Public Health and Environment Astute Physician Award for recognition of the association between acute flaccid myelitis and enterovirus D68 in Colorado children, and he is currently a member of the CDC Acute Flaccid Myelitis Task Force. In 2021, he received the Young Investigator Award from the Pediatric Infectious Disease Society for significant contributions to the field in the first 7 years of his career. He is currently the principal investigator of a multicenter pandemic preparedness clinical research study with the NIAID Vaccine Research Center called PREMISE, Pandemic REsponse REpository: Microbial and Immunologic Surveillance and Epidemiology.

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Guest Consultant

Robin Patel, MD

Robin Patel is the Elizabeth P. and Robert E. Allen Professor of Individualized Medicine and the Director of the Infectious Diseases Research Laboratory, Co-Director of the Clinical Bacteriology Laboratory, Vice Chair of Education in the Department of Laboratory Medicine and Pathology, and former Chair of the Division of Clinical Microbiology, at the Mayo Clinic.

Since the beginning of her tenure at the Mayo Clinic, Dr. Patel has focused her research on bacterial infections. Her work focuses on three major areas: (1) improvement of next-generation diagnostic techniques, (2) understanding the inherent biology of periprosthetic infection, and (3) understanding antibiotic resistance through a clinical lens. She has published over 540 peer-reviewed publications and is supported by the National Institutes of Health and the Centers for Disease Control and Prevention. She is the Director of the Laboratory Center of the Antibacterial Resistance Leadership Group of the National Institutes of Health.

Dr. Patel received an undergraduate degree in Chemistry from Princeton University, where she graduated magna cum laude. From there, she obtained a medical degree from McGill University. Afterwards, Dr. Patel completed Internal Medicine Residency and Fellowships in Medical Microbiology and Infectious Diseases at the Mayo Clinic. Since then, she has been involved in setting standards for diagnostic and clinical care of bacterial infections, as evidenced by the (select) positions she has held or holds within the American Society for Microbiology (President), American Board of Pathology (Microbiology Test Writing Committee Member), Clinical and Laboratory Standards Institute (Subcommittee on Antimicrobial Susceptibility Testing Voting Member), National Institutes of Allergy and Infectious Diseases (Council Member), National Board of Medical Examiners (Microbiology/Immunology Test Material Development Committee Chair), Journal of Clinical Microbiology (Associate Editor), and Clinical Infectious Diseases (Associate Editor).

In addition, Dr. Patel’s continued commitment to mentorship can be translated into a long list of trainees from around the world; she had dedicated hours of teaching to train the next generation of clinical and research laboratory scientists.

More information can be found at: https://journals.asm.org/doi/full/10.1128/JCM.01259-20.

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Consult Notes

We started with the basics. What is DNA? What is RNA? How do these relate to diagnostics of infectious diseases?

  • DNA = deoxyribonucleic acid.  We’ve learned about DNA as the hereditary material in humans, but it is also present in bacteria, fungi, parasites, and DNA viruses.  DNA makes a great target for detecting organisms because the DNA of each microbial species is fairly unique
  • RNA = ribonucleic acid.  This is what is transcribed from DNA in DNA containing organisms, but we also have certain organisms that have an RNA genome and no DNA (such as certain respiratory viruses).  So it’s important that we can use RNA as a target for detection as well

What is PCR?

  • PCR = polymerase chain reaction
  • Described back in the 1980s initially
  • A technique in molecular biology for amplifying small segments of DNA
      • Double stranded DNA is denatured first
      • Technique then involves the use of primers – typically a pair of primers that anneal and then synthesize the DNA between them using an enzyme called DNA polymerase
  • This cycle is repeated over and over again >> so whatever region of DNA is being targeted is amplified exponentially
  • Traditionally you would detect that amplified material with gels or Southern blots, but today we’re commonly using probes. Probes are hybridizing at the same time that the PCR is taking place so that we know we’ve amplified a very specific product, which is important for diagnosis of infectious diseases

Tik asked about different types of PCR “reactions”, such as qRT-PCR (quantitative real-time reverse transcriptase PCR), transcription-mediated amplification (TMA), nucleic acid amplification test (NAAT)

  • Robin explained that there are many ways of amplifying segments of DNA or RNA, and PCR as she described is like a classic or original way of doing so
  • Real-time PCR is based on generation of amplified target which is measured in “real time” with fluorescent probes
  • Reverse transcriptase PCR is a variation in which the starting material is RNA that must first be converted to DNA
  • There are many variations on how to do PCR that differ with the starting material and work on different principles – but the same idea is there that you’re amplifying a specific gene

What is a cycle threshold?

  • When you’re carrying out PCR, you’re amplifying exponentially whatever gene you’re targeting.  You have probes that are hybridizing or you can use specific stains that stain your amplified double stranded DNA, but you’re getting a signal from whatever is giving you the detection
  • That signal will increase over time as you amplify your target
  • At some point, that signal will cross over whatever threshold it is that you are defining as a positive result
  • So the cycle threshold refers to the number of cycles in a PCR assay needed to amplify until you reach that “detectable” threshold
      • CT values can thus indicate the relative amount/level in a specimen
      • A low CT means that you have a gene that you’re targeting that’s present in higher abundance
      • A high CT reflectsthat you have lower amount
  • *This is a general principle though*
      • People have tried to take cycle threshold values and transition what is a qualitative assay into a quantitative assay in ID – but its very important to note that clinical application of the non-standardized CT value is uncertain and not validated to guide management (such as in SARS-CoV2)
      • We do have quantitative assays (for example when we measure HIV viral load), but these are specifically validated to be quantitative and include standards in the assay that tell you when you get a certain quantity, that is the correct quantity.  So there are quality control measures incorporated into those assays

Kevin highlighted a few examples of specific tests that we use that are PCR based. This list is NOT comprehensive, but a few examples:

  • Traditionally we have used a very pathogen specific approach to identify if an organism is present or not (which required clinical suspicion).  Some examples include:
      • Influenza PCR
      • HSV PCR
      • Mycoplasma PCR
      • Group A Strep
      • Gonorrhea/chlamydia NAAT
      • Kingella kingae or Lyme PCR from joint fluid
      • And many more!
  • Kevin explained how there has been a bit of a transition in molecular testing for clinical infectious diseases in the past decade or so, which leaned more towards a syndromic approach.  This is often combining multiple PCR tests in one platform known as a syndromic panel (typically multiplex PCR, so semi nested PCR that can contain multiple targets which can include viruses, bacteria, parasites, fungi; can be DNA and/or RNA targets)
      • Many are familiar with the meningitis/encephalitis infection panels, but others exist for bloodstream infections, respiratory/pneumonia infection, GI illnesses

Next we moved to broad-range PCR

  • We had previously been discussing specific assays that target a single organism, but another approach is to go after a target that is present in every organism
  • Broad-range bacterial PCR testing is based on 16S rRNA
      • Think back to microbiology and genetics, all bacteria/prokaryotes have small and large subunit for their ribosomes which are responsible for making their proteins.  The predominant rRNA genes for the small and large subunits are called 16S and 23S respectively
      • 16S is present in all bacteria, so you can use this as a general indicator that there are bacteria there.  PCR primers can be used to amplify a fragment of the 16S rRNA gene from any bacterium that is present in the sample
      • 16S has areas of “conserved sequences” and then variable regions which are genus and species specific.  The sequence data can be compared to identify which bacterial species were are looking at
  • Broad-range fungal PCR testing
      • Fungi have larger subunits so the small subunit rRNA gene is 18S and large subunit is 28S. These follow the same pathway
  • Broad-range mycobacterial PCR testing
      • Robin explained that Mycobacteria are bacteria, so can still use 16S rRNA gene PCR sequencing assay.  Depending on how the assay is designed, you might be targeting different areas of the 16S rRNA gene to better separate different group of bacteria though
  • So once the relevant genes are amplified by PCR, how does the identification happen?
      • Robin explained Sanger sequencing, which is the most straightforward.  You have bidirectional sequencing (from both forward and reverse primers), which can overlap and provide same answer twice in both directions
      • The concatenated sequence is then run against a database, where you can have some variability
          • You might use a pre-constructed database or
          • Might use a public database such as NCBI, which is not completely curated but is more comprehensive
      • Robin explained that sometimes all related spp are in the analysis and in the database, but sometimes they are not.  Her example was how Brucella spp have pretty much the same sequence
  • In the US, University of Washington-Seattle Molecular Microbiology Diagnostics Lab and Mayo Clinic lab do this testing

What are some possible clinical scenarios where there is data or experience with broad-range PCR?



Next we unpacked metagenomic next-generation sequencing. We’ll include some reminders about the prior DNA sequencing technology as well

  • Sanger sequencing is considered the “first generation” of DNA sequencing.  This is also referred to as traditional or conventional sequencing, as Robin mentioned on the show
      • This determines the sequence of large DNA fragments by collecting and aligning different products polymerized along the DNA template (each nucleotide is tagged, usually by fluorescent tags now rather than radioactive markers)
      • This is used clinically when the sequence of a specific gene is being tested >> it often cannot provide info on multiple genes at a practical cost or within a reasonable timeframe
  • Next-generation sequencing (NGS), sometimes also called high throughput sequencing or deep sequencing
      • This sequences multiple DNA fragments in parallel with a much higher speed and amount and lower cost than Sanger. Robin explained that this could best be referred to as “massively parallel sequencing”
      • There are several platforms developed
  • Robin explained that the term “metagenomic” has been used in various ways, but typically a metagenomic assay is going to involve next generation sequencing
      • You can do this in a completely unbiased way where you just sequence everything in a sample >> everything (human genome, bacteria, fungi, parasites, DNA viruses) but RNA viruses
          • You could introduce a step where you take RNA and convert to DNA to sequence to detect RNA viruses
  • What commercial platforms can clinicians order this sequencing for ID purposes currently?
      • Blood: Karius platform
      • CSF: UCSF, UW

What are some possible clinical scenarios where there is data or experience with next generation metagenomic sequencing?

Here are some papers related to metagenomic and targeted next-generation sequencing

What are limitations of molecular testing as it relates to ID?

  • As discussed on the show, it all goes back to basic principles of diagnostic reasoning (and pretest probability).  It’s important to understand pathogens you may be suspicious of upfront, especially with syndromic panels.  In addition, results must be interpreted in the context of the patient’s presentation
  • Detection of nucleic acid or presence of organisms does not always equate to active infection that is causing illness/disease, especially in platforms that use cell-free DNA
      • We co-exist with microorganisms on and in our bodies, so these tests can detect background or commensal organisms (Kevin’s example was organisms found in immunocompromised patient populations that may be related to disrupted gut barrier)
      • We may also detect organism after the active infectious period is done 
          • Kevin provided the examples of Cdiff PCR testing and prolonged viral shedding after infection
      •  In addition, there is a risk of detecting contamination with this high level of sensitivity (that might be from time of sample collection, lab personnel, reagents used for sequencing, etc)
  • False negatives can occur with low amounts of organisms or nucleic acid to amplify or suboptimal specimen collection
  • It is also important to remember that multiplex PCR panels often have lower sensitivity compared to their respective singleplex tests, such as HSV and Cryptococcal testing with individual PCRs vs. meningitis-encephalitis panels
  • Detection of organisms does not give you susceptibility data for bacteria, mycobacteria, or fungi (yet…)
  • Finding localized infections from blood or other specimens may not be diagnostic
  • Here is a quick read about the human “contaminome” and limitations with high throughput genome sequencing: Simner PJ, Salzberg SL. The Human “Contaminome” and Understanding Infectious Disease. N Engl J Med. 2022;387(10):943-946. doi:10.1056/NEJMcibr2208625

Looking forward

  • Robin and Kevin emphasized how this is a great space for ID fellows to get involved with diagnostic stewardship and clinical research!
  • They emphasized the need for indication or disease driven data – so not just sending the test at any time, but trying to understand how does a particular platform work for say culture negative endocarditis or periprosthetic joint infection.  When is it clinically impactful?  Cost effective?
  • Robin discussed how maybe one day we hope to be able to sequence deep enough to recapitulate the bacteriums chromosomal genetic elements, plasmids, and so forth to analyze genomic data to phenotype susceptibility prediction!
  • Kevin is looking forward to when we might be able to use this testing +/- machine learning to understand or differentiate when something might be an autoimmune response vs pathogen response vs something else

Episode Art & Infographics

Infographics

Find all the Febrile infographics here!

Goal

Listeners will be able to understand the benefits and challenges of use of molecular diagnostics for detecting infectious diseases

Learning Objectives

After listening to this episode, listeners will be able to:

  • Describe the basic principles of polymerase chain reaction, Sanger sequencing, and next generation sequencing
  • Define a cycle threshold
  • Discuss the limitations and challenges of molecular diagnostics for infectious diseases

Disclosures

Our guests (Pratik Patel, Kevin Messacar, Robin Patel) as well as Febrile podcast and hosts report no relevant financial disclosures

Citation

Patel, P., Messacar, K., Patel, R., Dong, S. “#59: Hide & Seq: An ID Fellow Primer of Molecular Diagnostics.”  Febrile: A Cultured Podcast. https://player.captivate.fm/episode/4fbf54e8-5fbd-4dd0-9e58-3608c8855a66

Transcript

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